Production and Separation of 3-Hydroxypropionic Acid

Abstract

The present disclosure provides methods and apparatuses for producing 3-hydroxypropionic acid or its salts, for extracting 3-hydroxypropionic acid from an aqueous solution (e.g., an aqueous culture broth), and for using the same to manufacture various chemicals.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 62 / 577,361, filed on October 26, 2017, and U.S. Provisional Application No. 62 / 594,318, filed on December 4, 2017, under 35 U.S.C.§119(e), the entire contents of which are incorporated herein by reference. Technical field

[0003] The present disclosure relates to the production of 3 - hydroxypropionic acid, including production from glycerol by recombinant strains, extraction from an aqueous solution (e.g., an aqueous culture medium), and its use in the production of various chemicals.

[0004] Background

[0005] 3 - Hydroxypropionic acid (3 - HP) and 3 - hydroxypropionate salts (salts of 3 - HP) are used in the industrial production of various chemicals such as acrylic acid, which can be used as cross - linkers for polymer coatings, metal lubricants, and antistatic agents for textiles.

[0006] Summary

[0007] The present disclosure provides methods and apparatuses for producing 3 - HP or its salts, extracting 3 - HP from an aqueous solution (e.g., an aqueous culture medium), and for manufacturing various chemicals.

[0008] In some embodiments, the present disclosure provides a method for producing 3 - HP or its salts from glycerol using a genetically modified strain of Pseudomonas denitrificans.

[0009] In certain embodiments, the present disclosure provides a method for generating a Pseudomonas strain that can produce 3 - HP or its salts at a high titer without external supplementation of coenzyme B12.

[0010] In some embodiments, the present disclosure provides an expression system (e.g., an expression module or an expression construct) for Pseudomonas strains. Such an expression system can particularly include a tandem promoter system that includes two or more promoters operably linked to control downstream gene expression. Such promoters can be inducible promoters or constitutive promoters. Optionally, the expression system includes at least one inducible promoter. The expression system can be used to produce a target protein / enzyme or enhance a metabolic pathway to produce a target product, such as 3 - HP or its salts, at a high titer by minimizing toxic intermediates and / or increasing the production of vitamin B12 (a cofactor in the first reaction of the 3 - HP synthesis pathway).

[0011] In some embodiments, the present disclosure provides nucleic acids encoding these systems, recombinant bacteria expressing these systems, and / or methods of culturing bacteria to produce 3-HP or a salt thereof. These bacteria have been modified to include one or more expression systems that respond to the level of the target product (in this case 3-HP or a salt thereof) in the culture medium and express genes for producing the target product such as 3-HP or a salt thereof. In certain cases, these bacteria have also been modified to include one or more expression systems to increase the expression of genes for producing B12. Also provided herein are conditions for culturing the bacteria to increase the production of 3-HP (or a salt thereof).

[0012] In one aspect, the present disclosure provides an expression system comprising, consisting essentially of, or consisting of a first promoter that can be induced by a small molecule, a second promoter, a first gene encoding a protein involved in the synthesis of 3-HP (or a salt thereof) or B12, a modified UTR, and a native sequence encoding up to 20 amino acids of the N-terminus of a protein from Pseudomonas denitrificans, wherein the native sequence is operably linked to the 3'-end of the modified UTR and operably linked to the 5'-end of the first gene, and the first and second promoters are operably linked in tandem upstream of the modified UTR.

[0013] In some embodiments of all aspects, the expression system further comprises, consists essentially of, or consists of a third promoter and a second gene encoding a transcriptional regulator configured to regulate the expression of the first gene, wherein the third promoter is operably linked to the second gene. In certain cases, the second promoter can be induced by a small molecule. In certain cases, the native sequence is operably linked to the 5'-end of the first gene to define a fusion gene, the second promoter is the native promoter of a Pseudomonas denitrificans protein, and the second promoter is operably linked to the 5'-end of the fusion gene. In some embodiments, the first promoter is operably linked to the 5'-end of the second promoter, the second promoter is operably linked to the 5'-end of the modified UTR, and the modified UTR is operably linked to the 5'-end of the fusion gene.

[0014] In one aspect, the present disclosure provides a nucleic acid comprising, consisting essentially of, or consisting of a first promoter and a second promoter, wherein the first promoter is an inducible promoter and can be induced by a small molecule; the first and second promoters are operably linked to a first gene; and the first gene encodes a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP) (or a salt thereof) or coenzyme B12.

[0015] In some embodiments in all aspects, the first gene encodes a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP) (or its salt), and is selected from glycerol dehydratase, glycerol dehydratase reactivase, and aldehyde dehydrogenase. In some cases, the first gene comprises at least one gene selected from the group consisting of dhaB1, dhaB2, dhaB3, gdrA, gdrB, and kgsA. In certain cases, the first gene comprises the dhaB1 gene, the dhaB2 gene, the dhaB3 gene, the gdrA gene, and the gdrB gene.

[0016] In some embodiments, the first gene is dhaB1, and its sequence comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 1. In some cases, the first gene is dhaB2, and the sequence comprises, consists of, or consists essentially of a sequence that has at least 95% identity to SEQ ID NO: 2. In some cases, the first gene is dhaB3, and its sequence comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 3.

[0017] In some embodiments in all aspects, the first gene is gdrA, and the sequence comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 4. In some cases, the first gene is grdB, and the sequence comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 5.

[0018] In some embodiments in all aspects, the first gene comprises, consists of, or consists essentially of an aldehyde dehydrogenase gene. In certain cases, the aldehyde dehydrogenase is kgsA. In some cases, kgsA comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 6.

[0019] In some embodiments in all aspects, the small molecule is an acid or an alcohol. In certain cases, the small molecule acid or alcohol is selected from L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), 1,3-propanediol (1,3-PDO), 2,3-butanediol (2,3-BDO), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB) or its salt. In certain cases, the small molecule acid or alcohol is selected from 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB). In some cases, the small molecule is 3-hydroxypropionic acid (3-HP) (or its salt).

[0020] In some embodiments in all aspects, the nucleic acid comprises, consists of, or consists essentially of a sequence that is at least 85% identical to SEQ ID NO: 65. In some cases, the nucleic acid comprises, consists of, or consists essentially of SEQ ID NO: 65.

[0021] In some embodiments in all aspects, the second promoter is operably linked to the 3' end of the first promoter, and the first gene is operably located downstream of the second promoter. In some embodiments in all aspects, the first promoter is operably linked to the 3' end of the second promoter, and the first gene is operably located downstream of the second promoter.

[0022] In some embodiments in all aspects, the second promoter is a constitutive promoter. In some cases, the second promoter is a second inducible promoter. In some cases, the intergenic space between the first and second promoters does not include a terminator sequence. In some cases, the first and second promoters regulate the expression of the first gene.

[0023] In some embodiments in all aspects, the first promoter is derived from the PmmsA promoter, the PhpdH-1 promoter, the PhpdH-4 promoter, or the PhpdH promoter. In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 14 (PmmsA promoter). In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 13 (PhbdH-1 promoter). In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 11 (PhbdH-4 promoter). In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 12 (PhpdH promoter). In some cases, the first promoter comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0024] In some embodiments in all aspects, the first promoter comprises, consists of, or consists essentially of the PmmsA promoter, and the nucleic acid further comprises an operator site operably linked to the 5'-end of the PmmsA promoter. In some cases, the nucleic acids described herein comprise a sequence that is 95% identical to SEQ ID NO: 15, consists of such a sequence, or consists essentially of such a sequence. In certain cases, the nucleic acids described herein comprise a sequence that is 95% identical to SEQ ID NO: 16 (O1 of the MmsA operator) and a sequence that is 95% identical to SEQ ID NO: 17 (O2 of the MmsA operator), consists of such sequences, or consists essentially of such sequences. In some embodiments in all aspects, the nucleic acids described herein comprise a sequence that is 95% identical to SEQ ID NO: 18 (PmmsA and operator, Figure 3A - 3B )), consists of such a sequence, or consists essentially of such a sequence. In some cases, the nucleic acids described herein comprise a sequence selected from SEQ ID NO: 15, 18, 19 (PmmsA2a), 20 (PmmsA2b), and 21 (PmmsA2ab), consists of such a sequence, or consists essentially of such a sequence.

[0025] In some embodiments in all aspects, the second promoter is derived from the P mmsA promoter, the P hpdH-1 promoter, the P hpdH-4 promoter, the P hpdH promoter, or the P zwfPromoter. In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 11 (PhbdH-4 promoter). In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 12 (PhpdH promoter). In some embodiments in all aspects, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 14 (PmmsA promoter). In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 13 (PhbdH-1 promoter). In some embodiments in all aspects, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 7 (Pzwf). In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 8 (Pzwf-1). In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 9 (Pzwf-7). In some embodiments in all aspects, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 62 (shorter Pzwf-7). In some cases, the second promoter comprises a sequence that is at least 95% identical to, consists of, or consists essentially of SEQ ID NO: 10 (Pzwf-12). In some cases, the second promoter comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 52 - 60, 61, 62, and 63 (such as the Pzwf promoter).

[0026] In some embodiments in all aspects, the first promoter comprises, consists of, or consists essentially of the PmmsA promoter, and the second promoter comprises, consists of, or consists essentially of the PhbdH-4 promoter. In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 14 (PmmsA promoter sequence), and the second promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 11 (PhbdH-4 promoter). In some cases, the first promoter comprises, consists of, or consists essentially of the PhpdH-1 promoter, and the second promoter comprises, consists of, or consists essentially of the PhpdH promoter. In some cases, the first promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 13 (PhbdH-1 promoter sequence), and the second promoter comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: 12 (PhpdH promoter).

[0027] In some embodiments in all aspects, the second promoter comprises, consists of, or consists essentially of the PhbdH promoter, and the nucleic acid further comprises, consists of, or consists essentially of an operator site operably linked to the N-terminus of the PhpdH promoter. In some cases, the operator site comprises, consists of, or consists essentially of one or more sequences that are at least 95% identical to SEQ ID NO: RBS-1, RBS-2, ABS-1, ABS-2, and ABS-3. In some cases, the operator site comprises, consists of, or consists essentially of a sequence that is at least 95% identical to SEQ ID NO: RBS-1 site and ABS-2. In some cases, the operator site comprises, consists of, or consists essentially of SEQ ID NO: RBS-1 site and ABS-2.

[0028] In some embodiments in all aspects, the nucleic acid further comprises, consists of, or consists essentially of a gene encoding a transcriptional regulator that regulates the expression of a first gene. In certain cases, the transcriptional regulator binds to the first or second promoter. In certain cases, the transcriptional regulator is a LysR-type transcriptional regulator (LTTR). In some embodiments, the transcriptional regulator is MmsR or HpdR. In certain cases, the transcriptional regulator is MmsR.

[0029] In some embodiments in all aspects, the transcriptional regulator binds to a first promoter. In some cases, the transcriptional regulator is derived from the mmsR protein, and the first promoter is derived from the PmmsA promoter. In some cases, the transcriptional regulator comprises, consists of, or consists essentially of the mmsR protein, and the first promoter comprises, consists of, or consists essentially of the PmmsA promoter.

[0030] In some embodiments in all aspects, the nucleic acid described herein further comprises, consists of, or consists essentially of an operator site to which the MmsR protein binds. In some cases, the nucleic acid described herein comprises a sequence that is at least 95% identical to SEQ ID NO: 19 (PmmsA2a), consists of, or consists essentially of the same. In some cases, the nucleic acid described herein comprises a sequence selected from SEQ ID NOs: 19 - 21 (PmmsA2a, A2b, A2ab), consists of, or consists essentially of the same. In some cases, the transcriptional regulator binds to a second promoter. In some embodiments in all aspects, the second promoter is a constitutive promoter. In some cases, the transcriptional regulator is the HpdR protein, and the second promoter is derived from the hpdH promoter. In some cases, the second promoter comprises a sequence that is at least 95% identical to the sequence of hpdH SEQ ID NO: 12, consists of, or consists essentially of the same.

[0031] In some embodiments in all aspects, the transcriptional regulator binds to a first promoter and the binding to the first promoter is enhanced in the presence of the small molecule. In some cases, the transcriptional regulator binds to a second promoter and the binding to the second promoter is enhanced in the presence of the small molecule. In some cases, the transcriptional regulator is self - regulating. In some cases, the nucleic acid described herein further comprises, consists of, or consists essentially of a third promoter operably linked to the gene encoding the first transcriptional regulator.

[0032] In some embodiments in all aspects, the third promoter comprises a sequence that is 95% identical to a sequence selected from SEQ ID NOs: 7 - 10 and 52 - 63, consists of, or consists essentially of the same. In some cases, the second constitutive promoter is selected from SEQ ID NOs: 9, 10, 57 - 60, 62, and 63; the transcriptional regulator comprises, consists of, or consists essentially of the MmsR protein; and the first promoter comprises mmsA, consists of or consists essentially of the same. In some cases, the second constitutive promoter is selected from SEQ ID NO: 9, 10, 57 - 60, 62, and 63; the transcriptional regulator comprises, consists of, or consists essentially of the HpdR protein; and the first promoter comprises P hbdH , consists of or consists essentially of the same. In certain cases, the third promoter comprises, consists of, or consists essentially of SEQ ID NO: 10. In certain cases, the third promoter comprises SEQ ID NO: 62, consists of, or consists essentially of the same.

[0033] In some embodiments in all aspects, the first gene comprises, consists of, or consists essentially of a modified 5' UTR of the gene. In some cases, the first gene comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO: 22 - 28 and 64 (UTR 0 - 6). In certain cases, the 5' UTR comprises, consists of, or consists essentially of SEQ ID NO: 28 (UTR - 6). In certain cases, the first gene comprises, consists of, or consists essentially of a gene encoding kgsA, and the 5' UTR comprises, consists of, or consists essentially of SEQ ID NO: 28 (UTR - 6).

[0034] In some embodiments in all aspects, at least 10 codons at the 5′ end of the first gene are optimized for translation in Pseudomonas denitrificans. In certain cases, up to 10 codons at the 5' end of the first gene are optimized for translation in Pseudomonas denitrificans. In certain cases, codon optimization comprises, consists of, or consists essentially of the following: measuring the codon frequency of each amino acid in a gene encoding a native Pseudomonas denitrificans protein; replacing the 10 codons at the 5' end of the first gene with 10 optimized codons using the codon frequency of the native protein, wherein the codons of each amino acid of the 10 optimized codons are present at the same frequency as the codon frequency in the native protein. In certain cases, the codon frequency of each amino acid in the 10 optimized codons is greater than 0. In certain cases, the codon frequency of each amino acid in 10 codons at the 5' end of a gene encoding a native Pseudomonas denitrificans protein is measured.

[0035] In some embodiments in all aspects, the nucleic acid comprises, consists of, or consists essentially of a sequence that is 95% identical to a sequence selected from SEQ ID NOs: 29, 30, and 31 (Opt1-3). In some cases, the first gene comprises, consists of, or consists essentially of a gene encoding kgsA and comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 29-31. In certain cases, the first gene is fused at its 5' end to a sequence encoding up to 20 amino acids from the 5' end of a second gene encoding a native Pseudomonas denitrificans protein, thereby forming a fusion gene. In certain cases, the mRNA of the fusion gene is more stable than the mRNA of the first gene alone. In certain cases, the fusion gene increases gene translation compared to the first gene alone. In some embodiments in all aspects, the mRNA of the fusion gene is more resistant to ribonuclease degradation than the mRNA of the first gene alone. In certain cases, the fusion gene increases gene translation compared to the first gene alone.

[0036] In some embodiments in all aspects, the first gene is fused at its 5' end to a sequence encoding up to 20 (5, 10, 15, or 20) amino acids from the 5' end of a second gene encoding a native Pseudomonas denitrificans protein, thereby producing a fusion gene, and the second promoter is derived from the native promoter of the second gene. In some cases, the second promoter is derived from the P mmsA promoter, and the fusion gene comprises, consists of, or consists essentially of a sequence encoding at least 5, 10, 15, or 20 amino acids from the N-terminus of the native MmsA protein. In some cases, the second promoter comprises, consists of, or consists essentially of the PmmsA promoter, and the fusion gene comprises, consists of, or consists essentially of a sequence encoding five or more amino acids from the N-terminus of the native MmsA protein, and the fusion gene is operably linked to the 3' end of the PmmsA promoter. In some cases, the nucleic acid comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 32-35.

[0037] In some embodiments in all aspects, the first gene is fused at its 5' end to a sequence encoding up to 20 (5, 10, 15, or 20) amino acids from the N-terminus of a sequence encoding a native Pseudomonas denitrificans MmsA protein. In some cases, the nucleic acid described herein comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 32-35 (Pcm-mmsA(5)(10)(15)(20)).

[0038] In some embodiments in all aspects, the first gene encodes a protein involved in vitamin B12 synthesis. In certain cases, the first gene comprises, consists of, or consists essentially of a gene selected from the following: cobJ, cobI, cobH, cobG, cobL, cobF, cobK, gst, xre, chlD, chlI, dahp, cobNcobW, cbtBA, cobE, cobM, btuB, cobO, cob, cobR, cobD, cobC, cobQ, cobU, cobP, bgpM, and cobV. In some cases, the first gene comprises, consists of, or consists essentially of bgpM. In certain cases, the first gene is derived from a Pseudomonas species selected from the following: Pseudomonas denitrificans; Pseudomonas aeruginosa; Pseudomonas entomophila; Pseudomonas putida; Pseudomonas syringae; Pseudomonas fluorescens; Pseudomonas mendocina; and Pseudomonas stutzeri.

[0039] In another aspect, the present disclosure provides a nucleic acid comprising, consisting of, or consisting essentially of a first promoter operably linked to a first gene encoding a protein involved in coenzyme B12 synthesis. In certain cases, the first promoter is a constitutive promoter. In some cases, the nucleic acid comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 7-10 and 52-63. In some embodiments in all aspects, the first gene comprises, consists of, or consists essentially of one or more genes selected from the following: cobJ, cobI, cobH, cobG, cobL, cobF, cobK, gst, xre, chlD, chlI, dahp, cobN, cobW, cbtBA, cobE, cobM, btuB, cobO, cob, cobR, cobD, cobC, cobQ, cobU, cobP, bgpM, and cobV. In certain cases, the first gene is from a species of the genus Pseudomonas selected from the following: Pseudomonas denitrificans, Pseudomonas aeruginosa, Pseudomonas entomophila, Pseudomonas putida, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas mendocina, and Pseudomonas stutzeri.

[0040] In some embodiments in all respects, the nucleic acids described herein further comprise, consist of, or consist essentially of a second promoter operably linked to the first promoter. In certain cases, the first and second promoters regulate the expression of the first gene. In certain cases, the first promoter is not the native promoter of the first gene.

[0041] In another aspect, the present disclosure provides a nucleic acid comprising a cobG gene, a P edd promoter, a P sucA promoter, and a cobL gene; wherein the P edd promoter is operably linked to the cobG gene and the P sucA promoter, and the P sucA promoter is operably linked to the cobL gene. In some cases, the P edd promoter comprises the sequence SEQ ID NO: 36, consists of, or consists essentially of the same; the P sucA promoter comprises the sequence SEQ ID NO: 37, consists of, or consists essentially of the same. In some embodiments, the nucleic acids described herein comprise the sequence SEQ ID NO: 38, consist of, or consist essentially of the same.

[0042] In one aspect, the present disclosure provides a nucleic acid comprising a cobG gene, a P sp9 promoter, a P zwf promoter, and a cobL gene, consisting of, or consisting essentially of the same; wherein the P sp9 promoter is operably linked to the cobG gene and the P zwf promoter, and the P zwf promoter is operably linked to the cobL gene. In some cases, the P sp9 promoter comprises SEQ ID NO: 39, consists of, or consists essentially of the same; and the P zwf promoter comprises SEQ ID NO: 40. In certain cases, the nucleic acids described herein comprise SEQ ID NO: 41, consist of, or consist essentially of the same.

[0043] On the one hand, the present disclosure provides a nucleic acid comprising, consisting of, or consisting essentially of a cobW gene, a Pzwf promoter, a Psp9 promoter, and a cbtB gene, wherein the Pzwf promoter is operably linked to the cobW gene and the Psp9 promoter, and the Psp9 promoter is operably linked to the cbtB gene. In some cases, the Pzwf promoter comprises SEQ ID NO: 42; and the Psp9 promoter comprises SEQ ID NO: 43. In some cases, the nucleic acid described herein comprises SEQ ID NO: 44, consists of, or consists essentially of the same.

[0044] On the one hand, the present disclosure provides a nucleic acid comprising, consisting of, or consisting essentially of a cobW gene, a Ptkt promoter, a Psp2 promoter, and a cbtB gene, wherein the Ptkt promoter is operably linked to the cobW gene and the Psp2 promoter, and the Psp2 promoter is operably linked to the cbtB gene. In some cases, the Ptkt promoter comprises SEQ ID NO: 45, consists of, or consists essentially of the same; the Psp2 promoter comprises SEQ ID NO: 46, consists of, or consists essentially of the same. In some cases, the nucleic acid described herein comprises SEQ ID NO: 47, consists of, or consists essentially of the same.

[0045] On the one hand, the present disclosure provides a nucleic acid comprising, consisting of, or consisting essentially of a Pzwf promoter operably linked to a tonB gene (such as the butB gene). In some cases, the Pzwf promoter comprises SEQ ID NO: 48, consists of, or consists essentially of the same. In some cases, the nucleic acid described herein comprises SEQ ID NO: 49, consists of, or consists essentially of the same.

[0046] On the one hand, the present disclosure provides a nucleic acid comprising, consisting of, or consisting essentially of a Psp9 promoter operably linked to a tonB gene (such as the butB gene). In some cases, the Psp9 promoter comprises SEQ ID NO: 50, consists of, or consists essentially of the same. In some cases, the nucleic acid described herein comprises SEQ ID NO: 51, consists of, or consists essentially of the same.

[0047] In some embodiments in all aspects, the nucleic acid comprises, consists of, or consists essentially of one or more sequences derived from Pseudomonas denitrificans. In certain cases, the first or second promoter is derived from Pseudomonas denitrificans. In some cases, the nucleic acid comprises, consists of, or consists essentially of one or more sequences derived from Enterobacter, Lactobacillus, Pseudomonas, or Azospirillum bacteria. In certain cases, the first gene is derived from Enterobacter, Lactobacillus, Pseudomonas, or Azospirillum.

[0048] In one aspect, the present disclosure provides a nucleic acid comprising a DhaB expression module that comprises, consists of, or consists essentially of a sequence that is at least 85% identical to SEQ ID NO: 66 (DhaB expression module). In some cases, the sequence is at least 90% identical to SEQ ID NO: 66. In some cases, the nucleic acid described herein comprises, consists of, or consists essentially of SEQ ID NO: 66.

[0049] In one aspect, the present disclosure provides a nucleic acid comprising a DhaB expression module that comprises, consists of, or consists essentially of a sequence that is at least 85% identical to SEQ ID NO: 67 (kgsA expression module). In some cases, the sequence is at least 90% identical to SEQ ID NO: 67. In some cases, the sequence comprises, consists of, or consists essentially of SEQ ID NO: 67.

[0050] In one aspect, the present disclosure provides a nucleic acid comprising a DhaB expression module that comprises, consists of, or consists essentially of the UTR of the gdrA gene and the UTR of the gdrB gene, wherein the UTR of the gdrA gene comprises, consists of, or consists essentially of SEQ ID NO: 68, and the UTR of the gdrB gene comprises, consists of, or consists essentially of SEQ ID NO: 69. In certain cases, the nucleic acid comprises, consists of, or consists essentially of a sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 70 - 72. In some cases, the nucleic acid comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 70 - 72.

[0051] In one aspect, the present disclosure provides a nucleic acid comprising a coenzyme B12 sensor that comprises, consists of, or consists essentially of a sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 73 and 74. In some cases, the nucleic acid described herein comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 73 and 74.

[0052] In one aspect, the present disclosure provides a nucleic acid comprising, consisting essentially of, or consisting of a coenzyme B12 expression module, wherein the coenzyme B12 expression module comprises a first gene involved in the synthesis of coenzyme B12, a "first promoter" induced by a small molecule, and a second promoter, consisting essentially of, or consisting of, wherein the first and second promoters are operably linked in tandem upstream of the first gene such that they regulate the expression of the first gene. In some cases, the nucleic acid further comprises a second gene involved in coenzyme B12 synthesis, a third promoter induced by a small molecule, and a fourth promoter, consisting essentially of, or consisting of, wherein the third and fourth promoters are operably linked in tandem upstream of the second gene such that they regulate the expression of the second gene. In some embodiments of all aspects, the nucleic acid further comprises a third gene involved in coenzyme B12 synthesis, a fifth promoter induced by a small molecule, and a sixth promoter, consisting essentially of, or consisting of, wherein the fifth and sixth promoters are operably linked in tandem upstream of the third gene such that they regulate the expression of the third gene. In some cases, the nucleic acid further comprises a fourth gene involved in the synthesis of coenzyme B12, a seventh promoter induced by a small molecule, and an eighth promoter, consisting essentially of, or consisting of, wherein the seventh and eighth promoters are operably linked in tandem upstream of the fourth gene such that they regulate the expression of the fourth gene. In certain cases, each of the first, second, third, and fourth genes comprises, consists essentially of, or consists of one or more genes selected from the group consisting of: cobJ, cobI, cobH, cobG, cobL, cobF, cobK, gst, xre, chlD, chlI, dahp, cobN, cobW, cbtBA, cobE, cobM, btuB, cobO, cob, cobR, cobD, cobC, cobQ, cobU, cobP, bgpM, and cobV. In certain cases, the first gene comprises, consists essentially of, or consists of bgpM.

[0053] In some embodiments of all aspects, the nucleic acid does not include any native riboswitches that regulate genes involved in the production of coenzyme B12. In some cases, the nucleic acid does not include SEQ ID NO: 75 or 76.

[0054] In another aspect, the present disclosure provides a recombinant bacterium comprising, consisting essentially of, or consisting of the nucleic acid described herein. In certain cases, the bacterium is a species of the genus Pseudomonas. In certain cases, the bacterium is Pseudomonas denitrificans.

[0055] In some embodiments in all aspects, the nucleic acid is located on an expression plasmid. In some cases, the nucleic acid is integrated into the chromosome of the bacterium. In some cases, the nucleic acid is located on an episome.

[0056] In another aspect, the present disclosure provides a recombinant bacterium comprising, consisting of, or consisting essentially of a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises, consists of, or consists essentially of a DhaB expression module, and the second nucleic acid comprises, consists of, or consists essentially of an ALDH expression module. In some cases, the DhaB expression module comprises any one of the nucleic acids described herein, consists of, or consists essentially of the same. In some cases, the ALDH expression module comprises any one of the nucleic acids described herein, consists of, or consists essentially of the same.

[0057] In some embodiments in all aspects, the first and second nucleic acids are located on an expression plasmid. In some cases, the first and second nucleic acids are located on an episome. In some cases, the first and second nucleic acids are integrated into the chromosome of the bacterium. In some cases, the first nucleic acid is integrated into the chromosome of the bacterium and the second nucleic acid is located on an expression plasmid. In some cases, the first nucleic acid is located on an expression plasmid and the second nucleic acid is integrated into the chromosome of the bacterium. In some embodiments in all aspects, the first nucleic acid is integrated into the chromosome of the bacterium and the second nucleic acid is located on an episome. In some cases, the first nucleic acid is located on an episome and the second nucleic acid is integrated into the chromosome of the bacterium.

[0058] In some embodiments in all aspects, the first nucleic acid is integrated into the chromosome of the bacterium at a first position and the second nucleic acid is integrated into the chromosome of the bacterium at a second position, wherein the first and second positions are within 2500 kilobase pairs of each other. In some cases, the first and second positions are within 100 kilobase pairs of each other. In some cases, the first and second positions are within 50 kilobase pairs of each other. In some cases, the first and second positions are within 1000 base pairs of each other.

[0059] In some embodiments in all aspects, the first nucleic acid is integrated into the chromosome of the bacterium at a first position within 4000 base pairs of the origin of replication. In some cases, the first position is within 1000 base pairs of the origin of replication.

[0060] In another aspect, the present disclosure provides a recombinant bacterium comprising, consisting of, or consisting essentially of a coenzyme B12 expression module, wherein the expression module comprises a first gene involved in the synthesis of coenzyme B12, a first promoter induced by a small molecule, and a second promoter, consisting of, or consisting essentially of, wherein the first and second promoters are operably linked in tandem upstream of the first gene.

[0061] In some embodiments of all aspects, the bacterium is a Pseudomonas denitrificans bacterium. In some cases, the bacterium comprises, consists of, or consists essentially of a deletion at a first position of a first riboswitch that regulates the expression of the first gene, wherein the first and second promoters are integrated into the chromosome of the bacterium at the first position such that they regulate the expression of the first gene. In some cases, the bacterium further comprises a second deletion at a second position of a second riboswitch that regulates the expression of a second gene involved in the synthesis of coenzyme B12, a third deletion at a third position of a third riboswitch that regulates the expression of a third gene involved in the synthesis of coenzyme B12, and a fourth deletion at a fourth position of a fourth riboswitch that regulates the expression of a fourth gene involved in the synthesis of coenzyme B12, consisting of, or consisting essentially of. In some cases, the recombinant bacterium further comprises a third promoter and a fourth promoter that are operably linked in tandem and integrated into the chromosome at the second position such that they regulate the expression of the second gene, a fifth promoter and a sixth promoter that are operably linked in tandem and integrated into the chromosome at the third position such that they regulate the expression of the third gene, and a seventh promoter and an eighth promoter that are operably linked in tandem and integrated into the chromosome at the fourth position such that they regulate the expression of the fourth gene. In some embodiments of all aspects, the bacterium produces more coenzyme B12 than native Pseudomonas denitrificans. In some embodiments of all aspects, the recombinant bacterium described herein further comprises, consists of, or consists essentially of a coenzyme B12 expression module.

[0062] In another aspect, the present disclosure provides a method for producing 3-HP or a salt thereof, comprising culturing the recombinant bacterium described herein under conditions sufficient to produce 3-HP or a salt thereof, consisting of, or consisting essentially of. In certain cases, the conditions are sufficient to produce at least 85 g / L of 3-HP or a salt thereof. In certain cases, the conditions are sufficient to produce at least 90 g / L of 3-HP or a salt thereof. In some embodiments of all aspects, the conditions are sufficient to produce at least 95 g / L of 3-HP or a salt thereof. In certain cases, the conditions are sufficient to produce at least 100 g / L of 3-HP or a salt thereof.

[0063] In some embodiments in all aspects, culturing the bacteria does not include adding external coenzyme B12. In some cases, culturing the bacteria includes adding external coenzyme B12 to the culture.

[0064] In some cases, culturing includes growing the bacteria, consisting essentially of, or consisting of in a medium comprising a carbon source selected from glycerol, glucose, gluconate, glutamate, and citrate. In some cases, culturing includes inducing the bacteria to produce 3-HP or a salt thereof by adding glycerol, consisting essentially of, or consisting of. In some cases, glycerol is added in mid-log phase.

[0065] In some embodiments in all aspects, culturing includes growing the bacteria, consisting essentially of, or consisting of in a medium comprising a nitrogen source selected from yeast extract, corn steep liquor powder, and corn steep liquor paste.

[0066] In some embodiments in all aspects, culturing includes growing the bacteria, consisting essentially of, or consisting of at a pH between 6.8 and 7.8. In some cases, culturing includes adding one or more bases selected from NaOH, KOH, NaHCO3, NH4HCO3, NH4OH, (NH4)2CO3, and Na2CO3, consisting essentially of, or consisting of. In some cases, culturing includes adding at least one base selected from NaOH, KOH, NaHCO3, NH4HCO3, NH4OH, (NH4)2CO3, and Na2CO3, consisting essentially of, or consisting essentially of. In some cases, culturing includes diluting the base concentration and increasing the acid concentration, consisting essentially of, or consisting of (see Figure 46 and Figure 49 ).

[0067] In some embodiments in all aspects, culturing includes growing the bacteria, consisting essentially of, or consisting of at a temperature between 28 and 40 degrees Celsius. In some cases, culturing includes growing the bacteria, consisting essentially of, or consisting of at 33 degrees Celsius.

[0068] In another aspect, the present disclosure provides a method for producing 3-HP or a salt thereof, the method comprising: providing the recombinant bacteria described herein in a medium suitable for growth of the bacteria and production of 3-HP or a salt thereof, consisting essentially of, or consisting of. In some cases, the method described herein includes isolating 3-HP or a salt thereof from the bacteria or the medium, consisting essentially of, or consisting of.

[0069] In some cases, the culture medium comprises, consists of, or consists essentially of a dissolved oxygen concentration of about 2 - 20% dissolved oxygen. In some embodiments in all aspects, the dissolved oxygen concentration does not exceed 20%. In certain cases, the concentration of the dissolved oxygen is at least 2%.

[0070] In addition, the present invention relates to a method for providing and reacting 3 - hydroxypropionic acid.

[0071] The present disclosure provides a method for removing 3 - hydroxypropionic acid (3 - HP) from an aqueous solution. This method can be carried out relatively simply and inexpensively. This method can provide a relatively high yield of 3 - HP.

[0072] The present disclosure also provides a method for preparing acrylic acid. This method can be carried out relatively simply and inexpensively. This method can be carried out with little or no acrylic acid polymerization. This method can provide a relatively high yield of acrylic acid.

[0073] In one aspect, the present disclosure provides a method for removing 3 - HP from an aqueous solution without using a counter - current liquid flow.

[0074] In another aspect, the present disclosure provides a method that includes evaporating a first solvent, condensing the evaporated first solvent, and directing a flow of the first solvent to remove 3 - HP from the aqueous solution.

[0075] In another aspect, the present disclosure provides a method that includes using a water - immiscible solvent to remove 3 - HP from an aqueous solution with a yield of at least 50%.

[0076] In another aspect, the present disclosure provides a method that includes using an organic liquid comprising at least two solvents to remove 3 - hydroxypropionic acid (3 - HP) from an aqueous solution.

[0077] In another aspect, the present disclosure provides a method that includes using a liquid to remove 3 - HP from an aqueous solution, wherein the liquid comprises two different solvents (e.g., a water - miscible solvent and a water - immiscible solvent).

[0078] In another aspect, the present disclosure provides a method that includes removing 3 - HP from an aqueous solution, wherein the pH of the aqueous solution is at least about 3.

[0079] In another aspect, the present disclosure provides a method that includes reacting 3 - HP at a pressure of less than about one atmosphere to form acrylic acid.

[0080] In another aspect, the present disclosure provides a method that includes reacting 3 - HP to form acrylic acid in a reaction mixture and removing gaseous acrylic acid from the reaction mixture.

[0081] In another aspect, the present disclosure provides a method that includes reacting a liquid containing 3-HP to form acrylic acid.

[0082] In another aspect, the present disclosure provides a method that includes reacting 3-HP to form acrylic acid at a yield of at least about 70%.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials for the present disclosure are described herein. Other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present disclosure (including definitions) will control.

[0084] Other features and advantages of the present disclosure will become apparent from the following detailed description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1A - 1B is a schematic diagram showing the regulation of gene expression by the LysR-type transcriptional activator MmsR in the 3-hydroxypropionate degradation pathway of Pseudomonas denitrificans. ([ Figure 1A ) Hypothetical schematic diagram of regulation based on MmsR. hpdH, 3-hydroxypropionate dehydrogenase; mmsA, malonate semialdehyde dehydrogenase; hbdH-4, 3-hydroxyisobutyrate dehydrogenase IV. ([ Figure 1B ) Relative mRNA abundances of mmsA (gray bars) and hbdH-4 (black bars) in the presence of various inducer molecules.

[0086] Figure 2A - 2B is a graph showing transcriptional analysis of the mmsA (white bars) and hbdH-4 (black bars) genes in wild-type (WT) and mutant Pseudomonas denitrificans strains by real-time PCR. ([ Figure 2A ) mRNA levels in the Pseudomonas denitrificans ΔhpdHΔhbdH4ΔhbdH1 deletion mutant lacking 3-HP degrading enzymes (referred to as ΔΔΔ). ([ Figure 2B ) mRNA levels upon deletion and complementation of the transcriptional activator MmsR. ΔmmsR, Pseudomonas denitrificans mmsR deletion mutant; and mmsR-C, Pseudomonas denitrificans ΔmmsR with mmsR-complementation from a plasmid.

[0087] Figure 3A - 3B is a schematic diagram of the mmsR-mmsA intergenic region with the promoter region and the regulatory protein and MmsA binding sites identified. ([Figure 3A )DNA sequences between the mmsR and mmsA genes and mutations in the promoter region for in vivo and in vitro characterization.( Figure 3A )PmmsA( Figure 3B )Mapping of the 5'-ends of the MmsA binding sites (O1 and O2).

[0088] Figure 4A - 4B is a schematic diagram and graph showing in vivo studies to reveal the importance of the O1 and O2 sites for transcriptional activation in the mmsR promoter (PmmsR)( Figure 4A ) and the mmsA promoter (PmmsA)( Figure 4B )). The O1 or / and O2 sites were randomized, and the promoter strength with mutations within the O1 or / and O2 sites was examined using GFP as a reporter protein. These mutations were introduced into Pseudomonas denitrificans ΔhpdHΔhbdH1ΔhbdH4ΔmmsR and used as an expression host.

[0089] Figure 5A - 5E is a photograph of an SDS-PAGE analysis to study the solubility of recombinant C-His-tagged MmsR protein in recombinant Escherichia coli BL21 with various chaperone plasmids such as pG-KJE8( Figure 5A )、pGro7( Figure 5B )、pKJE7( Figure 5C )、pG-Tf2( Figure 5D ) and pTf16( Figure 5E ). The cell lysates (T) and cell-free extracts (S) of recombinant strains with MmsR and molecular chaperones were analyzed.

[0090] Figure 6A - 6C is a gel picture showing the expression, purification and oligomeric forms of recombinant MmsR protein. SDS-PAGE of recombinant MmsR with his tags at the N- or C-terminus( Figure 6A ), blue native-PAGE( Figure 6B ) and relative mRNA levels( Figure 6C ). Lane 1, host Escherichia coli BL21 (cell-free extract); Lane 2, recombinant Escherichia coli BL21 cultured without IPTG induction (cell-free extract); Lanes 3, 4, 5 and 7 correspond to the cell-free extract, soluble fraction, insoluble fraction and purified protein of recombinant Escherichia coli BL21, respectively; Lanes 9, 11 and 13 correspond to purified MmsR proteins at 65, 220, 550 nM, respectively.

[0091] Figure 7A - 7BThis is a picture of an electrophoretic mobility shift assay (EMSA) in vitro for studying the binding of the MmsR protein to the operator site. The DNA fragment containing the mmsA promoter (PmmsA) is intact (F12), or mutated at the O2 site (F12M) or the O1 site (F1M2). Experiments were performed in the absence (upper panel) and presence (lower panel) of 3-HP (25 mM). Lanes 1-10, increasing amounts of MmsR protein 0, 0.36, 0.73, 1.45, 2.9, 5.8, 11.6, 14.5, and 24.2 nM; and the DNA fragment was fixed at 0.4 nM( Figure 7A ), the electrophoretogram from the reaction with increasing amounts of MmsR( Figure 7B ). MmsR protects the operator region from DNase I digestion. The bottom is a scale that gives the nucleotide positions relative to the MmsR transcription start site.

[0092] Figure 8 This is a picture of electrophoretic mobility shift analysis at low DNA concentration to estimate the dissociation constant between MmsR and the PmmsA promoter. (F12) The DNA fragment with intact operators O1 and O2, (F12M) with a mutated O2 operator, and (F1M2) with a mutated O1 operator.

[0093] Figure 9 This is a graph and table showing the MmsR protein-DNA binding isotherm and the binding dissociation constant. DNA fragments containing the complete promoter sequence (F12) or mutated sequences were used.

[0094] Figure 10A - 10B This is a schematic diagram of the analysis of the hpdR-hpdH intergenic region.( Figure 10A ) The DNA sequence of the hpdR-hpdH intergenic region, showing the putative promoter and operator regions.( Figure 10B ) The consensus sequence of the operator palindromic site.

[0095] Figure 11A - 11B This is a graph showing the relative transcription of hpdR and hpdH.( Figure 11A ) HpdR is a transcription factor that controls the expression of hpdH,( Figure 11B ) and there is no crosstalk between the two 3-HP induction systems HpdR and MmsR.

[0096] Figure 12A - 12D This is a graph showing the relative inducibility of hpdH transcription and GFP fluorescence in the presence of various acids.( Figure 12A ) Transcription at the chromosomal level,( Figure 12B ) GFP fluorescence at the plasmid level,( Figure 12C ) GFP fluorescence at different time intervals, and( Figure 12D)Sensitivity at various 3-HP concentrations.

[0097] Figure 13A - 13B is a schematic diagram showing the positions where the PC33-HP-inducible promoter mutations were randomized, and graphs showing the normalized GFP levels of various promoters.

[0098] Figure 14A - 14B is a set of graphs showing the comparison of mmsA and kgsA transcription and enzyme activities at the plasmid and genomic levels.( Figure 14A )mRNA (transcription) and( Figure 14B )comparison of KgsA activity.

[0099] Figure 15A - 15C is a set of schematic diagrams showing how the randomization of the MmsR operator site was applied to KgsA overexpression.( Figure 15A )Schematic diagram of operon mutagenesis,( Figure 15B )kgsA mRNA expression level,( Figure 15C )GFP fluorescence with the selected operon mutation PmmsA2a.

[0100] Figure 16A - 16B is a set of graphs showing the effect of MmsR overexpression on KgsA transcription with various constitutive synthetic promoters,( Figure 16A )Pzwf promoter library,( Figure 16B )KgsA transcription.

[0101] Figure 17A - 17D is a set of graphs showing the effect of native MmsA protein fusion on the expression and enzyme activity of KgsA. Various lengths of the mmsA N-terminal sequences corresponding to 5, 10, 15, and 20 amino acids long (named Hyb-5, Hyb-10, Hyb-15, and Hyb-20, respectively) were ligated to the kgsA 5'-terminal DNA sequence.( Figure 17A )Enzyme activity of KgsA.( Figure 17B )SDS-PAGE analysis of the crude cell extracts expressing the fusion proteins. The stability of the transcripts was also measured( Figure 17C and 17D ).

[0102] Figure 18A - 18C is a set of schematic diagrams and graphs showing the effect of tandem promoters on kgsA expression at the mRNA and enzyme activity levels.( Figure 18A )Arrangement of tandem promoters and their transcripts; TSS, transcription start site; RBS, ribosome binding site; UTR, 5'-untranslated region,( Figure 18B )mRNA expression and( Figure 18C )KgsA activity.

[0103] Figure 19A - 19C Tables and figures showing the effect of the 5’UTR on kgsA expression.( Figure 19A )Predicting the 5’UTR strength of the kgsA expression system.( Figure 19B )KgsA specific activity of the UTR construct, induced differently at 0, 0.25, and 25 mM 3-HP.( Figure 19C )Comparison between the theoretically predicted and experimentally measured values of the 5’UTR strength at different 3-HP concentrations.

[0104] Figure 20A - 20B Schematic diagram showing the degradation of 3-HP to malonic semialdehyde by hbdH-4 and further degradation to acetyl-CoA by mmsA( Figure 20A ), and the genetic structure of the mmsR-mmsA intergenic region and its regulation( Figure 20B ).

[0105] Figure 21A - 21B Graphs and images showing the enzyme activity( Figure 21A ) and SDS-PAGE( Figure 21B ) analysis of the KgsA expression level of strains optimized for the first 10 codons of KgsA. Three different constructs were developed in terms of codon frequency levels (high, medium, and low), named Opt-1, Opt-2, and Opt-3, respectively.

[0106] Figure 22 A set of SDS-PAGE images showing the effect of UTR design on KgsA expression. UTRs with various predicted strengths by the UTR designer's computer were tested.

[0107] Figure 23 Schematic diagram of the vitamin B12 (coenzyme B12) gene cluster and riboswitch in Pseudomonas denitrificans.

[0108] Figure 24 Schematic diagram showing a comparative analysis of the organization of coenzyme B12 genes from various Pseudomonas species.

[0109] Figure 25A - 25D Schematic diagram showing the structure of the vitamin B12 riboswitch. Figure 25A Shows the structure of RS1 before cobG. Figure 25B Shows the structure of RS2 before cobW. Figure 25C Shows the structure of RS3 before cbtB. Figure 25D Shows the structure of RS4 before btuB.

[0110] Figure 26 Graph showing cob gene transcription at the chromosomal level in Pseudomonas denitrificans.

[0111] Figure 27 is a set of diagrams that describe the in vivo characteristics of the intergenic regions of the vitamin B12 genes in Pseudomonas denitrificans.

[0112] Figure 28 is a graph showing the correlation between B12 concentration and GFP fluorescence of the vitamin B12 ribosensor of the intergenic region of genes cobG and cbtB from Pseudomonas denitrificans.

[0113] Figure 29A - 29B is a set of graphs, ( Figure 29A ) showing gene essentiality profiles, which reveal that only the gene encoding bgpM is essential for coenzyme B12 biosynthesis. Different concentrations of cobalt chloride were used to understand the necessity of several uncharacterized or poorly understood genes in the cob gene cluster. ( Figure 29B ) The concentration of coenzyme B12 was quantified using Salmonella typhimurium metE-cbiB- and a B12 riboswitch-based biosensor.

[0114] Figure 30A - 30B is a set of schematic diagrams showing the development of the dhaB-gdrAB expression cassette. ( Figure 30A ) Schematic diagram of expression cassette construction, ( Figure 30B ) integrating the kgsA gene into the locus of the hbdH-4 gene in the ΔmmsA mutant strain.

[0115] Figure 31A - 31B is a schematic diagram showing the combination of transcriptional and translational modifications used for the development of the DhaB-GdrAB expression system ( Figure 31A ), and the schematic diagram of the DhaB-GdrAB (DhaB) expression system integrated into the chromosome for integration ( Figure 31B ).

[0116] Figure 32 is a set of animations that show the effect of genomic location on KgsA expression (e.g., the location of the kgsA gene after chromosomal integration and the activity of KgsA (P2-0, P2-10, P2-20, P2-30; strains without the DhaB plasmid). The numbers represent the KgsA activity 4 hours after induction with 25 mM 3-HP.

[0117] Figure 33 is a set of animations that show the genomic integration of the DhaB expression cassette into the chromosome of the P2-20 strain to generate P4-1, or into P2-10 to generate P4-2. Subsequently, the promoters of the DhaB-GdrAB expression cassette in P4-1 and P4-2 were attenuated (DhaB reduced) to generate strains P4-3 and P4-4.

[0118] Figure 34It is a schematic diagram showing strain development for 3-HPA channeling (P4-5). There is one copy of KgsA near DhaB in P4-5.

[0119] Figure 35 It is a graph showing the effects of various neutralizing bases on 3-HP production.

[0120] Figure 36 It is a graph showing the modification of recombinant strains in media supplemented with various 3-HP concentrations.

[0121] Figure 37 It is a graph showing the growth of 3-HP-modified recombinant strains (80 g / L) in media supplemented with various organic acids at a concentration of 50 g / L.

[0122] Figure 38 It is a graph showing the cultivation of 3-HP-modified recombinant strains (80 g / L) in media at different pH values.

[0123] Figure 39 It is a graph showing the modification of 3-HP (80 g / L)-tolerant recombinant strains to improve the specific growth rate at higher pH values.

[0124] Figure 40 It is a graph showing the cultivation of 3-HP (80 g / L)-tolerant recombinant strains adapted to pH 7.6 at different pH values.

[0125] Figure 41 It is a schematic diagram of the 3-HP synthesis pathway from glycerol-adapted recombinant strains. The first enzyme is oxygen-sensitive, while the second reaction is enhanced in the presence of oxygen.

[0126] Figure 42 It is a graph showing the effects of different dissolved oxygen concentrations on 3-HP production.

[0127] Figure 43 It is a graph showing the effects of different temperatures on 3-HP production.

[0128] Figure 44 It is a graph showing the effects of different induction OD600 on 3-HP production.

[0129] Figure 45 It is a graph showing the effects of various concentrations of NH4OH on 3-HP production and specific growth rate.

[0130] Figure 46 It is a graph showing the effects of NH4OH (%) on relative 3-HP titer (%), and amount (%).

[0131] Figure 47It is a schematic diagram illustrating anapleurotic reactions in central carbon metabolism. PPC, PEP carboxylase; PEPK, PEP carboxykinase, ME, malic enzyme; and PC, pyruvate carboxylase.

[0132] Figure 48 It is a graph showing the effect of various neutralizing agents on 3-HP production (%).

[0133] Figure 49 It is a graph showing the effect of various concentrations of NH4HCO3 on 3-HP titer and amount (%).

[0134] Figure 50 It is a graph showing the effect of sequentially disrupting the B12 riboswitch, promoter replacement, and ultimately using a tandem promoter to increase coenzyme B12 production and thereby increase 3-HP production to a commercial level.

[0135] Figure 51 It is a graph showing the effect of substrate channeling on 3-HP production.

[0136] Figure 52 It is a graph showing the effect of various neutralizing agents such as NH4OH and NaOH on 3-HP (%) production. Under one condition, NH4OH was used as the neutralizing agent until 24 hours of fermentation, and then NaOH until 48 hours of fermentation.

[0137] Figure 53 It is the in vitro enzyme activities of glycerol dehydratase (DhaB) and KgsA at different stages of the bioreactor.

[0138] Figure 54 It is a graph showing the effect of various toxic intermediates on the in vitro enzyme activity of glycerol dehydratase (DhaB).

[0139] Figure 55A - 55B Shows the development of a 3-HP-inducible tandem promoter library for controlled expression of DhaB at various expression levels. ( Figure 55A ) Schematic diagram of tandem promoter construction, ( Figure 55B ) Specific DhaB activity of various tandem promoter library constructs.

[0140] Figure 56 It is a schematic diagram illustrating the sequence of the PC3 promoter region.

[0141] Figure 57 It is a graph showing the role of natural and synthetic constitutive promoters in Pseudomonas denitrificans.

[0142] Figure 58An exemplary system for extracting 3-HP from an aqueous solution using a water-immiscible solvent with a density less than water is shown.

[0143] Figure 59 An exemplary system for extracting 3-HP from an aqueous solution using a water-immiscible solvent denser than water is shown.

[0144] Figure 60 An exemplary system for producing acrylic acid from 3-HP by reactive distillation is shown.

[0145] Figure 61 Is an exemplary line graph illustrating the increase in 3-HP concentration in a solvent container during the extraction of 3-HP from a cell-free fermentation using ethyl acetate.

[0146] Detailed description

[0147] The present disclosure provides industrial microbial strains (including, for example, Pseudomonas strains) having an expression system for enhanced production of 3-HP or its salts from glycerol. Enzymes involved in the production of 3-HP or its salts and their regulatory regions are integrated into the chromosome of the industrial strain. A 3-HP-inducible promoter has been identified, characterized, and operably linked to an enzyme involved in the production of 3-HP or its salts. By combining the 3-HP-inducible promoter with a constitutive, inducible, and / or synthetic promoter to establish a tandem promoter system, the expression of 3-HP enzymes can be increased. The tandem promoter system can be fused with UTRs (natural or synthetic) and other regulatory domains that control or enhance expression to improve system expression. The tandem promoter system (e.g., a tandem promoter system combined with UTRs) can also be fused with a sequence encoding the start amino acids of a highly expressed native gene (e.g., a sequence encoding up to twenty amino acids). These modifications can increase the expression of enzymes involved in 3-HP production (or the production of its salts), in this case the downstream genes. In addition, the localization of these expression systems in the industrial strain chromosome has been studied, and the impact on 3-HP (or its salts) production has been evaluated. The designed localization of the expression system can avoid the accumulation of toxic 3-HPA and can increase the yield of 3-HP by channeling.

[0148] In addition, the present disclosure provides industrial strains that can produce 3-HP at a high titer without external supplementation of coenzyme B12 (B12). Regulatory regions controlling the expression of enzymes in the B12 production pathway have been identified and characterized. Removal of many of these regulatory regions (including, for example, secondary structures) improves the expression of B12 enzymes. Integration of the constitutive or 3-HP-inducible expression systems described herein can increase the expression of native B12 enzymes and increase B12 production. This allows for the production of 3-HP (or its salts) without external addition of B12.

[0149] The present disclosure also provides a method for extracting 3-HP from an aqueous solution. For example, 3-HP can be extracted from an aqueous solution by solvent extraction. The present disclosure also provides a method for purifying 3-HP (e.g., after extraction from an aqueous solution). In addition, the present disclosure provides a method for preparing acrylic acid from 3-HP (e.g., after extraction from an aqueous solution and optionally purification). Further, the present disclosure provides a method for purifying acrylic acid.

[0150] Recombinant bacteria produce 3-hydroxypropionic acid

[0151] The compositions and methods described herein can be used to produce 3-hydroxypropionic acid (3-HP) (or its salts) from a carbon source using recombinant microorganisms. In some cases, the carbon source is glycerol. Glycerol / diol dehydratase (e.g., DhaB) converts glycerol to 3-hydroxypropanal (3-HPA) through a reaction involving coenzyme B12. Then, in a reaction requiring NAD(P)(+), 3-HPA is converted to 3-HP by aldehyde dehydrogenase (ALDH) (e.g., KgsA, EaldH, KaldH). The conversion of glycerol to 3-HP involves several rate-limiting factors that can impede the production of 3-HP (or its salts) at high titers. These factors include, for example, coenzyme B12, the complex nature of the enzymes in the pathway, NAD+ regeneration, and the toxicity of 3-hydroxypropanal (3-HPA) and 3-HP to cells. In this study, 3-HP (or its salts) is produced at a high titer, e.g., a titer sufficient to commercialize 3-HP.

[0152] The loss of enzyme activity due to the accumulation of toxic intermediates during the production of 3-HP (or its salts) is a challenging problem for the production of 3-HP (or its salts) at high titers. 3-HPA is a toxic intermediate that accumulates during the production of 3-HP. Studies have shown that when 3-HP pathway enzymes are incubated with 3-HPA, the enzyme activity decreases in a dose-dependent manner. Aldehydes are known to react with amino acid residues such as lysine, cysteine, and histidine by targeting the ε-amino (NH3+), sulfhydryl (-SH), and imidazole groups, respectively. The accumulation of 3-HPA disrupts proteins, including, for example, glycerol dehydratase or diol dehydratase, which is responsible for the synthesis of 3-HPA in the pathway for the synthesis of 3-HPA from glycerol. The inactivation of glycerol or diol dehydratase by 3-HPA results in a decrease in the production of 3-HP (or its salts) and a low titer. Potential methods for overcoming the inactivation of enzymes by toxic intermediates include, in particular, the development of enzymes highly resistant to the toxicity of 3-HPA or the continuous synthesis of new enzymes. The compositions and methods described herein can reduce the accumulation of toxic intermediates (e.g., 3-HPA) during the production of 3-HP from glycerol, thereby increasing the amount of 3-HP or its salts that can be produced by recombinant bacteria.

[0153] In some embodiments, a recombinant microorganism as described herein can express one or more genes encoding an enzyme that catalyzes the production of 3-HP or a salt thereof from a carbon source, such as one or more genes encoding glycerol dehydratase, diol dehydratase, and / or aldehyde dehydrogenase. In some embodiments, the protein involved in the synthesis of a compound can be one or more enzymes that catalyze the production of the compound from a carbon source. The protein involved in the synthesis of 3-HP or a salt thereof can be, for example, one or more proteins that constitute glycerol dehydratase, diol dehydratase, and / or aldehyde dehydrogenase. In some embodiments, the carbon source is glycerol, but other carbon sources can be used, such as glucose, glutamate, gluconate, and citrate. The recombinant organism can express glycerol dehydratase (e.g., DhaB) or diol dehydratase, which catalyzes glycerol into 3-hydroxypropionaldehyde (3-HPA). DhaB is a complex enzyme with three subunits, encoding the complex glycerol / diol dehydratase (dhaBCD). DhaB is complexed with coenzyme B12. In its catalytic activity (converting glycerol to 3-HPA), coenzyme B12 is damaged, and the damaged coenzyme B12 will be replaced by a new active coenzyme B12 by another enzyme, glycerol dehydratase reactivating factor (GdrAB) or diol dehydratase reactivating factor. GdrAB is a complex enzyme with two subunits, GdrA and GdrB. The recombinant organism can also express a gene encoding aldehyde dehydrogenase (ALDH), which then catalyzes 3-HPA to 3-HP. This catalysis involves NAD(P)(+). The expression of aldehyde dehydrogenase (ALDH enzyme) can include the expression of the kgsA gene. Thus, in some embodiments, the recombinant microorganism expresses the DhaB enzyme and the ALDH enzyme, where the DhaB enzyme catalyzes glycerol into 3-HPA, and then the ALDH enzyme converts 3-HPA into 3-HP.

[0154] The recombinant microorganisms described herein can be bacteria or fungi. In some embodiments, the recombinant microorganisms are bacteria. The recombinant bacteria can be any genetically engineered bacteria capable of producing 3-HP from a carbon source under culture conditions, such as in a bioprocessor or bioreactor. In some embodiments, the bacteria grow under aerobic conditions. In some embodiments, the bacteria grow under anaerobic conditions. In some embodiments, the genome of the bacteria naturally contains genes that can be used to produce 3-HP from a carbon source, such as glycerol, for example, genes encoding glycerol dehydratase or diol dehydratase (such as DhaB) and / or aldehyde dehydrogenase (such as ksgA). In some embodiments, the genome of the bacteria does not naturally contain genes that can be used to produce 3-HP from a carbon source, such as glycerol. In some embodiments, bacteria that do not naturally contain or express genes for producing 3-HP from glycerol can be genetically modified to express at least one gene for producing 3-HP. For example, bacteria that do not express glycerol dehydratase (DhaB), diol dehydratase, and / or aldehyde dehydrogenase (ALDH) can be genetically engineered to express DhaB and / or ALDH.

[0155] In some embodiments, the recombinant bacteria are strains of the genus Pseudomonas, Klebsiella, or Escherichia. In some embodiments, the recombinant bacteria are strains of Pseudomonas denitrificans, Klebsiella pneumonia, or Escherichia coli. In some embodiments, the recombinant bacteria are strains of Pseudomonas denitrificans. Pseudomonas denitrificans is an aerobic microorganism that can naturally synthesize the cofactor coenzyme B12. Coenzyme B12 is an important cofactor for the production of 3-HP from glycerol. In addition, Pseudomonas denitrificans can effectively regenerate NAD+. NAD+ regeneration is very important for the continuous production of 3-HP. Therefore, Pseudomonas denitrificans is presumed to be a suitable microorganism for the production of 3-HP (or the production of its salts).

[0156] In some embodiments, the recombinant bacterium expresses at least one glycerol dehydratase and / or diol dehydratase from Enterobacter bacteria (e.g., Klebsiella or Salmonella or Citrobacter), or Lactobacillus bacteria, or Propionibacterium, Proteus, or Serratia, or Clostridium, such as at least one glycerol dehydratase (e.g., DhaB) from a species of Enterobacter (e.g., Klebsiella, Salmonella, or Citrobacter), or Lactobacillus, or Propionibacterium, or Proteus, or Serratia, or Clostridium. In some embodiments, the recombinant bacterium expresses two or more glycerol dehydratases (e.g., DhaB) and / or diol dehydratases, wherein each dehydratase (e.g., DhaB) can be from a different species of Enterobacter (e.g., Klebsiella, or Salmonella, or Citrobacter), or Lactobacillus, or Propionibacterium, or Proteus, or Serratia, or Clostridium. In some embodiments, the recombinant bacterium expresses at least one aldehyde dehydrogenase (ALDH), such as at least one ALDH enzyme, from a species of Enterobacter bacteria (e.g., Klebsiella, Salmonella, or Citrobacter), or Lactobacillus, or Propionibacterium, or Proteus, or Serratia, or Clostridium. In some embodiments, the recombinant bacterium expresses two or more ALDH enzymes, wherein each ALDH enzyme can be from a different Enterobacter or Lactobacillus strain. In some embodiments, the recombinant bacterium expresses at least one glycerol dehydratase (e.g., DhaB) from Enterobacter bacteria (e.g., Klebsiella or Salmonella) or Lactobacillus, and / or a diol dehydratase from Enterobacter bacteria (e.g., Klebsiella or Salmonella) or Lactobacillus bacteria, and at least one aldehyde dehydrogenase (ALDH) enzyme from Enterobacter bacteria (e.g., Klebsiella or Salmonella) or Lactobacillus bacteria.

[0157] In some embodiments, the recombinant bacterium expresses at least one gene encoding at least one glycerol dehydratase and / or diol dehydratase from a bacterium, such as dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB from Klebsiella pneumoniae in the case of Klebsiella pneumoniae. In some embodiments, the recombinant bacterium expresses at least one gene encoding at least one aldehyde dehydrogenase from Azospirullum brasilense, such as kgsA from Azospirullum brasilense. In some embodiments, the recombinant bacterium expresses at least one gene encoding at least one glycerol dehydratase from Klebsiella pneumoniae, such as dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB from Klebsiella pneumoniae, or at least one gene encoding at least one diol dehydratase, and at least one gene encoding at least one aldehyde dehydrogenase from Azospirullum brasilense, such as kgsA from Azospirullum brasilense. In some embodiments, the recombinant bacterium is a Pseudomonas denitrificans strain that expresses at least one gene encoding at least one glycerol dehydratase and / or diol dehydratase from Klebsiella pneumoniae (such as dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB from Klebsiella) and at least one gene encoding at least one aldehyde dehydrogenase from Azospirullum brasilense (such as kgsA from Azospirullum brasilense).

[0158] In some embodiments, the recombinant bacterium has at least one nucleic acid comprising at least one glycerol dehydratase gene and / or diol dehydratase gene (such as DhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) from a bacterium of the genus Enterobacter (such as Klebsiella or Salmonella) or a bacterium of the genus Lactobacillus, for example, at least one glycerol / diol dehydratase gene (such as dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) from Klebsiella pneumoniae. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least two glycerol dehydratase genes and / or diol dehydratase genes (such as two or more of the dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) from a bacterium of the genus Enterobacter (such as Klebsiella or Salmonella) or a bacterium of the genus Lactobacillus, for example, at least two glycerol dehydratase genes (such as two or more of the dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) or diol dehydratase genes from Klebsiella pneumoniae. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one aldehyde dehydrogenase gene (kgsA gene) from a bacterium of the genus Azospirillum (such as at least one aldehyde dehydrogenase gene (kgsA gene) from an Azospirillum brasilense strain). In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least two aldehyde dehydrogenase genes (kgsA genes) from a bacterium of the genus Azospirillum (such as at least two aldehyde dehydrogenase genes (kgsA genes) from an Azospirillum brasilense strain). In some embodiments, the recombinant bacterium has at least one nucleic acid comprising at least one glycerol dehydratase gene and / or diol dehydratase gene (such as, DhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) (such as at least one glycerol dehydratase gene, such as dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes, from Klebsiella pneumoniae) from a bacterium of the genus Enterobacter (such as Klebsiella or Salmonella) or a bacterium of the genus Lactobacillus, and at least one nucleic acid comprising at least one aldehyde dehydrogenase gene (kgsA gene) from a bacterium of the genus Azospirillum (such as at least one aldehyde dehydrogenase gene (kgsA gene) from an Azospirillum brasilense strain).

[0159] dhaB1w SEQ ID NO:1

[0160]

[0161] DhaB1 SEQ ID NO:77

[0162] MKRSKRFAVLAQRPVNQDGLIGEWPEEGLIAMDSPFDPVSSVKVDNGLIVELDGKRRDQFDMIDRFIADYAINVERTEQAMRLEAVEIARMLVDIHVSREEIIAITTAITPAKAVEVMAQMNVVEMMMALQKMRARRTPSNQCHVTNLKDNPVQIAADAAEAGIRGFSEQETTVGIARYAPFNALALLVGSQCGRPGVLTQCSVEEATELELGMRGLTSYAETVSVYGTEAVFTDGDDTPWSKAFLASAYASRGLKMRYTSGTGSEALMGYSESKSMLYLESRCIFITKGAGVQGLQNGAVSCIGMTGAVPSGIRAVLAENLIASMLDLEVASANDQTFSHSDIRRTARTLMQMLPGTDFIFSGYSAVPNYDNMFAGSNFDAEDFDDYNILQRDLMVDGGLRPVTEAETIAIRQKAARAIQAVFRELGLPPIADEEVEAATYAHGSNEMPPRNVVEDLSAVEEMMKRNITGLDIVGALSRSGFEDIASNILNMLRQRVTGDYLQTSAILDRQFEVVSAVNDINDYQGPGTGYRISAERWAEIKNIPGVVQPDTIE

[0163] dhaB2 SEQ ID NO:2

[0164] ATGCAACAGACAACCCAAATTCAGCCCTCTTTTACCCTGAAAACCCGCGAGGGCGGGGTAGCTTCTGCCGATGAACGCGCCGATGAAGTGGTGATCGGCGTCGGCCCTGCCTTCGATAAACACCAGCATCACACTCTGATCGATATGCCCCATGGCGCGATCCTCAAAGAGCTGATTGCCGGGGTGGAAGAAGAGGGGCTTCACGCCCGGGTGGTGCGCATTCTGCGCACGTCCGACGTCTCCTTTATGGCCTGGGATGCGGCCAACCTGAGCGGCTCGGGGATCGGCATCGGTATCCAGTCGAAGGGGACCACGGTCATCCATCAGCGCGATCTGCTGCCGCTCAGCAACCTGGAGCTGTTCTCCCAGGCGCCGCTGCTGACGCTGGAAACCTACCGGCAGATTGGCAAAAACGCCGCGCGCTATGCGCGCAAAGAGTCACCTTCGCCGGTGCCGGTGGTGAACGATCAGATGGTGCGGCCGAAATTTATGGCCAAAGCCGCGCTATTTCATATCAAAGAGACCAAACATGTGGTGCAGGACGCCGAGCCCGTCACCCTGCACGTCGACTTAGTTAGGGAGTAA

[0165] DhaB2 SEQ ID NO:78

[0166] MQQTTQIQPSFTLKTREGGVASADERADEVVIGVGPAFDKHQHHTLIDMPHGAILKELIAGVEEEGLHARVVRILRTSDVSFMAWDAANLSGSGIGIGIQSKGTTVIHQRDLLPLSNLELFSQAPLLTLETYRQIGKNAARYARKESPSPVPVVNDQMVRPKFMAKAALFHIKETKHVVQDAEPVTLHVDLVRE

[0167] dhaB3 SEQ ID NO:3

[0168] ATGAGCGAGAAAACCATGCGCGTGCAGGATTATCCGTTAGCCACCCGCTGCCCGGAGCATATCCTGACGCCTACCGGCAAACCATTGACCGATATTACCCTCGAGAAGGTGCTCTCTGGCGAGGTGGGCCCGCAGGATGTGCGGATCTCCTGCCAGACCCTTGAGTACCAGGCGCAGATTGCCGAGCAGATGCAGCGCCATGCGGTGGCGCGCAATTTCCGCCGCGCGGCGGAGCTTATCGCCATTCCTGACGAGCGCATTCTGGCTATCTATAACGCGCTGCGCCCGTTCCGCTCCTCGCAGGCGGAGCTGCTGGCGATCGCCGACGAGCTGGAGCACACCTGGCATGCGACAGTGAATGCCGCCTTTGTCCGGGAGTCGGCGGAAGTGTATCAGCAGCGGCATAAGCTGCGTAAAGGAAGCTAA

[0169] DhaB3 SEQ ID NO:79

[0170] MSEKTMRVQDYPLATRCPEHILTPTGKPLTDITLEKVLSGEVGPQDVRISCQTLEYQAQIAEQMQRHAVARNFRRAAELIAIPDERILAIYNALRPFRSSQAELLAIADELEHTWHATVNAAFVRESAEVYQQRHKLRKGS

[0171] gdrA SEQ ID NO:4

[0172]

[0173] GdrA SEQ ID NO:80

[0174] MPLIAGIDIGNATTEVALASDDPQARAFVASGIVATTGMKGTRDNIAGTLAALEQALAKTPWSMSDVSRIYLNEAAPVIGDVAMETITETIITESTMIGHNPQTPGGVGVGVGTTIALGRLATLPAAQYAEGWIVLIDDAVDFLDAVWWLNEALDRGINVVAAILKKDDGVLVNNRLRKTLPVVDEVTLLEQVPEGVMAAVEVAAPGQVVRILSNPYGIATFFGLSPEETQAIVPIARALIGNRSAVVLKTPQGDVQSRVIPAGNLYISGEKRRGEADVAEGAEAIMQAMSACAPVRDIRGEPGTHAGGMLERVRKVMASLTDHEMSAIYIQDLLAVDTFIPRKVQGGMAGECAMENAVGMAAMVKADRLQMQVIARELSARLQTEVVVGGVEANMAIAGALTTPGCAAPLAILDLGAGSTDAAIVNAEGQITAVHLAGAGNMVSLLIKTELGLEDLSLAEAIKKYPLAKVESLFSIRHENGAVEFFREALSPAVFAKVVYIKEGELVPIDNASPLEKIRLVRRQAKEKVFVTNCLRALRQVSPGGSIRDIAFVVLVGGSSLDFEIPQLITEALSHYGVVAGQGNIRGTEGPRNAVATGLLLAGQAN

[0175] gdrB SEQ ID NO:5

[0176] ATGTCGCTTTCACCGCCAGGCGTACGCCTGTTTTACGATCCGCGCGGGCACCATGCCGGCGCCATCAATGAGCTGTGCTGGGGGCTGGAGGAGCAGGGGGTCCCCTGCCAGACCATAACCTATGACGGAGGCGGTGACGCCGCTGCGCTGGGCGCCCTGGCGGCCAGAAGCTCGCCCCTGCGGGTGGGTATTGGGCTCAGCGCGTCCGGCGAGATAGCCCTCACTCATGCCCAGCTGCCGGCGGACGCGCCGCTGGCTACCGGACACGTCACCGATAGCGACGATCATCTGCGTACGCTCGGCGCCAACGCCGGGCAGCTGGTTAAAGTCCTGCCGTTAAGTGAGAGAAACTGA

[0177] GdrB SEQ ID NO:81

[0178] MSLSPPGVRLFYDPRGHHAGAINELCWGLEEQGVPCQTITYDGGGDAAALGALAARSSPLRVGIGLSASGEIALTHAQLPADAPLATGHVTDSDDHLRTLGANAGQLVKVLPLSERN

[0179] kgsA SEQ ID NO:6

[0180]

[0181] KgsA SEQ ID NO:82

[0182] MANVTYTDTQLLIDGEWVDAASGKTIDVVNPATGKPIGRVAHAGIADLDRALAAAQSGFEAWRKVPAHERAATMRKAAALVRERADAIAQLMTQEQGKPLTEARVEVLSAADIIEWFADEGRRVYGRIVPPRNLGAQQTVVKEPVGPVAAFTPWNFPVNQVVRKLSAALATGCSFLVKAPEETPASPAALLRAFVDAGVPAGVIGLVYGDPAEISSYLIPHPVIRKVTFTGSTPVGKQLASLAGLHMKRATMELGGHAPVIVAEDADVALAVKAAGGAKFRNAGQVCISPTRFLVHNSIRDEFTRALVKHAEGLKVGNGLEEGTTLGALANPRRLTAMASVIDNARKVGASIETGGERIGSEGNFFAPTVIANVPLDADVFNNEPFGPVAAIRGFDKLEEAIAEANRLPFGLAGYAFTRSFANVHLLTQRLEVGMLWINQPATPWPEMPFGGVKDSGYGSEGGPEALEPYLVTKSVTVMAV

[0183] In some embodiments, the diol dehydratase gene is from a bacterium of the genus Klebsiella, such as Klebsiella pneumoniae. In some embodiments, the diol dehydratase is from Klebsiella pneumoniae subsp. pneumoniae MGH78478. In some embodiments, the diol dehydratase gene can be one or more of pduC, pduD, pduE, pduG, and / or pduH. In some embodiments, the recombinant bacterium expresses at least one diol dehydrogenase, including, for example, PduC, PduD, PduE, PduG, and / or PduH.

[0184] In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence selected from SEQ ID NO: 1-6 or SEQ ID NO: 83-87. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence selected from SEQ ID NO: 1-5. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence selected from SEQ ID NO: 83-87. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having SEQ ID NO: 6. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence selected from SEQ ID NO: 1-5 and / or SEQ ID NO: 83-87, and another nucleic acid having SEQ ID NO: 6.

[0185] In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence that is at least 80% identical to any one of SEQ ID NO: 1-6 and SEQ ID NO: 83-87. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1-6 and SEQ ID NO: 83-87. In some embodiments, the recombinant bacterium comprises at least one nucleic acid having at least one sequence that differs from the nucleic acid sequence of any one of SEQ ID NO: 1-6 and SEQ ID NO: 83-87 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides.

[0186] In some embodiments, the recombinant bacterium expresses at least one protein having an amino acid sequence selected from SEQ ID NOs: 77 - 82 and 88 - 92. In some embodiments, the recombinant bacterium expresses at least one protein having an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 77 - 82 and 88 - 92. In some embodiments, the recombinant bacterium expresses at least one protein having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 77 - 82 and 88 - 92. In some embodiments, the recombinant bacterium expresses at least one protein having an amino acid sequence that differs from the amino acid sequence of any one of SEQ ID NOs: 77 - 82 and 88 - 92 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more amino acids.

[0187] Promoter

[0188] As used herein, the term "promoter" refers to a DNA sequence that contains regulatory nucleic acid sequences for expressing an operably linked gene. The core promoter contains nucleotide sequences necessary for promoter function, including the TATA box and the transcription start site. By this definition, the core promoter may or may not have detectable activity in the absence of specific sequences, such as those that may enhance activity or confer tissue - specific activity. A promoter can be constitutive or inducible. A constitutive promoter controls the transcription of a gene at a constant rate throughout the life of the cell. The activity of an inducible promoter is determined by the presence (or absence) of a specific inducer, such as the presence (or absence) of an extracellular or environmental factor. A "3 - HP inducible promoter" is a promoter that increases the expression of one or more operably linked genes upon exposure of a cell bearing such a promoter to 3 - HP (e.g., exposure of a cell bearing a 3 - HP promoter to 3 - HP present in the cell culture medium or bioreactor). In certain cases, a promoter includes one or more operator sites.

[0189] The recombinant microorganisms described herein express at least one gene for the production of 3-HP (or its salt) from glycerol under the control of at least one promoter. In some embodiments, the expression system comprises at least one inducible promoter that increases gene expression in response to a stimulus (i.e., an inducer, such as 3-HP). Once the stimulus contacts the inducible promoter, the promoter turns on or upregulates the expression of the gene. In some embodiments, the inducible promoter is induced by 3-HP. The stimulus or inducer that causes upregulation of gene expression by at least one inducible promoter in the expression system is 3-HP. In some embodiments, the stimulus or inducer that causes upregulation of gene expression by at least one inducible promoter in the expression system is chemically and / or structurally similar to 3-HP. In some embodiments, the stimulus or inducer that causes upregulation of gene expression by at least one inducible promoter in the expression system is a small molecule, such as a small organic molecule, such as an acid or an alcohol. In some embodiments, the small molecule is structurally similar to 3-hydroxypropionic acid (3-HP) or 3-hydroxypropionaldehyde (3-HPA). 3-HP is a carboxylic acid with the chemical structure C3H6O3 and a molecular weight of 90.08. In some embodiments, the small various acids structurally similar to 3-HP or intermediates that occur in the L-valine degradation pathway and central carbon metabolism. In some embodiments, the small molecule acid or alcohol can be, but is not limited to, any one of L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxybutyric acid (3-HB), 1,3-propanediol (1,3-PDO), 2,3-butanediol (2,3-BDO), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB). In some embodiments, the inducible promoter can be induced by two or more inducers.

[0190] The present invention provides novel promoters that can be induced by the C3 platform chemical 3-HP. The present disclosure reports the mechanism by which 3-HP activates / starts gene expression through these new promoters. In our experiments, we observed that when Pseudomonas strains were provided with 3-HP as the sole carbon source, these strains actively consumed 3-HP and showed growth. We identified several enzymes, namely putative 3-hydroxyisobutyrate dehydrogenase IV (HbdH-4), 3-hydroxypropionate dehydrogenase (HpdH), and / or methylmalonatesemialdehyde dehydrogenase (MmsA) involved in 3-HP degradation. Interestingly, we found that the transcription of the genes encoding these enzymes was essentially upregulated at a high level, but only in the presence of 3-HP or similar small acids. Analysis of the gene arrangement near these 3-HP degradation genes revealed the presence of putative transcriptional regulatory proteins, namely the corresponding mmsA, hbdH-4, or hpdH. These transcriptional regulatory proteins activate the transcription of mmsA, hpdH, and other proteins after binding to 3-HP.

[0191] The inducible nature and high induction efficiency of the promoters described herein make them useful for expressing pathway enzymes for 3-HP synthesis, developing 3-HP-responsive biosensors, and / or for expressing pathway enzymes for coenzyme B12 production. Here, we elucidated different 3-HP-inducible gene regulatory systems. At the cellular level, the specificity and / or spectrum of these small molecule inducers were studied using various acids structurally similar to 3-HP or intermediates emerging in the L-valine degradation pathway and central carbon metabolism.

[0192] Using these promoters, a promoter library was generated, in which the promoters are inducible by small acids and show different expression levels. This library was used in combination with other natural or synthetic promoters to develop a tandem promoter system. Here, we attempted to mimic the tandem promoter system by combining a small acid-inducible system and a natural / synthetic promoter library to express target genes (such as 3-HP synthesis pathway genes).

[0193] In some embodiments, the inducible promoter is induced by 3-HP. In some embodiments, the inducible promoter is a naturally occurring promoter, such as a promoter that drives gene expression in bacteria such as Pseudomonas bacteria. In some embodiments, the inducible promoter is a naturally occurring 3-HP inducible promoter present in bacteria such as Pseudomonas bacteria (e.g., Pseudomonas denitrificans). In Pseudomonas bacteria such as Pseudomonas denitrificans, some natural promoters that drive the expression of 3-HP degradation-related proteins are upregulated by 3-HP, including promoters that drive the expression of 3-hydroxyisobutyrate dehydrogenase I (HbdH-1), 3-hydroxyisobutyrate dehydrogenase IV (HbdH-4), 3-hydroxypropionate dehydrogenase (HpdH), and methylmalonate semialdehyde dehydrogenase (MmsA). In some embodiments, the recombinant bacteria described herein express at least one gene for producing 3-HP from glycerol under the control of at least one natural promoter of the mmsA gene (PmmsA promoter), hpdH gene (PhpdH promoter), or hbdH-4 gene (PhbdH-1 promoter) of Pseudomonas denitrificans. In some embodiments, the inducible promoter is a synthetic promoter.

[0194] P hbdH -4 promoter (SEQ ID NO: 11)

[0195] GCCCCCGCCTACCCGCCCCAGCCACCCCGGACTCAGCAAGGATGCTGGCCCGGGCCTGGGCGGAGACGTCTTTCGCGCCCGACCATCAGAACAAGAGGACAACCCC

[0196] P hpdH promoter (SEQ ID NO: 12)

[0197] TGTGGGAGCGGGCGTGCCCGCGAAGAGGCCAGCACAGACTTACCACTGTGCTAAAACGCACAGCGGCTGCGCGAAATCTCGTGTTTCCTCCACGAAATTACTCACTAAGATGGATCGGGACAAGAATAATAATCAGGCCCGAGGTTGCAC

[0198] P hbdH -1 promoter (SEQ ID NO: 13)

[0199] TGCGTAATGCCCCACCGTTCTGCCAGGCAACGCGAAACCTGTAGGAGCGGCCTTGTGTCGCGATGGGCTGCGCAGCAGCCCCGGCATTTTTTGCATCGATGCGGAGATCTGGGGCTGCTGCGCAGCCCATCGCGACACAAGGCCGCTCCTACAGGTTCCTGGCCCGCATGGGTAAAGTTCGAACCAGTCAGGAGTCATTG

[0200] P mmsA Promoter (SEQ ID NO: 14)

[0201] CCTCGAATGTGCAAAAACGCAGACCATACTTGCACATCACCGCATTGAGTACATCAAAAATGCACTGTTAGGATCGATCCAGACAACAAAAAAGCCACAGGCTGGGAGAATCCCG

[0202] In some embodiments, the recombinant bacterium expresses at least one gene for producing 3-HP (or a salt thereof) from glycerol under the control of at least one promoter represented by any one of SEQ ID NOs: 11-14. In some embodiments, the recombinant bacterium expresses at least one gene from any one of dhaB1, dhaB2, dhaB3, gdrA, gdrB, and / or kgsA under the control of at least one promoter represented by SEQ ID NOs: 11-14.

[0203] In some embodiments, the recombinant bacterium expresses at least one gene for producing 3-HP (or a salt thereof) from glycerol under the control of at least one promoter, wherein the promoter has a sequence that is at least 80% identical to any one of SEQ ID NOs: 11-14. In some embodiments, the recombinant bacterium expresses at least one gene for producing 3-HP or a salt thereof from glycerol under the control of at least one promoter, and the promoter has a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 11-14. In some embodiments, the recombinant bacterium expresses at least one gene for producing 3-HP or a salt thereof from glycerol under the control of at least one promoter, and the promoter has a sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides from any sequence of SEQ ID NOs: 11-14.

[0204] In some embodiments, the expression system described herein includes at least two promoters in tandem that control at least one gene involved in the production of 3-HP or a salt thereof from a carbon source such as glycerol. Tandem promoter systems have been observed in microorganisms and control the expression of downstream genes under various physiological or environmental conditions. Such systems facilitate the controlled regulation of target gene expression at different stages of cell growth. The tandem promoters can be natural or endogenous to the microorganism, such as promoters that are naturally present in a bacterial strain, or can be synthetic. In some embodiments, one or more of the promoters can be natural promoters. In some embodiments, one or more of the promoters can be synthetic promoters.

[0205] The tandem promoters of the expression system described herein can include one or more inducible promoters, and / or one or more constitutive promoters. In some cases, the tandem promoter includes two or more inducible promoters. A tandem promoter that includes two or more inducible promoters can have two identical inducible promoters, i.e., the inducible promoters are induced by the same inducer or stimulus, or two different inducible promoters, i.e., the inducible promoters are induced by different inducers or stimuli. In some embodiments, the tandem promoter includes at least one inducible promoter and at least one constitutive promoter. For example, the tandem promoter can combine a 3-HP inducible promoter and a constitutive promoter to regulate the expression of genes involved in 3-HP synthesis.

[0206] In some embodiments, there is no terminator sequence between the tandem promoters, for example, between any of the two or more promoters in an expression system, such that each promoter can regulate the expression of a gene located downstream of the tandem promoters.

[0207] In some embodiments, the tandem promoters include at least two 3-HP inducible promoters. In some embodiments, the tandem promoters include a first promoter that is a 3-HP inducible promoter and a second promoter that is a 3-HP inducible promoter. In some embodiments, each of the first 3-HP inducible promoter and the second 3-HP inducible promoter can be P mmsA promoter (SEQ ID NO: 14), P hpdH-1 promoter (SEQ ID NO: 13), P hbdH-4 promoter (SEQ ID NO: 11), or P hpdH promoter (SEQ ID NO: 12).

[0208] In some embodiments, the tandem promoters include a first promoter that is a 3-HP inducible promoter and a second promoter that is a constitutive promoter, wherein the first 3-HP inducible promoter is located 5' or upstream of the second constitutive promoter. In some embodiments, the first 3-HP inducible promoter is P mmsA promoter (SEQ ID NO: 14), P hpdH-1 promoter (SEQ ID NO: 13), P hpdH-4 promoter (SEQ ID NO: 11), or P hpdH promoter (SEQ ID NO: 12). In some embodiments, the second constitutive promoter is P zwf(SEQ ID NO: 7) Promoter. In some embodiments, the sequence of the second constitutive promoter is at least 80% identical to SEQ ID NO: 7. In some embodiments, the sequence of the second constitutive promoter is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7. In some embodiments, the sequence of the second constitutive promoter differs from SEQ ID NO: 7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. In some embodiments, the sequence of the second constitutive promoter is SEQ ID NO: 8 (Pzwf-1), SEQ ID NO: 9 (Pzwf-7), SEQ ID NO: 10 (Pzwf-12), SEQ ID NO: 52 (Pzwf-2), SEQ ID NO: 53 (Pzwf-3), SEQ ID NO: 54 (Pzwf-4), SEQ ID NO: 55 (Pzwf-5), SEQ ID NO: 56 (Pzwf-6), SEQ ID NO: 57 (Pzwf-7), SEQ ID NO: 58 (Pzwf-8), SEQ ID NO: 59 (Pzwf-10), SEQ ID NO: 60 (Pzwf-11), SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63.

[0209] Pzwf Constitutive Promoter (SEQ ID NO: 7)

[0210] GGCGACCAACAACGGCGCGAGGTGGCAAAAATATCTTGTTTAATTACTACATATTTGTCTTAATGCCGGCGTGTAAGGCTAACTATCGTTCAAAATTTAGTTGGTAACAACAA

[0211] Pzwf-1 Constitutive Promoter (SEQ ID NO: 8)

[0212] GGCGACCAACAACGGCGCGAGGTGGCAAAAACGGTTTGACACAGTAATTAAAAAGACGTATAATTGCGTTGTGTAAGGCTAACTATCGTTCAAAATTTAGTTGGTAACAACAA

[0213] The shorter constitutive promoter of Pzwf-1 (SEQ ID NO: 61)

[0214] ACGGTTTGACACAGTAATTAAAAAGACGTATAATTGCGTT

[0215] The constitutive promoter of Pzwf-7 (SEQ ID NO: 9)

[0216] GGCGACCAACAACGGCGCGAGGTGGCAAAAGTATATTGACATTCCATGCGAAGGTCGTTATAATACAGTAGTGTAAGGCTAACTATCGTTCAAAATTTAGTTGGTAACAACAA

[0217] The shorter Pzwf-7 (SEQ ID NO: 62)

[0218] GTATATTGACATTCCATGCGAAGGTCGTTATAATACAGTA

[0219] The constitutive promoter of Pzwf-12 (SEQ ID NO: 10)

[0220] GGCGCGAGGTGGCAAAATAATCTTGACAACTGGAGAGAATTGTGGTATAATGGGAGCGTGTAAGGCTAACTATCGTTCAAAATTTAGTTGGTAACAACAA

[0221] The shorter constitutive promoter of Pzwf-12 (SEQ ID NO: 63)

[0222] TAATCTTGACAACTGGAGAGAATTGTGGTATAATGGGAGC

[0223] The constitutive promoter of Pzwf#2 (SEQ ID NO: 52)

[0224] TCCACTTGACATACCCTAACATCGGGATTATAATGTCTGC

[0225] The constitutive promoter of Pzwf#3 (SEQ ID NO: 53)

[0226] GTTGGTTGACATGGCGCTGTCGATCGGATATAATGTTTGT

[0227] Pzwf#4 Constitutive Promoter (SEQ ID NO: 54)

[0228] GCGTCTTGACATCTTACTAGATTGTGCGTATAATAGTCGC

[0229] Pzwf#5 Constitutive Promoter (SEQ ID NO: 55)

[0230] GGCGTTTGACATGTGGATGTAATCCTGTTATAATTTTTTA

[0231] Pzwf#6 Constitutive Promoter (SEQ ID NO: 56)

[0232] GATCCTTGACAGCGAGGTATGAGTGAGGTATAATGTAACC

[0233] Pzwf#7 Constitutive Promoter (SEQ ID NO: 57)

[0234] TGCGTTTGACAATTTGTTACGTTAGTGCTATAATCTAGTT

[0235] Pzwf#8 Constitutive Promoter (SEQ ID NO: 58)

[0236] TACCCTTGACATAACGGCATTCTGGTGGTATAATCATGCC

[0237] Pzwf#10 Constitutive Promoter (SEQ ID NO: 59)

[0238] GGGGGTTGACAACTGCGTGTTTGTCTGTTATAATATCCCG

[0239] Pzwf#11 Constitutive Promoter (SEQ ID NO: 60)

[0240] GAGAATTGACAGATGACTTATTTCGTTGAATTCCTGC

[0241] In some embodiments, the sequence of the tandem promoter in the expression cassette can be SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In some embodiments, the sequence of the tandem promoter in the expression cassette can be at least 80% identical to SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In some embodiments, the sequence of the tandem promoter in the expression cassette can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In some embodiments, the sequence of the tandem promoter in the expression cassette can differ from any of the sequences of SEQ ID NO: 70 - 72 by 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides.

[0242] Pcm - Pc4 tandem promoter (SEQ ID NO: 65)

[0243] CCTCGAATGTGCAAAAACGCAGACCATACTTGCACATCACCGCATTGAGTACATCAAAAATGCACTGTTAGGATCGATCCAGACAACAAAAAAGCCACAGGCTGGGAGAATCCCGGCCCCCGCCTACCCGCCCCAGCCACCCCGGACTCAGCAAGGATGCTGGCCCGGGCCTGGGCGGAGACGTCTTTCGCGCCCGACCATCAGAACAAGAGGACAACCCC

[0244] Pc3 - Pc1 tandem promoter (SEQ ID NO: 70)

[0245] CGTGGCGACTCTCATTGTTAGAAAACGCACAGCAGGTGACTTTAAACGTTCGTATTTTTATCGCGAACGAACGACTAGGCTCCATCGTCATACCCAAAAGAACAAGAACGACGAGGGACTTTCCGGCGTTTGACATGTGGATGTAATCCTGTTATAATTTTTTAGTGTAAGGCTAACTATCGTTCAAAATTTAGGTGGTAACAACAAATG

[0246] Pc1-Pc3 tandem promoter (SEQ ID NO: 71)

[0247] GGCGTTTGACATGTGGATGTAATCCTGTTATAATTTTTTAGTGTAAGGCTAACTATCGTTCAAAATTTAGGTGGTAACAACAACGTGGCGACTCTCATTGTTAGAAAACGCACAGCAGGTGACTTTAAACGTTCGTATTTTTATCGCGAACGAACGACTAGGCTCCATCGTCATACCCAAAAGAACAAGAACGACGAGGGACTTTCCATG

[0248] Pc3-Pzwf tandem promoter (SEQ ID NO: 72)

[0249] CGTGGCGACTCTCATTGTTAGAAAACGCACAGCAGGTGACTTTAAACGTTCGTATTTTTATCGCGAACGAACGACTAGGCTCCATCGTCATACCCAAAAGAACAAGAACGACGAGGGACTTTCCGGCGACCAACAACGGCGCGAGGTGGCAAAAATATCTTGTTTAATTACTACATATTTGTCTTAATGCCGGCGTGTAAGGCTAACTATCGTTCAAAATTTAGTTGGTAACAACAAATG

[0250] Transcription regulatory factor

[0251] The regulatory region of the 3-HP degrading gene is located near the sequence encoding the natural LysR family transcriptional regulator (LTTR) protein. When complexed with 3-HP, the LysR family transcriptional regulator protein activates transcription. In bacteria, the LTTR protein upregulates downstream genes by interacting with RNA polymerase. Thus, the amount of the LTTR protein affects the transcriptional level of downstream target genes.

[0252] In some embodiments, at least one transcriptional regulator protein is expressed in a recombinant microorganism such as a bacterium with at least one 3-HP producing gene described herein. In some cases, at least one transcriptional regulator protein is an LTTR protein under the control of at least one constitutive or inducible promoter. In some cases, the transcriptional regulator protein binds to a site upstream of the at least one 3-HP producing gene and regulates the expression of the at least one gene involved in 3-HP synthesis.

[0253] In some embodiments, the recombinant bacteria as described herein express at least one LTTR protein and at least one gene for producing 3-HP or a salt thereof, such as at least one glycerol dehydratase, diol dehydratase, and / or aldehyde dehydrogenase. In some embodiments, the LTTR protein is MmsR. In some embodiments, the MmsR promoter is expressed in the recombinant bacteria under the control of a constitutive promoter. In some embodiments, the constitutive promoter is the Pzwf promoter (SEQ ID NO: 7). In some embodiments, the constitutive promoter has been mutated to increase or decrease the expression of MmsR relative to the Pzwf promoter. In some embodiments, the constitutive promoter is the Pzwf (SEQ ID NO: 7) promoter. In some embodiments, the sequence of the constitutive promoter is at least 80% identical to SEQ ID NO: 7. In some embodiments, the sequence of the constitutive promoter is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In some embodiments, the sequence of the constitutive promoter differs from SEQ ID NO: 7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides. In some embodiments, the constitutive promoter is SEQ ID NO: 8 (Pzwf-1), SEQ ID NO: 9 (Pzwf-7), SEQ ID NO: 10 (Pzwf-12), SEQ ID NO: 52 (Pzwf-2), SEQ ID NO: 53 (Pzwf-3), SEQ ID NO: 54 (Pzwf-4), SEQ ID NO: 55 (Pzwf-5), SEQ ID NO: 56 (Pzwf-6), SEQ ID NO: 57 (Pzwf-7), SEQ ID NO: 58 (Pzwf-8), SEQ ID NO: 59 (Pzwf-10), SEQ ID NO: 60 (Pzwf-11), SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the mmsR gene is located on the chromosome, such as under the control of a constitutive promoter expressed on the chromosome of the recombinant bacteria.

[0254] 5’UTR

[0255] In some embodiments, the expression systems described herein can have a 5'UTR operably linked to a gene of interest (e.g., a gene involved in 3-HP synthesis), wherein the 5'UTR is from a native gene regulated by a 3-HP inducible promoter in a bacterium that naturally produces 3-HP, such as Pseudomonas denitrificans. In some embodiments, the expression systems described herein have the 5'UTR of the mmsA gene of Pseudomonas denitrificans (SEQ ID NO: 64).

[0256] In some cases, mutations in the 5'UTR can increase the translation initiation rate of the mRNA transcript and / or increase the stability of the mRNA transcript produced by the expression system. In some embodiments, the expression systems described herein can have a 5'UTR whose sequence is at least 80% identical to SEQ ID NO: 64. In some embodiments, the expression systems described herein can have a 5'UTR whose sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 64. In some embodiments, the expression systems described herein can have a 5'UTR whose sequence differs from SEQ ID NO: 64 by 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. In some embodiments, the expression systems described herein can have a 5'UTR comprising any sequence selected from SEQ ID NOs: 22-28.

[0257] In a non-limiting example, an expression system having the PmAdH-4 tandem promoter, Opt-3, Hyb-20, and chromosomal MmsR has the 5′UTR of the mmsA gene. In some embodiments, the mmsA 5’UTR can be mutated.

[0258] mmsA 5’UTR (SEQ ID NO: 64)

[0259] CGACGGCAAGCCCGTCGAGTCCACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0260] mmsA 5’UTR mutant UTR-0 (SEQ ID NO: 22)

[0261] CGACGGCAAGCCCAAGCAGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0262] mmsA 5’UTR mutant UTR-1 (SEQ ID NO: 23)

[0263] CGACGGCAAGCCAACTGAACCCACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0264] mmsA 5’UTR mutant UTR-2 (SEQ ID NO: 24)

[0265] CGACGGCAAGCCCTAACTGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0266] mmsA 5’UTR mutant UTR-3 (SEQ ID NO: 25)

[0267] CGACGGCAAGCCCTAACAGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0268] mmsA 5’UTR mutant UTR-4 (SEQ ID NO: 26)

[0269] CGACGGCAAGCCCAAGCTGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0270] mmsA 5’UTR mutant UTR-5 (SEQ ID NO: 27)

[0271] CGACGGCAAGCCCTAGCAGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0272] mmsA 5’UTR mutant UTR-6 (SEQ ID NO: 28)

[0273] CGACGGCAAGCCCAAGCAGGACACCATGGCTAACGTGACTTACACCGATACCCAACTGCT

[0274] Optimizing gene expression in Pseudomonas denitrificans by fusing the first few amino acids of a native protein

[0275] To optimize the expression of heterologous proteins in Pseudomonas denitrificans, a new method was adopted. The heterologous protein was fused at the N-terminus of the target protein with some initial amino acids (5 - 20 AA) of a highly expressed native enzyme (for example, fusing the 5 - 20 amino acids at the N-terminus of the native protein with the N-terminus of the heterologous protein). For example, the N-terminus of mmsA of various lengths (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long; named Hyb-5, Hyb-6, Hyb-7, Hyb-8, Hyb-9, Hyb-10, Hyb-11, Hyb-12, Hyb-13, Hyb-14, Hyb-15, Hyb-16, Hyb-17, Hyb-18, Hyb-19 and Hyb-20 respectively) was ligated to the full-length heterologous kgsA gene and expressed by a multi-copy plasmid ( Figure 17A - 17D ). The highest activity was obtained with Hyb-20 (+3-HP, 9.3 U / mg protein; -3-HP, 3.0 U / mg protein). On the other hand, neither the expression nor the enzyme activity of Hyb-5 showed any change ( Figure 17A ). SDS-PAGE analysis showed that when kgsA was fused with the N-terminal mmsA fragment (more than five amino acids were added), the production of the protein increased ( Figure 17B ). To understand the mechanism of improved expression and mRNA stability in wild-type and Hyb-20 kgsA recombinant strains, transcripts were measured and compared. The stability of mmsA and mmsR transcripts was also determined ( Figure 17C and 17D ). The half-life of the wild-type kgsA transcript (2.7 minutes) was 2.3 times shorter than that of mmsA (6.1 minutes); however, by fusing the initial 20 aa of mmsA (20), the half-life of the mRNA of the fusion protein was greatly increased to 5.7 minutes, which was comparable to the half-life of the mRNA of native mmsA. This indicates that the fusion protein (mRNA transcript) became less vulnerable to endogenous nuclease attack. The transcription level of the hybrid mmsA(20)kgsA gene determined by RT-PCR was also twice as high as that of the wild-type kgsA gene (data not shown). We believe that the higher mRNA stability as well as the improved transcription should contribute to the improved KgsA activity of the hybrid enzyme. Among the several genes examined, mmsA showed the highest mRNA stability with a half-life of 9.5 minutes (data not shown).

[0276] Recombinant bacteria produce coenzyme B12

[0277] In the absence of externally added coenzyme B12, the production of 3-HP from glycerol by microorganisms that produce 3-HP or its salts is limited. Coenzyme B12 is an important cofactor for glycerol / diol dehydratase activity and is used in the first reaction in the 3-HP synthesis pathway, where glycerol is catalytically converted to 3-hydroxypropanal. Thus, continuous supply of coenzyme B12 is essential for the continuous production of 3-HP or its salts from glycerol by microorganisms such as bacteria. Coenzyme B12 can be naturally synthesized by a limited number of microorganisms under aerobic or anaerobic conditions (anaerobic producers of coenzyme B12 include, for example, species of Klebsiella, Streptococcus, and Salmonella. Aerobic producers of coenzyme B12 include, for example, species of Pseudomonas, Rhizobium, and Rhodobacter). However, microorganisms that have been evaluated as potential hosts for the production of 3-HP or its salts from glycerol do not appear to produce sufficient amounts of coenzyme B12 to produce 3-HP at a high titer. Further analysis has shown that the production of coenzyme B12 is subject to transcriptional and translational regulation. These processes can be genetically modified in recombinant microorganisms such as bacteria to increase the production of coenzyme B12 and thus increase the titer of 3-HP (or its salts) without the need to supplement the bacteria with external coenzyme B12.

[0278] As Figure 23 shown, two gene clusters (cluster I and cluster II) are responsible for the production of proteins used to generate coenzyme B12 in Pseudomonas denitrificans. Riboswitches and other secondary structures have been identified in the promoter regions of the operons present in cluster I and cluster II (see Figure 23 ). These riboswitches appear to regulate the expression of the B12 biosynthesis operon and may repress gene expression. In some embodiments described herein, one or more riboswitches or other secondary structures can be removed from the promoter region of one or more operons of cluster I and / or cluster II in the genome of Pseudomonas denitrificans. In some embodiments, one or more or portions of riboswitch 1, 2, 3, and / or 4 are deleted in the genome of Pseudomonas denitrificans, as Figure 23 and Figure 25A - 25D shown. In some embodiments, all or part of the DNA sequence of riboswitch 1 (SEQ ID NO: 75) or riboswitch 2 (SEQ ID NO: 76) is removed from the genome of Pseudomonas denitrificans. In some embodiments, all or part of the DNA sequences of riboswitch 1 (SEQ ID NO: 75) and riboswitch 2 (SEQ ID NO: 76) are removed from the genome of Pseudomonas denitrificans.

[0279] Any expression system described herein can be used to increase the expression of genes involved in coenzyme B12 production and / or increase the production of coenzyme B12. The 3-HP inducible promoter system described herein can be used to replace the regulatory regions of B12-producing genes (e.g., translational and / or transcriptional regulators). The present disclosure provides methods for increasing coenzyme B12 production such that the supplementation of coenzyme B12 is not used by the organism to produce 3-HP. Recombinant organisms are provided herein that can include an expression system for controlling and / or upregulating the expression of genes for producing 3-HP and an expression system for controlling and / or upregulating the expression of genes for producing coenzyme B12.

[0280] Riboswitch 1 (RS1) (SEQ ID NO: 75)

[0281] Ccagatgccgacgatggtcagccagggcgtcatggggattcctctgaagggatgcgatgatcgatccgacgggcaggcttgttccgccctcgggcaaagcaggcataatacccgcatcgtcggtgctcgtaggacgcttcgctgtttgaaggcgatgcatccgagagccgaagagggaacacggaaaaaccgtggctgcccccgcaactgtaagcagcgagtccgcgcacttcgaccacagcgtctgttgtcgtcgatctggccactgggcaaccgggaaggccgtgccggatgaggacctgccagccaggagacctgccgacgaaaccagtcgcgcgtgcagacatcgagcggggtgtatcggtgtcgtgaagtcccctgtggggattcgcaggtcaccgcgacccagccctggtgacctca

[0282] Riboswitch 2 (RS2) (SEQ ID NO: 76)

[0283] ggcggatgaagggcgcgcgcgcgggttcgccgcgcaacgaaggtcgccaccggatcaccccgcccggttgtctgttgataacgaaccggttccggatctcgcgagctagagccaggcaaggcggaggatcgtcggggacgcggagttgactgtagtcaatgagcagtccacgacgatccgccaacgcagcatggccgacgcgcagcagatcgaaaaccagtcacgaggcaggtctcctggctcacagcccttgatcgtcctttcgccttcccgccgtagtcggcagtggcgtgtgaaagaacaggctgttcacagttgcgggggcagccgtggcgcatccgaagatttccacgttccctcttagctccggccagtgccggagaacctcgaagggaggaaggctacgcagcgtggccggggcggtcaatcgccggggatcgggcgacgcgcagttgacgctgcgggactgccgtggtcagctagcgcggtttcaggtgtctcgcgccgacgcgcgcgaggtgaaacgggaagccggtgcgtccgcaaggaccagtccggcgctgcccccgcaacggtaagcgcatcgagggtcgtcagtagccactgtgccaaggcatgggaaggctggcccatccggcgagagtctctcgctggcgtcgcaagcccggagaccggcctggaatcctcacattttggcaaacccgcggtgggcgggcgcaggccgtggcgcgaccgatccggcgcgttcgaatgcgttcaacctctgcgttctcactcttttcagagggaacgttcatgtccagcagcatcctgacgcaacaggcg

[0284] Expression system placement for Channeling

[0285] Any method well-known in the art can be used to genetically introduce the expression systems described herein into a host microorganism, such as a bacterium. In certain cases, plasmids, artificial chromosomes, or other vectors can be used to introduce the expression systems into the microorganism. In some embodiments, the expression systems can be integrated into the genome of the microorganism, such as into a chromosome.

[0286] Multiple copies of the expression systems can be introduced into a microorganism such as a bacterium. Multiple copies of the expression systems can be introduced into a microorganism such as a bacterium on at least one plasmid, artificial chromosome, or other vector. For example, multiple copies of the expression systems can be introduced into a bacterium on a single plasmid or on two or more different plasmids (i.e., plasmids having different sequences, selectable markers, etc.). Multiple copies of the expression systems can be integrated into the genome of the microorganism. In some embodiments, multiple copies of the expression systems can be integrated at the same location in the genome, such as at the same chromosomal location. In some embodiments, multiple copies of the expression systems can be integrated at different locations, such as at different chromosomal locations. In some embodiments, multiple copies of the expression systems are integrated at different locations in a Pseudomonas bacterium such as Pseudomonas denitrificans. In some embodiments, two or more expression systems that regulate different genes involved in the production of 3-HP from a carbon source (e.g., one or more glycerol dehydratase or diol dehydratase genes and aldehyde dehydrogenase genes) are integrated into the genome of a Pseudomonas bacterium. In some embodiments, the two or more expression systems are integrated at the same location in the genome. In some embodiments, the two or more expression systems are integrated at different chromosomal locations.

[0287] In some embodiments, one or more of the expression systems described herein are introduced into a bacterium, such as a Pseudomonas denitrificans bacterium. In some embodiments, an expression system that regulates one or more genes encoding a glycerol dehydratase gene and / or a diol dehydratase gene (e.g., at least one of the dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) and an expression system that regulates one or more genes encoding an aldehyde dehydrogenase (e.g., at least one ksgA gene) are introduced into a Pseudomonas denitrificans bacterium and optionally integrated into the genome of the bacterium. In some embodiments, the expression systems that regulate the glycerol dehydratase (and / or diol dehydratase) and aldehyde dehydrogenase genes are integrated at different chromosomal locations.

[0288] In some embodiments, expression systems that regulate the expression of glycerol dehydratase (and / or diol dehydratase) and aldehyde dehydrogenase genes are integrated at different chromosomal locations. The specific chromosomal integration sites can significantly affect the expression levels of the expression systems or modules, such that the gene expression levels at one integration site will be different from those at another integration site. Additionally, the positions at which specific genes in a pathway are inserted into the chromosome can balance the activities of the synthetic pathway enzymes and avoid the accumulation of toxic intermediates (such as 3-hydroxypropanal). For example, in many prokaryotes, genes encoding the enzymes of a pathway are clustered together. Such a clustered arrangement has been observed in bacteria, and it is believed that this avoids the accumulation of intermediates produced by the pathway in the cell. In prokaryotes, transcription and translation occur simultaneously, and in many cases, the product of the first enzyme encoded by the first gene will be the substrate for the second enzyme encoded by the second gene in the pathway. If these genes are close to each other, the substrate for the second enzyme encoded by the second gene in the pathway (which is produced by the enzyme encoded by the first gene) will be located near the second enzyme. This mechanism increases the efficiency of the pathway by allowing the intermediate to be immediately consumed by the next enzyme in the pathway and avoids the accumulation of potentially toxic intermediates. Such clustering of genes that are very close to each other is called channeling.

[0289] Thus, the levels of glycerol dehydratase (and / or diol dehydratase) and aldehyde dehydrogenase can be regulated by integrating expression systems that regulate these enzymes at different chromosomal locations, thereby regulating the levels of 3-HPA and 3-HP in recombinant bacteria. Thus, the localization of glycerol dehydratase and aldehyde dehydrogenase in the chromosome will produce a channeling effect, such as by causing the rapid conversion of 3-HPA to 3-HP. By producing a channeling effect by localizing different expression systems that respectively regulate glycerol dehydratase (and / or diol dehydratase) and aldehyde dehydrogenase, the accumulation of 3-HPA, which is toxic to cells, in bacterial cells can be prevented, thereby enabling the cells to survive and grow for a longer time and produce a higher titer of 3-HP or its salt.

[0290] In some cases, the interrelationship between the first integration position of the first expression system and the second integration position of the second expression system affects the production level of 3-HP or its salt. In certain cases, the first expression system can be an expression system that expresses glycerol dehydratase or diol dehydratase (e.g., one or more dhaB and / or gdrAB genes). In certain cases, the second expression system can be an expression system that expresses aldehyde dehydrogenase (e.g., one or more ALDH genes, such as the kgsA, ealdH, and / or kaldH genes). In certain cases, a system integrated in such a way that the two expression systems are closely related to each other can reduce the accumulation of toxic intermediates (such as 3-HPA) and reduce the exposure of other enzymes to these toxic intermediates, thereby increasing the yield of 3-HP or its salt.

[0291] In some embodiments, as described herein, an expression system or module encoding at least one glycerol dehydratase (and / or one or more diol dehydratase genes) is located within about 500 - 2,500,000 nucleotide base pairs (e.g., between about 500 base pairs and 2,500 kilobase pairs) (e.g., nucleotides or base pairs), such as within about 500 - 2,500,000 base pairs of an expression system or module encoding at least one aldehyde dehydrogenase as described herein, e.g., in a recombinant bacterium such as Pseudomonas denitrificans. In some embodiments, as described herein, the expression systems encoding at least one glycerol dehydratase (or diol dehydratase) and at least one aldehyde dehydrogenase are located within about 2,500 kilonucleotides, 1,500 kilonucleotides, 500 kilonucleotides, 250 kilonucleotides, 150 kilonucleotides, 50,000 nucleotides, 25,000 nucleotides, 15,000 nucleotides, 10,000 nucleotides, 8,000 nucleotides, 6,000 nucleotides, 4,000 nucleotides, 3,800 nucleotides, 3,600 nucleotides, 3,400 nucleotides, 3,200 nucleotides, 3,000 nucleotides, 2,800 nucleotides, 2,600 nucleotides, 2,400 nucleotides, 2,200 nucleotides, 2,000 nucleotides, 1,800 nucleotides, 1,600 nucleotides, 1,400 nucleotides, 1,200 nucleotides, 1,000 nucleotides, 900 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 50 nucleotides, or fewer nucleotides of each other. In some embodiments, the distance between the integration sites of the genes or expression systems is about 4000 base pairs. In some embodiments, the integration sites are at least about 1500 base pairs apart (e.g., about 1500 base pairs; 2000 base pairs; 2500 base pairs; 3000 base pairs; 3500 base pairs; 4000 base pairs; 4500 base pairs; 5000 base pairs; 5500 base pairs; 6000 base pairs; 6500 base pairs; 7000 base pairs; 7500 base pairs; 8000 base pairs; 8500 base pairs; 9000 base pairs; 9500 base pairs; 10,000 base pairs; 20,000 base pairs; 50,000 base pairs; 100,000 base pairs; 200,000 base pairs; or 500,000 base pairs).In some embodiments, an expression system encoding at least one glycerol dehydratase or diol dehydratase as described herein and an expression system encoding at least one aldehyde dehydrogenase as described herein are within about 500 nucleotides of each other in the bacterial chromosome (e.g., the distance between the integration sites of the genes or expression systems is about 500 base pairs).

[0292] In some embodiments, the location in the bacterial chromosome into which an expression system is integrated affects the expression level of the expression system. In certain cases, the closer to the origin of replication, the higher the expression level of the expression system. In some embodiments, the expression system is integrated at a position between about 500 and 4000 nucleotides (e.g., nucleotides or base pairs) from the origin of replication. In some embodiments, the expression system is integrated at about 4000 nucleotides, about 3000 nucleotides, about 2000 nucleotides, about 1000 nucleotides, about 750 nucleotides, or about 500 nucleotides from the origin of replication. In some embodiments, the integration site closest to the origin of replication is about 500 nucleotides from the origin of replication. In some embodiments, the integration site closest to the origin of replication is at least 500 nucleotides (e.g., about 500 nucleotides; 600 nucleotides; 700 nucleotides; 800 nucleotides; 900 nucleotides; 1000 nucleotides; 1100 nucleotides; 1200 nucleotides; 1300 nucleotides; 1400 nucleotides; 1500 nucleotides; 2000 nucleotides; 2500 nucleotides; 4000 nucleotides; or 5000 nucleotides). In some embodiments, the expression system is integrated at about 500 nucleotides from the origin of replication.

[0293] As described above, a system was created that can channelize 3-HPA through DhaB and ALDH enzymes, thereby increasing 3-HP production and cell viability. The system reduces the exposure of other enzymes and cellular components to toxic 3-HPA and increases the production of 3-HP or its salts. In one embodiment, the dhaB, gdrAB genes, and kgsA are placed adjacent to each other. In another case, the position of kgsA is more than 2000 bp away from the DhaB and GdrAB encoding genes. The integration positions of the genes relative to each other and to the origin of replication affect expression and activity.

[0294] When kgsA is placed in different positions (close to DhaB, GdrAB (strain P4-20), 2000 bp away from DhaB, GdrAB (strain P4-10)), we notice differences in enzyme activity. In order to study the channelization effect, the enzyme activities were synchronized in strains P4-10 and P4-20 (by changing gene regulatory modules such as promoters, UTRs). After these modifications, both strains (with (P4-20) and without (P4-10) channelization effect) showed similar KgsA and DhaB activities. The only difference between the two strains (with and without channelization) is the position of the kgsA gene relative to the dhaB and gdrAB genes ( Figure 51 ).

[0295] In these identical strains (P4-10 and P4-20), the yield of 3-HP was compared. Compared with the P4-10 strain in which the genes (kgsA gene and dhaB and gdrAB genes) were not channelized (e.g., 2000bp apart on the chromosome), the P4-20 strain in which the genes (kgsA gene and dhaB and gdrAB genes) were channelized (e.g., located close to each other) showed higher 3-HP productivity and improved 3-HP titer. In the P4-20 strain, the accumulation level of 3-HPA was lower than that of P4-10. In some cases, this was due to the consumption of 3-HPA by the KgsA enzyme next to it. In the P4-20 strain, the DhaB enzyme (producing 3-HPA) was close to the KgsA enzyme, so 3-HPA was rapidly consumed by the KgsA enzyme before the KgsA enzyme accumulated and / or affected the enzyme.

[0296] Biological production

[0297] Optimal physiological parameters, such as culture medium, aeration, temperature, pH, and induction time of the target product, either alone or in combination, can have a substantial impact on cell growth and production of the target molecule.

[0298] The culture medium is an important parameter in the bioprocess, which plays a vital role in maintaining cell viability and improving the titer of the target product, and therefore must be properly formulated to be suitable for the efficient production of 3-HP. Bioprocesses that use analytical grade chemicals to produce 3-HP or its salts are not commercially viable. Therefore, it is very important to carefully select and formulate the culture medium components. Among these components, coenzyme B12, carbon source and nitrogen source for DhaB enzyme activity are expensive components supplemented in the culture medium for 3-HP production, therefore, it is very important to identify and examine cheaper culture medium components and formulate suitable culture medium for 3-HP production and commercialization.

[0299] In addition to the industrial medium formulation, bioprocess conditions under bioreactor conditions, such as temperature, pH, aeration, etc., were also studied and optimized. Cell metabolism and NAD+ regeneration, which are crucial for 3-HP production, were analyzed by changing physiological parameters under controlled conditions. The effect of pH, which plays a crucial role in enhancing the acid tolerance of the recombinant strain and the enzyme efficiency of the 3-HP production pathway, was investigated. The optimization of aeration, temperature, and induction time was carefully studied because each physiological parameter significantly affects 3-HP production. Through bioprocess optimization, the performance of the recombinant strain for producing 3-HP from glycerol was improved.

[0300] As described in the examples, various carbon sources that can support the growth of the recombinant strain to a higher cell density and produce 3-HP at a high titer were studied, including, for example, glucose, glutamate, gluconate, and citrate. The pH of the growth conditions was also studied, and in some cases, neutralizing bases were used in the bioreactor to maintain the pH. Bacterial strains that can grow at a higher acidic concentration and / or a higher 3-HP concentration are also provided herein. In some cases, the bicarbonate level in the growth culture can be controlled.

[0301] Nitrogen is another important component in the medium. As shown in the following examples, the use of various nitrogen sources for 3-HP production was also studied, including, for example, corn steep liquor, yeast extract, etc.

[0302] Aeration is an important characteristic for cell growth. Aeration of the culture conditions is used for cell growth, but excessive aeration will reduce the level of 3-HP production. The following examples illustrate the appropriate aeration for 3-HP bioproduction.

[0303] The present disclosure also provides the identification of the optimal temperature for recombinant microorganisms for cell growth and target molecule production. This involves a balance between the optimal temperature for cell growth and the optimal temperature for target molecule production.

[0304] The induction time schedule for 3-HP production can also be adjusted according to the culture conditions and the recombinant bacterium. For example, by adding glycerol (as a source of 3-HP) at the mid-log phase for induction, a high level of 3-HP was produced by the recombinant strain. Other culture conditions and / or recombinant strains can produce a high level of 3-HP by inducing at the early or late log phase.

[0305] Removing 3-HP from the aqueous solution

[0306] In some embodiments, a method for extracting 3-HP from an aqueous solution includes: evaporating a water-immiscible solvent; condensing the vaporized water-immiscible solvent into a liquid state; extracting 3-HP from an aqueous phase containing 3-HP using a water-immiscible liquid solvent to provide a solution of 3-HP in the water-immiscible solvent; separating the solution of 3-HP in the water-immiscible solvent from the aqueous phase. Typically, prior to contact with the water-immiscible liquid solvent, the aqueous phase containing 3-HP further contains a water-miscible solvent.

[0307] Generally, any suitable water-miscible or water-immiscible solvent, or any combination thereof, can be used to remove 3-HP from an aqueous solution. Exemplary embodiments of these solvents and their combinations are described in the corresponding "water-immiscible solvent", "water-miscible solvent", and "solvent combination" sections of the present disclosure.

[0308] Generally, any appropriate process conditions can be used in the method for removing 3-HP from an aqueous solution. Exemplary process parameters are disclosed in the section titled "Process Parameters" below.

[0309] Generally, 3-HP is a polar organic acid that is highly soluble in water and poorly soluble in organic solvents. 3-HP is a commercially available product. For example, 3-HP can be purchased from Sigma-Aldrich in the form of a 30 wt% aqueous solution (catalog number 792659), while a 5-6 wt% solution of 3-HP in ethyl acetate is difficult to obtain. Without wishing to be bound by theory, it is believed that adding a water-miscible solvent (such as methanol) to an aqueous solution of 3-HP reduces the solubility of 3-HP in the resulting aqueous solution, thereby facilitating the extraction of 3-HP from the aqueous solution using a water-immiscible solvent (such as ethyl acetate).

[0310] Aqueous solution

[0311] As used herein, the term "aqueous solution" refers to a solution of at least one solute in a liquid comprising one or more solvents (e.g., a mixture of solvents), wherein at least one solvent is water and the weight percentage of water in the solvent or solvent mixture is at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least 90%). In some embodiments, the aqueous solution is a solution in which water is the only solvent.

[0312] In some embodiments, the amount of 3-HP in the aqueous solution is from about 10 g / L to about 150 g / L (e.g., from about 20 g / L to about 140 g / L, from about 30 g / L to about 130 g / L, from about 40 g / L to about 120 g / L, from about 50 g / L to about 110 g / L, from about 50 g / L to about 100 g / L, from about 60 g / L to about 100 g / L, from about 60 g / L to about 80 g / L, or from about 80 g / L to about 120 g / L). The concentration of 3-HP in the solution can be expressed as the mass (titer) of 3-HP in a specific volume of the solution containing 3-HP, the amount (molarity) of 3-HP in a specific mass of the solvent, or the amount (molarity) of 3-HP in a specified volume of the solution. Any other way of expressing the concentration of the solute in the solution can be used additionally or alternatively to describe the concentration of 3-HP in the aqueous solution. For example, in some embodiments, the titer of 3-HP in the aqueous solution is at least about 40 g / L (e.g., about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, or about 120 g / L).

[0313] In some embodiments, the aqueous solution is the fermentation broth obtained after decellularization. Any method for removing whole cells from the fermentation broth can be used for decellularization. For example, a centrifuge can be used to decellularize the fermentation broth. In some embodiments, the aqueous solution is the fermentation broth (e.g., culture medium) obtained after culturing or growing microorganisms. In certain cases, the aqueous solution is decellularized. In some embodiments, a fermentation broth containing 3-HP can be obtained by culturing a microorganism that produces 3-HP. Illustrative examples of such microorganisms include Gram-negative bacteria such as Escherichia coli, Oligotropha carboxidovorans, Klebsiella pneumoniae, or species of the genus Pseudomonas; and Gram-positive bacteria such as Bacillus subtilis, species of the genus Lactobaccilus, or species of the genus Lactococcus. Microorganisms that may produce 3-HP by fermentation include the genus Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, or Saccharomyces. In some embodiments, the microorganism that produces 3-HP is the recombinant bacterium described herein. Some examples of recombinant bacteria that produce 3-HP are strains of the genus Pseudomonas, strains of the genus Klebsiella, or strains of the genus Escherichia coli.Some examples of microorganisms that may produce 3-HP by fermentation include Alcaligenes eutrophus (Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis, and Saccharomyces cerevisiae.

[0314] The fermentation medium in the fermentation broth may contain a variety of nutrients and components (e.g., carbon and / or nitrogen sources) that are typically useful in bacterial or fungal growth media. Illustrative and non-limiting examples of such nutrients and components are described herein. An example of a carbon source is sugar, such as glucose, glutamate, gluconate, fructose, arabinose, or galactose, citric acid, or citric acid cycle intermediates such as pyruvate or succinate, or combinations thereof. Another example of a carbon source is glycerol. Exemplary nitrogen sources include ammonium salts, corn steep liquor, yeast extract, and nitrates. Illustrative examples of other components that can be used in the fermentation medium also include serum proteins, vitamins, nucleic acids, and amino acids.

[0315] In some embodiments, the cell-free fermentation broth containing 3-HP after fermentation contains from about 0.5 wt% to about 5 wt% (e.g., from about 1 wt% to about 3 wt%) of a carbon source, such as glycerol.

[0316] In some embodiments, the cell-free fermentation broth contains additional components (such as those described herein) used by the microorganism to produce 3-HP in a total amount of from about 0.5 wt% to about 20 wt% (e.g., from about 1 wt% to about 10 wt%).

[0317] In some embodiments, the pH of the fermentation broth after fermentation and decellularization is from about 7 to about 8 (e.g., from about 7 to about 7.5). Such a fermentation broth may contain a salt of 3-HP, such as a sodium salt or a potassium salt. In some embodiments, an acid may be added to the decellularized fermentation broth to lower the pH and obtain the free acid form of 3-HP in the fermentation broth. Any suitable inorganic or organic acid can be used to adjust the pH of the fermentation broth. Exemplary inorganic acids include HCl, H2SO4, HNO3, and H3PO4. Exemplary organic acids include formic acid, oxalic acid, acetic acid, tartaric acid, malonic acid, glutaric acid, succinic acid, and trifluoroacetic acid. In some embodiments, a saturated aqueous solution of oxalic acid is used to adjust the pH of the aqueous solution. The concentration of the saturated solution of oxalic acid at about room temperature is, for example, from about 40 g / L to about 50 g / L (e.g., about 45 g / L).

[0318] In some embodiments, the pH of the aqueous solution containing 3-HP is from about 3 to about 7 (e.g., from about 4 to about 7, from about 4 to about 5, from about 4 to about 6, from about 4.1 to about 4.9, from about 4.2 to about 4.7, from about 4.2 to about 4.8, from about 4.3 to about 4.6, or from about 4.3 to about 4.5). In some embodiments, the pH of the aqueous solution is at least about 3 (e.g., at least about 3.5, at least about 4, or at least about 4.1) and / or at most about 7 (e.g., at most about 6.5, at most about 6, at most about 5.5, or at most about 5.0). In some embodiments, the pH of the aqueous solution is about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, or about 4.7.

[0319] In some embodiments, as 3-HP is removed from the aqueous solution, the pH of the aqueous solution gradually increases. In such embodiments, during the removal process, the pH value of the aqueous solution can be adjusted (continuously or discontinuously) by continuously or discontinuously adding one or more acids to the aqueous solution to address any gradual increase in the pH value due to the removal of 3-HP as needed.

[0320] In some embodiments, it can be at least about 20% (e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%). In some embodiments, 3-HP can be removed from the aqueous solution such that only trace amounts of 3-HP can be detected in the aqueous solution (e.g., by HPLC or LCMS). In some embodiments, 3-HP can be completely removed from the aqueous solution (i.e., the yield of the removal process is 100%).

[0321] As used herein, the term "yield" refers to the ratio (expressed as a percentage) of the total amount of product obtained, based on the amount of starting material used in the process, to the theoretical amount of product that could be obtained by the method used to obtain the product.

[0322] In some embodiments, 3-HP can be removed from an aqueous solution without using a counter-current fluid flow. As used herein, the term "counter-current fluid flow" refers to a two-way flow of two immiscible liquids after contact and phase separation. In some embodiments, the flow rates of the liquids are equal. In other embodiments, the flow rate of one liquid is greater than the flow rate of the other liquid. The counter-current extraction of a solute from an aqueous solvent by a water-immiscible solvent is just one example of counter-current fluid flow. In embodiments where 3-HP is removed from an aqueous solution without using counter-current, 3-HP can be extracted from the aqueous solution with an organic solvent, for example, without using any techniques and equipment for counter-current flow of the aqueous solution and the organic solvent. Counter-current techniques and equipment are described, for example, in PCT Publication Nos. WO 2005 / 003074, WO 2013 / 192450, and WO 2013 / 192451.

[0323] In some embodiments, 3-HP can be removed from an aqueous solution by using an organic solvent or a combination of two or more organic solvents. In some embodiments, the organic solvent used to remove 3-HP from an aqueous solution can be used after (1) evaporating the solvent; and (2) condensing the evaporated solvent. In some embodiments, 3-HP can be, for example, extracted from the aqueous phase by the solvent by directing a solvent stream into the aqueous solution to achieve effective mixing of the solvent and the aqueous phase, and then the condensed solvent stream can be directed to remove 3-HP from the aqueous solution.

[0324] As used herein, the term "evaporation" refers to the conversion of a liquid to the gas phase (vapor). The evaporation of a liquid is called "boiling" when the vapor pressure equals the pressure exerted on the liquid by the surrounding atmosphere. In some embodiments, evaporation occurs when the liquid is heated. Generally, boiling of a liquid occurs when the liquid is heated at or above its boiling point. It should be understood that when a liquid containing more than one solvent is heated, the solvent with the lower boiling point evaporates first, and then the solvent with the higher boiling point evaporates, unless the two solvents form an azeotropic mixture and evaporate simultaneously.

[0325] As used herein, the term "condensation" refers to the process of converting a vapor to a liquid. Generally, condensation of a vapor occurs when the vapor is cooled below the boiling point of the liquid.

[0326] a water-immiscible solvent

[0327] In some embodiments, a water-immiscible solvent (e.g., an organic solvent immiscible with water) can be used in the process of extracting 3-HP from an aqueous solution. That is, the water-immiscible solvent and the solution can be combined to form a solution of 3-HP in the water-immiscible solvent. Then the solution of 3-HP in the water-immiscible solvent can be separated from the aqueous phase.

[0328] As used herein, the term "water-immiscible solvent" refers to a solvent that cannot mix with water to form a homogeneous liquid. For example, a water-immiscible solvent has a solubility in water of less than about 3 wt% (e.g., less than about 2 wt% or less than about 1 wt%). For example, less than about 3 g (e.g., less than about 2 g, or less than about 1 g) of the water-immiscible solvent is soluble in 100 mL of water.

[0329] In some embodiments, the water-immiscible solvent has a density of less than about 1 g / mL. In such embodiments, the water-immiscible solvent has a density less than that of water and, when mixed with water, forms an organic phase above the aqueous phase. In some embodiments, the water-immiscible solvent has a density of from about 0.5 g / mL to about 1 g / mL (e.g., about 0.5 g / mL, about 0.6 g / mL, about 0.75 g / mL, about 0.85 g / mL, about 0.9 g / mL, or about 0.95 g / mL).

[0330] Illustrative examples of water-immiscible solvents having a density lower than water include C 5-10 alkanes, C 5-8 cycloalkanes, aromatic hydrocarbon solvents, C 1-6 alkyl acetates, C 4-6 alcohols, and C 1-6 alkyl ethers. Combinations of such water-immiscible solvents can be used.

[0331] Illustrative C 5-10 alkanes include n-pentane, n-hexane, n-heptane, n-octane, and isooctane.

[0332] Illustrative C 5-8 cycloalkanes include cyclopentane and cyclohexane.

[0333] Exemplary aromatic hydrocarbon solvents include benzene, toluene, o-xylene, m-xylene, p-xylene, and cumene.

[0334] Illustrative C 1-6 alkyl acetates include methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, tert-butyl acetate, n-pentyl acetate, and n-hexyl acetate.

[0335] Illustrative C 4-6The alcohols include n-butanol, isobutanol, tert-butanol, n-pentanol and n-hexanol.

[0336] Illustrative C 1-6 The alkyl ethers include diethyl ether, dipropyl ether, ethyl propyl ether, methyl tert-butyl ether (MTBE) and methyl hexyl ether.

[0337] In some embodiments, the water-immiscible solvent has a density greater than about 1 g / mL. In such embodiments, the water-immiscible solvent has a greater density than water and, when mixed with water, forms an organic phase below the aqueous phase. In some embodiments, the density of the water-immiscible solvent is from about 1 g / mL to about 1.5 g / mL (such as about 1.1 g / mL, about 1.15 g / mL, about 1.2 g / mL, about 1.25 g / mL, about 1.3 g / mL or about 1.4 g / mL).

[0338] In some embodiments, the water-immiscible solvent having a greater density than water is a C 1-4 alkane, a C 1-4 alkene, a C 4-6 cycloalkane, a C 4-6 cycloalkene, an aromatic hydrocarbon solvent, an acetic acid C 1-6 alkyl ester, a C 2-6 alcohol or a C 1-6 alkyl ether. Optionally, such a solvent is substituted with one or more (such as 1, 2, 3, 4 or 5) independently selected halogen atoms, such as one or more Cl atoms, one or more Br atoms, one or more F atoms or a combination thereof. Illustrative embodiments of such a water-immiscible solvent include C 1-4 haloalkanes, C 1-4 haloalkenes, C 2-6 haloalcohols and haloaromatic hydrocarbon solvents. Combinations of the water-immiscible solvents described in this paragraph can be used.

[0339] C 1-6 Illustrative examples of C haloalkanes include chloroform, bromoform, chlorofluorocarbons, dichloromethane, carbon tetrachloride, 1,1-dichloro-1-fluoroethane, 1,1,1-trichloroethane and perfluorodecalin.

[0340] C 1-4 Illustrative examples of C haloalkenes include 1,2-dichloroethylene, 1,1-dichloroethylene and trichloroethylene.

[0341] Illustrative examples of haloaromatic hydrocarbon solvents include chlorobenzene, 1,2-difluorobenzene, 1,2,4-trichlorobenzene and trifluorotoluene.

[0342] C 2-6 Illustrative examples of C haloalcohols include hexafluoro-2-propanol, 2,2,2-trifluoroethanol and trichloro-2-methyl-2-propanol.

[0343] When extracting 3-HP from an aqueous solution using a water-immiscible solvent, conditions can be selected such that the concentration of 3-HP in the water-immiscible solvent can be from about 1 g / L to about 300 g / L (e.g., from about 5 g / L to about 250 g / L, from about 10 g / L to about 200 g / L, from about 20 g / L to about 150 g / L, from about 30 g / L to about 100 g / L, from about 40 g / L to about 90 g / L, or from about 50 g / L to about 80 g / L).

[0344] Generally, the amount of the water-immiscible solvent can be appropriately selected relative to the amount of the aqueous solution. In some embodiments, based on the amount of the aqueous solution, the amount of the water-immiscible solvent is from about 50 v / v% to about 150 v / v% (e.g., from about 80 v / v% to about 120 v / v% or from about 90 v / v% to about 110 v / v%). For example, based on the amount of the aqueous solution, the amount of the water-immiscible solvent can be about 50 v / v%, about 60 v / v%, about 70 v / v%, about 80 v / v%, about 90 v / v%, about 100 v / v%, about 105 v / v%, about 110 v / v%, about 120 v / v%, about 125 v / v%, about 130 v / v%, or about 140 v / v%.

[0345] Water-miscible solvent

[0346] In some embodiments, a water-miscible solvent (e.g., a water-miscible organic solvent) can be used in the process of extracting 3-HP from the aqueous solution. As used herein, the term "water-miscible" refers to a solvent that can be combined with water in any proportion to form a homogeneous liquid.

[0347] Generally, in embodiments where a water-miscible solvent is used in the process of extracting 3-HP from an aqueous solution, the water-miscible solvent is mixed with the aqueous solution to form a homogeneous aqueous solution. For example, the water-miscible solvent can be poured into the aqueous solution to form a uniform liquid. As another example, the water-miscible solvent can be added to the aqueous solution after the following steps to form a homogeneous liquid: (1) evaporating the water-miscible solvent; (2) condensing the evaporated water-miscible solvent; (3) guiding the condensed water-miscible solvent stream into the aqueous solution.

[0348] In embodiments where a water-miscible solvent is used in the process of extracting 3-HP from an aqueous solution, a single water-miscible solvent can be used, or a combination of water-miscible solvents can be used.

[0349] In some embodiments, the water-miscible solvent is C 1-3 alcohol. Illustrative examples of C 1-3 alcohols include methanol, ethanol, n-propanol, and isopropanol.

[0350] In some embodiments, the water-miscible solvent is a polar aprotic solvent. Illustrative examples of polar aprotic solvents include tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPT), dimethylformamide (DMF), acetonitrile, dioxane, and acetone.

[0351] Generally, the amount of the water-miscible solvent can be appropriately selected relative to the amount of the aqueous solution. In some embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent is from about 1 v / v% to about 50 v / v% (e.g., from about 1 v / v% to about 40 v / v%, from about 1 v / v% to about 30 v / v%, from about 5 v / v% to about 50 v / v%, from about 10 v / v% to about 40 v / v%, from about 15 v / v% to about 35 v / v%, or from about 20 v / v% to about 30 v / v%). For example, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be about 5 v / v%, about 10 v / v%, about 15 v / v%, about 20 v / v%, about 25 v / v%, about 30 v / v%, or about 40 v / v%.

[0352] Combination of a water-immiscible solvent and a water-miscible solvent

[0353] In some embodiments, both the water-miscible solvent and the water-immiscible solvent are used in the process of extracting 3-HP from the aqueous solution. In such embodiments, generally, the volume ratio of the water-immiscible solvent to the water-miscible solvent can be appropriately selected. In some embodiments, the volume ratio of the water-immiscible solvent to the water-miscible solvent is from about 10:1 to about 1:1 (e.g., from about 9:1 to about 2:1, from about 8:1 to about 2:1, from about 7:1 to about 2:1, from about 6:1 to about 2:1, or from about 5:1 to about 3:1). For example, the volume ratio of the water-immiscible solvent to the water-miscible solvent is about 2:1, about 3:1, about 4:1, about 4.5:1, about 5:1, about 8:1, or about 10:1.

[0354] Generally, the ratio of 1) the total volume of the water-miscible solvent and the water-immiscible solvent to 2) the volume of the aqueous solution can be appropriately selected. In some embodiments, the ratio of 1) the combined volume of the water-miscible solvent and the water-immiscible solvent to 2) the volume of the aqueous solution can be from about 1:1 to about 2:1 (e.g., from about 1:1 to about 1.5:1), such as about 1:1, about 1.2:1, about 1.3:1, about 1.4:1, or about 1.5:1.

[0355] Generally, the relative boiling points of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. In some embodiments, the boiling point of the water-miscible solvent is lower than the boiling point of the water-immiscible solvent. In some embodiments, the boiling point of the water-miscible solvent is higher than the boiling point of the water-immiscible solvent.

[0356] Generally, the water-immiscible solvent, the water-miscible solvent, and their respective amounts can be appropriately selected.

[0357] In some embodiments, the water-miscible solvent is selected from methanol, ethanol, acetone, acetonitrile, THF, dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPT), and dimethylformamide (DMF), and the water-immiscible solvent is selected from methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, tert-butyl acetate, benzene, chlorobenzene, toluene, o-xylene, m-xylene, and p-xylene, n-butanol, isobutanol, tert-butanol, diethyl ether, methyl tert-butyl ether (MTBE), methyl hexyl ether, chloroform, dichloromethane, and carbon tetrachloride. In such embodiments, the amounts of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. For example, in such embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be from about 5 v / v% to about 50 v / v%, the volume ratio of the water-miscible solvent to the water-immiscible solvent can be from about 1:10 to about 1:2, and / or the ratio of 1) the total volume of the water-miscible solvent and the water-immiscible solvent to 2) the volume of the aqueous solution can be from about 1:1 to about 2:1.

[0358] In some embodiments, the water-miscible solvent is a C1-3 alcohol (such as methanol, ethanol, or isopropanol), and the water-immiscible solvent is an aromatic solvent (such as benzene, toluene, o-xylene, m-xylene, p-xylene, or chlorobenzene). In such embodiments, the amounts of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. For example, in such embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be from about 5 v / v% to about 50 v / v%, the volume ratio of the water-miscible solvent to the water-immiscible solvent can be from about 1:10 to about 1:2, and / or the ratio of 1) the total volume of the water-miscible solvent and the water-immiscible solvent to 2) the volume of the aqueous solution can be from about 1:1 to about 2:1.

[0359] In some embodiments, the water-miscible solvent is C 1-3 alcohol (such as methanol, ethanol, or isopropanol), and the water-immiscible solvent is C 1-6Alkyl esters (e.g., methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate or tert-butyl acetate). In such embodiments, the amounts of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. For example, in such embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be from about 5 v / v% to about 50 v / v%, the volume ratio of the water-miscible solvent to the water-immiscible solvent can be from about 1:10 to about 1:2, and / or 1) the total volume of the water-miscible solvent and the water-immiscible solvent and 2) the volume of the aqueous solution can be in a ratio from about 1:1 to about 2:1.

[0360] In some embodiments, the water-miscible solvent is a C 1-3 alcohol (e.g., methanol, ethanol or isopropanol), and the water-immiscible solvent is a C 1-6 alkyl ether (e.g., diethyl ether, methyl tert-butyl ether (MTBE) or methyl hexyl ether). In such embodiments, the amounts of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. For example, in such embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be from about 5 v / v% to about 50 v / v%, the volume ratio of the water-miscible solvent to the water-immiscible solvent can be from about 1:10 to about 1:2, and / or 1) the total volume of the water-miscible solvent and the water-immiscible solvent and 2) the volume of the aqueous solution can be in a ratio from about 1:1 to about 2:1.

[0361] In some embodiments, the water-miscible solvent is a C 1-3 alcohol (e.g., methanol, ethanol or isopropanol), and the water-immiscible solvent is a C 1-6 haloalkane (e.g., chloroform, dichloromethane or carbon tetrachloride). In such embodiments, the amounts of the water-miscible solvent and the water-immiscible solvent can be appropriately selected. For example, in such embodiments, based on the amount of the aqueous solution, the amount of the water-miscible solvent can be from about 5 v / v% to about 50 v / v%, the volume ratio of the water-miscible solvent to the water-immiscible solvent can be from about 1:10 to about 1:2, and / or 1) the total volume of the water-miscible solvent and the water-immiscible solvent and 2) the volume of the aqueous solution can be in a ratio from about 1:1 to about 2:1.

[0362] In some embodiments, the water-miscible solvent is methanol, and the water-immiscible solvent is butyl acetate. In such embodiments, the amounts of methanol and butyl acetate can be appropriately selected. For example, in such embodiments, the amount of methanol is about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to butyl acetate can be about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and butyl acetate to 2) the volume of the aqueous solution can be about 1:1 to about 2:1.

[0363] In some embodiments, the water-miscible solvent is methanol, and the water-immiscible solvent is MTBE. In such embodiments, the amounts of methanol and MTBE can be appropriately selected. For example, in such embodiments, the amount of methanol can be about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to MTBE can be about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and MTBE to 2) the volume of the aqueous solution can be about 1:1 to about 2:1.

[0364] In some embodiments, the water-miscible solvent is methanol, and the water-immiscible solvent can be isobutanol. In such embodiments, the amounts of methanol and isobutanol can be appropriately selected. For example, in such embodiments, the amount of methanol can be about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to isobutanol can be about 1:10 to about 1:2, and / or the ratio of 1) the combined volume of methanol and isobutanol to 2) the volume of the aqueous solution can be about 1:1 to about 2:1.

[0365] In some embodiments, the water-miscible solvent is methanol, and the water-immiscible solvent is tert-butanol. In such embodiments, the amounts of methanol and tert-butanol can be appropriately selected. For example, in such embodiments, the amount of methanol can be about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to tert-butanol can be about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and tert-butanol to 2) the volume of the aqueous solution can be about 1:1 to about 2:1.

[0366] In some embodiments, the water-miscible solvent is methanol, and the water-immiscible solvent is benzene. In such embodiments, the amounts of methanol and benzene can be appropriately selected. For example, in such embodiments, the amount of methanol can be about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to benzene can be about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and benzene to 2) the volume of the aqueous solution is about 1:1 to about 2:1.

[0367] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is toluene. In such embodiments, the amounts of methanol and toluene can be appropriately selected. For example, in such embodiments, the amount of methanol can be from about 5 v / v% to about 50 v / v% of the aqueous solution, the volume ratio of methanol to toluene can be from about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and toluene to 2) the volume of the aqueous solution is from about 1:1 to about 2:1.

[0368] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is chloroform. In such embodiments, the amounts of methanol and chloroform can be appropriately selected. For example, in such embodiments, the amount of methanol can be from about 5 v / v% to about 50 v / v% of the aqueous solution, and the volume ratio of methanol to chloroform can be from about 1:10 to about 1:2, and / or the ratio of 1) the total volume of methanol and chloroform to 2) the volume of the aqueous solution is from about 1:1 to about 2:1.

[0369] In certain exemplary embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is ethyl acetate. In such embodiments, the amounts of methanol and ethyl acetate can be appropriately selected. For example, in such embodiments, the amount of methanol can be from about 15 v / v% to about 35 v / v% of the aqueous solution, and the volume ratio of methanol to ethyl acetate is from about 1:9 to about 3:7, and / or the ratio of 1) the combined volume of methanol and ethyl acetate to 2) the volume of the aqueous solution can be from about 1:1 to about 2:1. Also, in such embodiments, the concentration of 3-HP in the aqueous solution can be from about 50 g / L to about 80 g / L, the pH of the aqueous solution can be from about 4 to about 5, and the temperature of the aqueous solution during the extraction of 3-HP from the aqueous solution is from about 15 °C to about 40 °C.

[0370] In some embodiments, a method for extracting 3-HP from an aqueous solution includes: (1) providing an extraction container containing a cell-free fermentation broth containing 3-HP, a solvent container containing ethyl acetate at about 90 v / v% to about 110 v / v% (based on the amount of the fermentation broth in the extraction container), and a condenser; (2) evaporating ethyl acetate from the solvent container; (3) condensing the vaporized ethyl acetate into a liquid state in the condenser; (4) guiding the ethyl acetate stream from the condenser to the extraction container, thereby forming an ethyl acetate solution of 3-HP in the extraction container; (5) separating the fermentation broth and the solution of 3-HP in ethyl acetate in the extraction container; (6) guiding the solution stream of 3-HP in ethyl acetate from the extraction container to the solvent container. In some embodiments, the extraction container further contains methanol at about 20 v / v% to about 30 v / v% based on the amount of the fermentation broth. In such embodiments, methanol is completely miscible with the fermentation broth and remains in the solvent container during the extraction process. The ratio of the amount of ethyl acetate to the amount of methanol in this system is about 5:1 to about 3:1.

[0371] Process parameters

[0372] Generally, any suitable flow rate can be used for the solvent (e.g., an organic solvent) during the process of removing 3-HP from an aqueous solution. For example, in some embodiments, the flow rate of the solvent during the process of removing 3-HP from an aqueous solution is about 0.1 L / h to about 10 L / h (e.g., about 0.5 L / h to about 8 L / h, about 1 L / h to about 5 L / h, or about 1 L / h to about 3 L / h).

[0373] Generally, any suitable temperature can be used during the process of removing 3-HP from an aqueous solution. For example, in some embodiments, the process of removing 3-HP from an aqueous solution is carried out at a temperature of the aqueous solution of about 15 °C to about 50 °C (e.g., about 15 °C to about 40 °C, about 20 °C to about 30 °C). In some embodiments, the process of removing 3-HP from an aqueous solution is carried out at a temperature of the aqueous solution of at most about 50 °C (e.g., about 40 °C or about 30 °C). Optionally, 3-HP is removed from the aqueous solution at room temperature.

[0374] In some embodiments, as shown in Scheme 1, heating 3-HP in a polar protic solvent such as water or a lower alcohol causes 3-HP to decompose.

[0375] Scheme 1

[0376]

[0377] Referring to Scenario 1, the hydrogen atom of the hydroxyl group at the 3-position can form a hydrogen bond with the carbonyl group of 3-HP, thereby forming a six-membered ring. A polar protonic solvent can promote the formation of the six-membered ring and the decomposition of 3-HP to form ethylene, carbon dioxide, and water. In one example, when a solution of 3-HP in a polar protonic solvent (such as water) is heated at a temperature above 50 °C, decomposition may occur. Thus, without wishing to be bound by theory, in some embodiments, maintaining the temperature at 50 °C or below when retrieving 3-HP from an aqueous solution can reduce the formation of unwanted ethylene and / or carbon dioxide.

[0378] In certain embodiments, the temperature of a solution of 3-HP in a water-immiscible solvent is equal to or close to the boiling point of the water-immiscible solvent. For example, when ethyl acetate is used in the method, the temperature of a solution of 3-HP in ethyl acetate can be from about 78 °C to about 80 °C. As another example, when isobutanol is used in the method, the temperature of a solution of 3-HP in isobutanol can be from about 105 °C to about 110 °C. Advantageously, the temperature is selected such that 3-HP hardly decomposes or does not decompose into unwanted products when the solution of it in the water-immiscible solvent is heated during retrieval from the aqueous solution.

[0379] In some embodiments, the process of retrieving 3-HP from an aqueous solution results in a relatively high yield of the recovered 3-HP, such as at least about 70% (such as at least about 80%, at least about 90%, at least about 95%, or at least about 99%).

[0380] Exemplary Systems and Methods for Retrieving 3-HP from an Aqueous Solution

[0381] Figure 58 An exemplary system for retrieving 3-HP from an aqueous solution using a water-immiscible solvent having a density less than that of water is shown. Referring to Figure 58 , the system includes a solvent container 100 and a condenser 110 fluidly connected to the solvent container 100 via a side tube 108. The condenser 110 is also fluidly connected to an extraction container 116 via a siphon tube 112. The solvent container 100 includes a stirrer 102, a thermometer 124, and a heating element 104. The extraction container 116 includes a stirrer 120, a pH meter 122, and an inlet tube 114. The inlet tube 114 is connected to a peristaltic pump 126 configured to add acid from an acid storage container 130 to the extraction container 116 through the inlet tube 114.

[0382] In some embodiments, both the water-miscible solvent and the water-immiscible solvent are placed in solvent container 100. Typically, an aqueous solution of 3-HP (e.g., a cell-free fermentation broth containing 3-HP) is placed in extraction container 116 after the water-miscible solvent and the water-immiscible solvent are placed in solvent container 100, although optionally the aqueous solution of 3-HP can be placed in extraction container 116 before and / or simultaneously with the placement of the water-miscible solvent in solvent container 100 and / or the placement of the water-immiscible solvent in solvent container 100. The amount of the aqueous solution of 3-HP initially placed in extraction container 116 is selected such that it does not exceed side arm 108, so that the aqueous solution of 3-HP does not transfer into solvent container 100. Solvent container 100 is heated using heating element 104 until the temperature of the liquid contained in solvent container 100 reaches the boiling point of the solvent with the lower boiling point contained in solvent container 100 (the water-immiscible solvent or the water-miscible solvent), as determined, for example, by thermometer 124. Typically, the boiling point of the water-miscible solvent is lower than that of the water-immiscible solvent. The water-miscible solvent in solvent container 100 evaporates (the water-immiscible solvent in solvent container 100 does not evaporate), and the water-miscible solvent vapor flows from solvent container 100 through side tube 108 to condenser 110. Condenser 110 uses a coolant at a temperature lower than the boiling point of the water-miscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Thus, the vaporized water-miscible solvent condenses into a liquid state in condenser 110, and the liquid water-miscible solvent (e.g., at about room temperature) flows via siphon 112 to the bottom of extraction container 116. The condensed water-miscible solvent combines with the aqueous solution of 3-HP to form an aqueous phase in extraction container 116. The amount of the water-miscible solvent initially placed in solvent container 100 and then transferred to extraction container 116 is selected such that the aqueous phase in container 116 does not exceed side arm 108, so that the aqueous phase does not transfer into solvent container 100. Solvent container 100 is then heated to a higher temperature using heating element 104 until the temperature of the liquid contained in solvent container 100 reaches the boiling point of the water-immiscible solvent, causing the water-immiscible solvent in solvent container 100 to evaporate. The vapor of the water-immiscible solvent flows from solvent container 100 through side tube 108 to condenser 110. Condenser 110 uses a coolant at a temperature lower than the boiling point of the water-immiscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Thus, the vaporized water-immiscible solvent condenses into a liquid state in condenser 110, and the liquid water-immiscible solvent (e.g., at about room temperature) flows via siphon 112 to the bottom of extraction container 116.The condensed water-immiscible solvent is mixed with the aqueous phase in the extraction vessel 116 such that the water-immiscible solvent extracts 3-HP from the aqueous phase, thereby forming a solution of 3-HP (organic phase) in the water-immiscible solvent. As a result, the extraction vessel 116 contains an aqueous phase and an organic phase. Since the density of the water-immiscible solvent is less than that of water, the organic phase is above the aqueous phase. The total amount of the aqueous 3-HP solution, the water-miscible solvent, and the water-immiscible solvent initially placed in the system is selected such that the organic phase in the extraction vessel 116 reaches the side arm 108. Thus, the organic phase flows from the extraction vessel 116 through the side arm 108 into the solvent vessel 100. In this way, 3-HP is transferred from the extraction vessel 116 to the solvent vessel 100. As the amount of 3-HP in the extraction vessel 116 decreases, the pH of the aqueous phase in the extraction vessel 116 tends to increase, as detected, for example, by the pH meter 122. In response to this increase in pH, the peristaltic pump 126 can pump acid from the acid storage vessel 130 via the inlet tube 114 into the extraction vessel 116, thereby adjusting the pH of the aqueous phase in the extraction vessel 116. The aqueous phase in the extraction vessel 116 can be stirred (e.g., continuously) by the stirrer 120 to enhance the pH homogeneity of the aqueous phase. The temperature of the aqueous phase in the extraction vessel 116 and the organic phase in the extraction vessel 116 can be substantially the same and is typically about 15 °C to about 40 °C (e.g., about room temperature). Generally, the pH of the aqueous phase in the extraction vessel 116 is adjusted to within the above range. For example, in some embodiments, the pH of the aqueous phase is about 4 to about 7 (e.g., about 4 to about 5, about 4.2 to about 4.7, about 4.3 to about 4.4, about 4.3 or about 4.4).

[0383] In certain embodiments, the water-miscible solvent is initially placed in the extraction vessel 116, and the water-immiscible solvent is initially placed in the solvent vessel 100. Generally, after placing the water-miscible solvent in the extraction vessel 116 and the water-immiscible solvent in the solvent vessel 100, an aqueous solution of 3-HP (e.g., a cell-free fermentation broth containing 3-HP) is placed in the extraction vessel 116, although optionally the aqueous solution of 3-HP can be placed in the extraction vessel 116 before and / or simultaneously with placing the water-miscible solvent in the extraction vessel 116 and / or placing the water-immiscible solvent in the solvent vessel 100. The aqueous solution of 3-HP and the water-miscible solvent in the extraction vessel 116 combine to form an aqueous phase in the extraction vessel 116. The total amounts of the aqueous solution of 3-HP and the water-miscible solvent are selected such that the aqueous phase does not extend upward into the side arm 108, so that the aqueous phase does not transfer into the solvent vessel 100. The solvent vessel 100 is heated using the heating element 104 until the temperature of the water-immiscible solvent contained in the solvent vessel 100 reaches its boiling point, as measured by the thermometer 124, for example. The water-immiscible solvent in the solvent vessel 100 evaporates, and the vapor of the water-immiscible solvent flows from the solvent vessel 100 through the side tube 108 to the condenser 110. The condenser 110 uses a coolant at a temperature below the boiling point of the water-immiscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Thus, the vaporized water-immiscible solvent condenses into a liquid state in the condenser 110, and this liquid water-immiscible solvent (e.g., at about room temperature) flows via the siphon 112 to the bottom of the extraction vessel 116. The condensed water-immiscible solvent mixes with the aqueous phase in the extraction vessel 116 such that the water-immiscible solvent extracts 3-HP from the aqueous phase, thereby forming a solution of 3-HP (organic phase) in the water-immiscible solvent. As a result, the extraction vessel 116 contains an aqueous phase and an organic phase. Since the density of the water-immiscible solvent is less than that of water, the organic phase is above the aqueous phase. The total amounts of the aqueous solution of 3-HP, the water-miscible solvent, and the water-immiscible solvent initially placed in the system are selected such that the organic phase in the extraction vessel 116 reaches the side arm 108. Thus, the organic phase flows from the extraction vessel 116 through the side arm 108 into the solvent vessel 100. In this way, 3-HP is transferred from the extraction vessel 116 to the solvent vessel 100. As the amount of 3-HP in the extraction vessel 116 decreases, the pH of the aqueous phase in the extraction vessel 116 tends to increase, as detected by the pH meter 122, for example. In response to this increase in pH, the peristaltic pump 126 can pump acid from the acid storage container 130 via the inlet tube 114 into the extraction vessel 116, thereby adjusting the pH of the aqueous phase in the extraction vessel 116. The aqueous phase in the extraction vessel 116 can be stirred (e.g., continuously) using the stirrer 120 to enhance the pH homogeneity of the aqueous phase.The temperature of the aqueous phase in the extraction vessel 116 and the organic phase in the extraction vessel 116 can be substantially the same and is typically from about 15 °C to about 40 °C (e.g., about room temperature). Generally, the pH of the aqueous phase in the extraction vessel 116 is adjusted to be within the above range. For example, in some embodiments, the pH of the aqueous phase is from about 4 to about 7 (e.g., from about 4 to about 5, from about 4.2 to about 4.7, from about 4.3 to about 4.4, about 4.3 or about 4.4).

[0384] Figure 59 An exemplary system for removing 3-HP from an aqueous solution using a water-immiscible solvent having a higher density than water is shown. The system includes a solvent vessel 300, a condenser 310, and an extraction vessel 316. The solvent vessel 300 is fluidly connected to the condenser 310 via a side tube 308. The solvent vessel 300 is also fluidly connected to the extraction vessel 316 via a side tube 312. The condenser 310 is fluidly connected to the extraction vessel 316 via a connecting tube 334. The solvent vessel 300 includes a stirrer 302, a heating element 304, and a thermometer 332. The extraction vessel 316 includes a pH meter 320 and an inlet tube 324. The inlet tube 324 is connected to a peristaltic pump 326 configured to add acid from an acid storage vessel 330 to the extraction vessel 316 via the inlet tube 324.

[0385] In some embodiments, both the water-miscible solvent and the water-immiscible solvent are placed in extraction vessel 300. Typically, an aqueous solution of 3-HP (e.g., a cell-free fermentation broth containing 3-HP) is placed in extraction vessel 316 before the water-miscible solvent and the water-immiscible solvent are placed in solvent vessel 300. However, optionally, the aqueous solution of 3-HP can be placed in extraction vessel 316 after and / or simultaneously with placing the water-miscible solvent in solvent vessel 300 and / or placing the water-immiscible solvent in solvent vessel 300. The volume of the aqueous 3-HP solution placed in extraction vessel 316 is chosen such that the solution does not extend high enough to flow into solution vessel 300 through side tube 312. Solvent vessel 300 is heated using heating element 304 until the temperature of the liquid contained in solvent vessel 300 reaches the boiling point of the solvent with the lower boiling point contained in solvent vessel 300 (the water-immiscible solvent or the water-immiscible solvent), as determined, for example, by thermometer 332. Typically, the boiling point of the water-miscible solvent is lower than that of the water-immiscible solvent. The water-miscible solvent in solvent vessel 300 evaporates (the water-immiscible solvent in solvent vessel 300 does not evaporate), and the vapor of the water-miscible solvent flows from solvent vessel 300 through side tube 308 to condenser 310. Condenser 310 uses a coolant at a temperature below the boiling point of the water-miscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Thus, the vaporized water-miscible solvent condenses to a liquid state in condenser 310, and the liquid water-miscible solvent (e.g., at about room temperature) flows via connecting tube 334 to extraction vessel 316. The condensed water-miscible solvent combines with the aqueous 3-HP solution to form an aqueous phase in extraction vessel 316. The amount of the water-miscible solvent initially placed in solvent vessel 300 and subsequently transferred to extraction vessel 316 is chosen such that the aqueous phase in vessel 316 does not extend high enough to flow into solution vessel 300 through side tube 312. Solvent vessel 300 is then heated to a higher temperature using heating element 304 until the temperature of the liquid contained in solvent vessel 300 reaches the boiling point of the water-immiscible solvent, causing the water-immiscible solvent in solvent vessel 300 to evaporate. The vapor of the water-immiscible solvent flows from solvent vessel 300 through side tube 308 to condenser 310. Condenser 310 uses a coolant at a temperature below the boiling point of the water-immiscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Thus, the vaporized water-immiscible solvent condenses to a liquid state in condenser 310, and the liquid water-immiscible solvent (e.g., at about room temperature) flows via connecting tube 334 to extraction vessel 316.The condensed water-immiscible solvent is mixed with the aqueous phase in the extraction vessel 316 such that the water-immiscible solvent extracts 3-HP from the aqueous phase, thereby forming a solution of 3-HP (organic phase) in the water-immiscible solvent. As a result, the extraction vessel 316 contains an aqueous phase and an organic phase. Since the water-immiscible solvent is denser than water, the organic phase is below the aqueous phase. The total amounts of the 3-HP aqueous solution, the water-miscible solvent, and the water-immiscible solvent initially placed in the system are selected such that the organic phase in the extraction vessel 316 is large enough so that the organic phase can be transferred from the extraction vessel 316 to the solvent vessel 300 through the side tube 312. In this way, 3-HP is transferred from the extraction vessel 316 to the solvent vessel 300. As the amount of 3-HP in the extraction vessel 316 decreases, the pH of the aqueous phase in the extraction vessel 316 tends to increase, as detected, for example, by the pH meter 320. In response to this increase in pH, the peristaltic pump 326 can pump acid from the acid storage vessel 330 to the extraction vessel 316 via the inlet tube 324, thereby adjusting the pH of the aqueous phase in the extraction vessel 316. The temperature of the aqueous phase in the extraction vessel 316 and the temperature of the organic phase in the extraction vessel 316 can be substantially the same and are typically about 15 °C to about 40 °C (e.g., about room temperature). Generally, the pH of the aqueous phase in the extraction vessel 316 is adjusted to be within the above range. For example, in some embodiments, the pH of the aqueous phase is about 4 to about 7 (e.g., about 4 to about 5, about 4.2 to about 4.7, about 4.3 to about 4.4, about 4.3 or about 4.4).

[0386] In certain embodiments, a water-miscible solvent is placed in extraction vessel 316 and a water-immiscible solvent is placed in extraction vessel 300. Typically, prior thereto, an aqueous solution of 3-HP (e.g., a cell-free fermentation broth containing 3-HP) is placed in extraction vessel 316. Optionally, however, the aqueous solution of 3-HP may be placed in extraction vessel 316 after and / or simultaneously with placing the water-miscible solvent in extraction vessel 316 and / or placing the water-immiscible solvent in solvent vessel 300. The aqueous solution of 3-HP and the water-miscible solvent in extraction vessel 316 are combined in extraction vessel 316 to form an aqueous phase. The combined amount of the aqueous solution of 3-HP and the water-miscible solvent in extraction vessel 316 is selected such that the aqueous phase in extraction vessel 316 does not extend high enough to flow into solution vessel 300 via side tube 312. Solvent vessel 300 is heated using heating element 304 until the temperature of the liquid contained in solvent vessel 300 reaches the boiling point of the water-immiscible solvent, thereby causing the water-immiscible solvent in solvent vessel 300 to evaporate. The vapor of the water-immiscible solvent flows from solvent vessel 300 through side tube 308 to condenser 310. Condenser 310 uses a coolant having a temperature below the boiling point of the water-immiscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Accordingly, the vaporized water-immiscible solvent condenses into a liquid state in condenser 310, and the liquid water-immiscible solvent (e.g., at about room temperature) flows via connecting tube 334 to extraction vessel 316. The condensed water-immiscible solvent is mixed with the aqueous phase in extraction vessel 316, whereby the water-immiscible solvent extracts 3-HP from the aqueous phase, thereby forming a solution of 3-HP in the water-immiscible solvent (organic phase). As a result, extraction vessel 316 contains an aqueous phase and an organic phase. Since the water-immiscible solvent has a higher density than water, the organic phase is beneath the aqueous phase. The total amounts of the aqueous solution of 3-HP, the water-miscible solvent, and the water-immiscible solvent initially placed in the system are selected such that the organic phase in extraction vessel 316 is large enough so that the organic phase can be transferred from extraction vessel 316 through side tube 312 to solvent vessel 300. In this manner, 3-HP is transferred from extraction vessel 316 to solvent vessel 300. As the amount of 3-HP in extraction vessel 316 decreases, the pH of the aqueous phase in extraction vessel 316 tends to increase, as detected, for example, by pH meter 320. In response to this increase in pH, peristaltic pump 326 can pump acid from acid storage vessel 330 via inlet tube 324 to extraction vessel 316, thereby adjusting the pH of the aqueous phase in extraction vessel 316. The temperature of the aqueous phase in extraction vessel 316 and the temperature of the organic phase in extraction vessel 316 may be substantially the same and are typically about 15°C to about 40°C (e.g., about room temperature).Typically, the pH of the aqueous phase in extraction vessel 316 is adjusted to within the above range. For example, in some embodiments, the pH of the aqueous phase is from about 4 to about 7 (e.g., from about 4 to about 5, from about 4.2 to about 4.7, from about 4.3 to about 4.4, about 4.3 or about 4.4).

[0387] Purification of 3-HP

[0388] In some embodiments, 3-HP can be purified after removal from the aqueous solution. For example, crude 3-HP (3-HP after removal from the aqueous solution but before purification) can be converted to a salt, such as an alkali metal salt, and the salt can then be washed with an organic solvent to provide the pure alkali metal salt of 3-HP. The pure alkali metal salt of 3-HP can be converted to the pure 3-HP free acid. In such embodiments, the alkali metal salt of 3-HP can be formed by treating crude 3-HP with an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide. To conduct such a reaction, a solution of crude 3-HP in an organic solvent can be formed, and the resulting solution can be treated with an alkali metal hydroxide. In some embodiments, the organic solvent is at least one of acetone, methanol, or isopropanol. In some embodiments, the pH of the solution of crude 3-HP in the organic solvent is from about 4 to about 5 (e.g., about 4.3 or about 4.4). In some embodiments, the alkali metal hydroxide is added to the reaction mixture until the pH of the reaction mixture is about 7. The alkali metal salt of 3-HP precipitated from the reaction mixture can be collected by filtration and further washed with the organic solvent as described above.

[0389] To obtain the pure 3-HP free acid, the salt of 3-HP, such as the sodium salt, can be dissolved in water, and the resulting aqueous solution can be treated with any one of the acids described herein. For example, the aqueous solution of the 3-HP salt can be treated with hydrochloric acid or oxalic acid until the pH is from about 4 to about 5, and the pure 3-HP free acid can be removed from the resulting aqueous solution by any one of the methods described herein.

[0390] Production of acrylic acid

[0391] In some embodiments, the method for preparing acrylic acid from 3-HP includes reacting 3-HP to form acrylic acid. The method can provide a yield of acrylic acid of at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%). In certain embodiments, the method provides a quantitative yield of acrylic acid (i.e., 100% yield).

[0392] In some embodiments, the method includes reacting liquid 3-HP. In such embodiments, the 3-HP can be pure or can be in solution in water or in one or more organic solvents. Exemplary organic solvents include DMSO, DMF, and water.

[0393] Generally, the method for preparing acrylic acid can be carried out at any suitable temperature. In some embodiments, the method for preparing acrylic acid includes heating 3-HP at a temperature of at least about 50 °C (e.g., about 60 °C, about 70 °C, or about 80 °C). In some embodiments, the reaction of 3-HP occurs at a temperature of at most about 200 °C (e.g., about 190 °C or about 180 °C). In some embodiments, reacting 3-HP to produce acrylic acid is carried out at a temperature of about 50 °C to about 200 °C (e.g., about 60 °C to about 190 °C, or about 80 °C to about 180 °C).

[0394] Generally, 3-HP can be reacted at any suitable pressure to form acrylic acid. In some embodiments, when the pressure adjacent to the reaction mixture containing 3-HP is below atmospheric pressure, acrylic acid evaporates from the reaction mixture at the reaction temperature. That is, acrylic acid can be removed from the reaction mixture in gaseous form. In some embodiments, reacting 3-HP to form acrylic acid occurs under reduced pressure, for example, at a pressure less than one atmosphere adjacent to the reaction mixture containing 3-HP. In certain embodiments, the pressure adjacent to the reaction mixture containing 3-HP is less than about 700 mbar (e.g., less than about 500 mbar, less than about 400 mbar, less than about 300 mbar, less than about 200 mbar, less than about 150 mbar, less than about 120 mbar, or less than about 100 mbar). In some embodiments, the pressure adjacent to the reaction mixture containing 3-HP is from about 50 mbar to about 200 mbar (e.g., about 60 mbar to about 150 mbar, about 70 mbar to about 130 mbar, or about 70 mbar to about 100 mbar). In some embodiments, the pressure adjacent to the reaction mixture containing 3-HP is about 70 mbar, about 74 mbar, about 75 mbar, about 80 mbar, about 90 mbar, about 100 mbar, or about 120 mbar.

[0395] In some embodiments, the reaction mixture contains a catalyst to catalyze the conversion of 3-HP to acrylic acid. Generally, an acid catalyst can be used to catalyze the conversion of 3-HP to acrylic acid.

[0396] Scheme 2

[0397]

[0398] Referring to Scheme 2, when 3-HP contacts an acid, the hydroxyl group at position 3 is protonated, and then an elimination reaction occurs to produce acrylic acid and water. Suitable examples of the reaction catalyst include any of the organic acids and inorganic acids described herein. For example, hydrochloric acid, sulfuric acid, polyphosphoric acid, oxalic acid, or acetic acid can be used to catalyze the reaction of 3-HP to form acrylic acid. In some embodiments, zeolite, silica, or sea sand can be used as the catalyst for the reaction. Suitable examples of zeolite include molecular sieves, such as 3A molecular sieve, 4A molecular sieve, or 5A molecular sieve.

[0399] Generally, the amount of the catalyst can be appropriately selected. In some embodiments, based on the amount of 3-HP in the reaction mixture, the reaction mixture can contain about 1 wt% to about 25 wt% (e.g., about 1 wt% to about 20 wt%, about 2 wt% to about 20 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 5 wt%, or about 2 wt% to about 4 wt%) of the catalyst. In some embodiments, based on the amount of 3-HP in the reaction mixture, the amount of the catalyst in the reaction mixture is about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 10 wt%, about 20 wt%, or about 25 wt%.

[0400] The acrylic acid formed from 3-HP in the reaction mixture is prone to polymerization, thus forming polyacrylic acid. The method for preparing acrylic acid described herein advantageously avoids the formation of polyacrylic acid and provides the desired product in a high yield, such as a yield of at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%).

[0401] To reduce unwanted polymerization, in some embodiments, 3-HP is reacted to form acrylic acid in the presence of a polymerization inhibitor. Illustrative examples of the polymerization inhibitor include phenothiazine, hydroquinone, 4-tert-butylcatechol, tert-butylhydroquinone, 1,4-benzoquinone, 6-tert-butyl-2,4-xylenol, 2,6-di-tert-butyl-p-cresol, 2,6-di-tert-butylphenol, or 4-methoxyphenol.

[0402] Generally, the amount of the polymerization inhibitor can be appropriately selected. In some embodiments, based on the amount of 3-HP in the reaction mixture, the polymerization inhibitor is present in the reaction mixture in an amount of about 1 wt% to about 20 wt% (e.g., about 1 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 5 wt% to about 15 wt%). In some embodiments, based on the amount of 3-HP in the reaction mixture, the amount of the polymerization inhibitor is about 1 wt%, about 2 wt%, about 3 wt%, about 5 wt%, about 10 wt%, or about 15 wt%.

[0403] In some embodiments, acrylic acid formed from 3-HP is removed from the reaction mixture. The removal of acrylic acid can be carried out continuously throughout the reaction, optionally until all of the 3-HP has reacted to form acrylic acid, or the removal of acrylic acid can be carried out discontinuously during the reaction, optionally until all of the 3-HP has reacted to produce acrylic acid.

[0404] As described above, acrylic acid can be removed from the reaction mixture as gaseous acrylic acid. In such embodiments, the gaseous acrylic acid that has been removed from the reaction mixture can be condensed into a liquid, collected, and used for the intended purpose. In some examples, a coolant is used to condense the gaseous acrylic acid. For example, the temperature of the coolant used to condense acrylic acid at a pressure below atmospheric pressure is from about -5 °C to about 10 °C (e.g., from about 0 °C to about 5 °C).

[0405] Exemplary systems and methods for preparing acrylic acid

[0406] Figure 60 An exemplary system for reacting 3-HP to form acrylic acid and distilling acrylic acid from the reaction mixture is shown. The system includes a reaction vessel 200, a distillation head 214 connected to the reaction vessel 200 via a solvent line 212, a solvent collector 224, a condenser 226 above the distillation head 214, and a collection vessel 238 connected to the distillation head 214 via a valve 236 for collecting the distilled acrylic acid. The reaction vessel 200 includes a stirrer 206, a heating element 202, a thermometer 208 for controlling the temperature inside the reaction vessel 200, and an inlet tube 210 through which a reaction mixture containing 3-HP (and optionally a solvent and a catalyst) can be introduced into the reaction vessel 200. The distillation head 214 includes a cooling jacket 218 having a coolant inlet tube 220 and a coolant outlet tube 222. The collection vessel 238 includes an inlet tube 242 through which other components (such as a polymerization inhibitor) can be introduced into the collection vessel 238. The collection vessel 238 also includes an outlet tube 244 for removing acrylic acid from the system. The inlet tube 242 can also be used to connect the collection vessel 238 to an additional vacuum line. The condenser 226 includes a cooling jacket 228 having a coolant inlet tube 230 and a coolant outlet tube 232. The condenser 226 also includes a fitting 234 through which the system can be connected to a main vacuum line.

[0407] Before the extraction process, 3-HP and optionally a catalyst, such as 4A molecular sieve, are charged to the reaction vessel 200. Then a vacuum is applied to the system to create a pressure of about 70 - 100 mbar inside the system. Then the reaction mixture is heated to a temperature of about 80 °C as determined by the thermometer 208 using the heating mantle 202. At this temperature, 3-HP starts to react to form the reaction products acrylic acid and water. At a pressure of about 70 - 100 mbar, the acrylic acid and water formed during the reaction evaporate, and the vapor stream flows through the solvent line 212 to the distillation head 214. Some of the vapor containing acrylic acid and water condenses in the distillation head 214, thus forming a liquid product contained in the distillation head 214. The remaining vapor reaches the condenser 226 through the valve 224, where the remaining gaseous acrylic acid and water condense and flow downward through the valve 224 to the distillation head 214. If a sufficient amount of liquid is collected in the distillation head 214, the liquid flows through the open valve 236 to the collection container 238. The valve 236 can be closed to prevent the liquid contained in the distillation head 214 from undesirably flowing to the collection container 238.

[0408] In some embodiments, the present disclosure provides a method for reacting 3-HP to form acrylic acid, comprising: (1) providing a reaction vessel, a collection container, and a condenser containing 3-HP; (2) reacting 3-HP in the reaction vessel to produce acrylic acid; (3) evaporating acrylic acid from the reaction vessel; (3) condensing the vaporized acrylic acid into a liquid state in the condenser; and (4) directing an acrylic acid stream from the condenser to the collection container. In some embodiments, the reaction vessel further contains a catalyst. In some embodiments, the reaction vessel further contains a polymerization inhibitor. Exemplary embodiments of the catalyst, the polymerization inhibitor, the amounts of the catalyst and the polymerization inhibitor, and the reaction conditions are described in the "Making Acrylic Acid" section of this application.

[0409] Purification of Acrylic Acid

[0410] In some embodiments, acrylic acid prepared by any of the methods described in the "Making Acrylic Acid" section of the present disclosure can be further purified to obtain acrylic acid free of any polymeric products and unreacted 3-HP. Conventional methods of purifying acrylic acid include distilling acrylic acid at atmospheric pressure (the boiling point of acrylic acid is about 141 °C at about 760 mmHg) in the presence of a polymerization inhibitor such as 4-methoxyphenol (MEHQ). When heated at or near its boiling point, acrylic acid polymerizes rapidly even in the presence of a polymerization inhibitor. Thus, the yield of conventional methods is only satisfactory (e.g., 40 - 60%). The methods of the present disclosure advantageously avoid polymerization and purify acrylic acid in a yield of at least about 50% (e.g., from about 75% to about 95%) based on the amount of crude acrylic acid before purification. In some embodiments, pure acrylic acid is obtained in the form of an aqueous mixture. The concentration of acrylic acid in such mixtures is from about 70 wt% to about 90 wt% (e.g., about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt% or about 90 wt%).

[0411] In some embodiments, the present disclosure provides a method of purifying acrylic acid by distilling acrylic acid under reduced pressure. In such embodiments, the pressure exerted by the atmosphere around the acrylic acid during distillation is generally from about 70 mbar to about 100 mbar (e.g., from about 75 mbar to about 105 mbar, or from about 80 mbar to about 100 mbar), and the temperature is generally from about 80 °C to about 120 °C (e.g., from about 90 °C to about 110 °C, or from about 80 °C to about 100 °C). For example, in some embodiments, the pressure exerted by the atmosphere around the acrylic acid during distillation is about 70 mbar, about 75 mbar, about 80 mbar, about 85 mbar, about 90 mbar, about 95 mbar or about 100 mbar, and / or the temperature is about 80 °C, about 85 °C, about 90 °C, about 95 °C or about 100 °C. In some embodiments, the distillation is carried out in the presence of a polymerization inhibitor, where the amount of the polymerization inhibitor can be appropriately selected (e.g., from about 1 wt% to about 20 wt%). The polymerization inhibitor can be any one or a combination of the polymerization inhibitors described herein.

[0412] In some embodiments, the present disclosure provides a method for purifying acrylic acid by purging a container containing crude acrylic acid with a gas. In some embodiments, the gas is air, nitrogen, or argon. In some embodiments, the temperature of the gas is lower than the boiling point of acrylic acid. In such embodiments, the temperature is from about 40 °C to about 80 °C (e.g., about 50 °C, about 60 °C, or about 70 °C). In some embodiments, the purging is carried out at approximately atmospheric pressure. During this process, acrylic acid slowly evaporates (without boiling) at the temperature of the warm gas, and the flow of the warm gas carries the pure acrylic acid vapor out of the container. In some embodiments, the system for purging acrylic acid with a gas includes a condenser, and the temperature of the coolant in the condenser is from about 5 °C to about 25 °C. When the warm gas containing acrylic acid vapor passes through the condenser, acrylic acid condenses into a liquid state, and the carrier gas does not remain in the condenser but leaves the system. The pure acrylic acid can be collected from the condenser and used for the intended purpose. Examples

[0413] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0414] Example 1. Specificity of Small Molecule Inducers for the MmsR Transcriptional Activator

[0415] The 3-HP or similar small acid-inducible gene expression system is a novel expression system identified in Pseudomonas denitrificans. This expression system regulates the expression of 3-HP-degrading enzymes. Analysis of the gene arrangement near the 3-HP degradation genes in the Pseudomonas denitrificans genome indicated the presence of a putative LysR family transcriptional regulator, MmsR. It is speculated that the transcriptional regulator protein activates the transcription of mmsA, hpdH, etc. after complexing with 3-HP. The putative transcriptional activator MmsR consists of an N-terminal helix-turn-helix domain for DNA binding, a C-terminal domain for binding an inducer responsive to 3-HP, and a linker connecting these two domains. A study was conducted to determine the range of molecules that can induce MmsR. The ability of various acids and alcohols to induce MmsR was tested, including L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxybutyrate (3-HB), 1,3-propanediol (1,3-PDO), and 2,3-butanediol (2,3-BDO), as well as L-valine (L-val) and its degradation intermediate 3-hydroxyisobutyrate (3-HIB). Most of the acids and alcohols tested were selected mainly because of their size and / or structure similarity to 3-HP. However, L-val and 3-HIB were selected based on their similarity to methylmalonyl semialdehyde dehydrogenase and 3-hydroxyisobutyl dehydrogenase, since mmsA and hbdH-4, whose transcription is regulated by MmsR, encode methylmalonyl semialdehyde dehydrogenase and 3-hydroxyisobutyl dehydrogenase involved in L-val degradation, respectively.

[0416] Pseudomonas denitrificans was cultured on minimal medium in the presence and absence of each test compound, and the transcription of mmsA and hbdH-4 was determined by quantitative RT-PCR( Figure 2A ). The housekeeping gene rpoD encoding σ factor 70 was used as a reference. When Pseudomonas denitrificans was exposed to 3-HIB (mmsA, 154-fold; hbdH-4, 146-fold), 3-HB (mmsA, 38-fold; hbdH-4, 32-fold) and L-val (mmsA, 72-fold; hbdH-4, 68-fold) and 3-HP (mmsA, 134-fold; hbdH-4, 128-fold), the transcription of both genes (mmsA and hbdH-4) was significantly enhanced. In contrast, limited or no induction was observed when exposed to LAC, AcOH, PA, 1,3-PDO and 2,3-BDO. 3-HP, 3-HB and 3-HIB are structurally similar as they are all β-hydroxy acids. Both the carboxyl group and the β-hydroxy seem to be essential for the ability of 3-HP, 3-HB and 3-HIB to bind MmsR. L-val is structurally very different from these three compounds and is converted to 3-HIB. The induction by L-val can be attributed to L-val-derived 3-HIB rather than L-val itself.

[0417] The possibility that malonaldehyde (MSA) derived from 3-HP and methylmalonatesemialdhyde (MMSA) derived from L-val and 3-HIB rather than 3-HP and / or 3-HIB serve as physiological inducers was also examined (see Figure 2A and 2B ). In general, aldehydes are toxic and when aldehydes accumulate in cells, aldehyde degradation genes are usually upregulated. 3-HP is degraded by the enzymes encoded by hpdH, hbdH-4 and hbdH-1, and the deletion mutant Pseudomonas denitrificans ΔhpdHΔhbdH-4ΔhbdH-1 does not degrade 3-HP at an appreciable rate. In this triple mutant that does not produce MSA from 3-HP, the transcription of mmsA was still upregulated by 3-HP( Figure 3A - 3B ). This indicates that 3-HP can activate the MmsR protein without being converted to MSA. Similarly, mmsA transcription was upregulated by 3-HIB in the triple mutant, indicating that 3-HIB is also a true inducer of MmsR. Interestingly, the transcription of mmsA in the triple deletion mutant Pseudomonas denitrificans was higher than that in the wild-type strain in the absence or presence of 3-HP.

[0418] To confirm that MmsR is a transcriptional activator, deletion and subsequent complementation experiments were performed( Figure 2B)。In the deletion mutant (ΔmmsR), the transcription of both mmsA and hbdH-4 was low, and this level was not affected by 3-HP. However, when mmsR was reintroduced into the ΔmmsR mutant via a plasmid, the upregulation of mmsA and hbdH-4 by 3-HP was fully restored. These results confirmed that MmsR is a transcriptional activator protein for the expression of mmsA and hbdH-4. Interestingly, the basal level transcription of mmsA and hbdH-4 in the ΔmmsR mutant was approximately 2-fold higher than that in the wild type or the mmsR complemented recombinant (mmR-C). This suggests that the MmsR protein without an inducer may inhibit the transcription of mmsA and hbdH-4 to some extent.

[0419] Although the expression of hbdH-4 is regulated by 3-HP, the promoter sequence is very different from that of mmsA because the promoter lacks an operator site for binding the transcriptional activator protein. To confirm that the hbdH-4 promoter is inducible, the intergenic region between mmsA and hbdH-4 was cloned into a plasmid with green fluorescent protein (GFP) as a reporter gene. In addition, electrophoretic mobility shift assays (EMSA) were performed with purified MmsR protein and a DNA fragment of the hbdH-4 promoter region. Both experiments showed that the hbdH-4 promoter is constitutive and cannot be induced by 3-HP. GFP under the control of PhbdH-4 was constitutively expressed, and there was no binding between the MmsR protein and the hbdH-4 promoter region (data not shown). In addition, in qRT-PCR experiments on cells grown in the presence of 3-HP, a large number of polycistronic mRNA transcripts of mmsA and hbdH-4 were detected. These results suggest that the transcription of hbdH-4 is controlled by two independent promoters, namely the 3-HP inducible PmmsA and the constitutive PhbdH-4, and the significant upregulation of hbdH-4 after the addition of 3-HP (see Figure 1B and 2B ) is attributed to the transcriptional read-through from PmmsA. We also noticed that there is no terminator sequence in the intergenic region between mmsA and hbdH-4. Since PhbdH-4 is not inducible, PmmsA was studied in detail.

[0420] Example 2. In silico analysis of the mmsR-mmsA intergenic operon-promoter region

[0421] In silico analysis of the intergenic region between two genes with distinct transcription of mmsR and mmsA was performed. This study characterized cis-acting elements, including: (i) a putative promoter upstream of the mmsA transcription start site (TSS), (ii) two putative tandem operator sites O1 and O2, and (iii) three T-N11-A motifs, two of which include the two half-sites of O1 and the last one includes the half-site of O2 ( Figure 3A ). Each operator contains a dyad-symmetric DNA sequence with centers located at positions -81 and -33 upstream of the putative TSS of the mmsA gene, respectively. Inverted repeats are common in many prokaryotic operators recognized by regulatory proteins. The distance between the centers of the two palindromic regions is approximately 50 bp, corresponding to five turns of the helical DNA. The nucleotide sequence in the dyad of the O1 site consists of two 9-bp segments (separated by 15 bp), highly symmetric with only one mismatch. The inverted repeat sequences of the O2 site are separated by 11 bp, less symmetric, with six mismatches in nine bases. Alignment of the four palindromic segments of the O1 and O2 operators revealed the presence of the consensus sequence AACGTGTAA ( Figure 3B ). In all segments, the three bases A, G, and T ( Figure 3B , bold) located at positions 2, 4, and 5, respectively, are completely conserved, while the three bases C, T, and A (underlined) at positions 3, 7, and 8 are highly conserved in the three segments, respectively. It should be noted that the identity of the putative O2 region is somewhat questionable. One of its half-sites shows very poor conservation (3 / 9), and the spacer is 4 bp shorter than that of O1. Additional evidence confirming the identity of the O2 site should be obtained from in vivo and in vitro experiments (see the following examples).

[0422] LysR-type transcriptional regulators (LTTR) are the largest family of transcription factors in prokaryotes. LTTR binds to DNA sequences with two symmetric operator regions, called the RBS (regulatory binding site) and the ABS (activator binding site), where the RBS shows a greater degree of symmetry relative to the ABS. In addition, it is known that the symmetric half-sites of the RBS and ABS are often included in the T-(N11)-A motif. These sequence features, along with the structural features of MmsR, suggest that MmsR belongs to the LTTR family of transcriptional regulators. Our analysis of the sequence of the intergenic region (see Figure 3A) also indicates that the transcription of mmsR is inhibited by its own product, MmsR. The O1 site is located in the putative -10 region upstream of mmsR, and the O2 site completely overlaps with the -35 promoter region upstream of mmsR. The binding of the MmsR protein to the O1 and O2 sites interferes with the binding of RNA polymerase to the mmsR promoter and / or its movement along the DNA strand during transcription. We also note that the transcription of mmsA may be interfered with by the binding of the MmsR protein, because the -35 region of mmsA is located in the 11-bp long spacer region between the two half-sites of the O2 site. This may explain why the basal level transcription of mmsA and hbdH-4 in the ΔmmsR mutant is 2-fold higher than that of the wild-type counterpart (see Figure 2A and Figure 2B ). Extensive in vivo and in vitro studies were carried out on the intergenic region to verify the in silico predictions (see Examples below).

[0423] Example 3. In vivo characterization of the mmsR-mmsA intergenic operator-promoter region

[0424] The promoter region controlling the expression of the mmsA gene was characterized by 5'-end mapping and in vivo random mutagenesis. To identify the -10 and -35 regions, various mutations were made in the P mmsA promoter region, including serial deletions in the upstream region or randomization of the putative -10 and -35 regions, and then the mutant promoters were fused with the gfp reporter gene ( Figure 4A - 4B ). The ammonia-oxidizing Pseudomonas lacking the mmsR gene was used as the host to test the mutant promoters, because MmsR can bind to the promoter and affect the expression of the gfp reporter gene. When the promoter region between -117 and -60 (P mmsA _Δ1) or -117 and -37 (P mmsA _Δ2) was deleted, the gfp expression decreased by about 16% compared with the control containing the full-length promoter (P mmsA _wt) (Table 1). In contrast, when longer upstream sequences before -27 (P mmsA _Δ3) or -14 (P mmsA _Δ4) were deleted, the promoter strength decreased significantly by 39% or 58%. These results indicate that the sequences deleted in P mmsA _Δ3 and P mmsA _Δ4 contain important transcriptional elements, most likely the -10 and -35 regions as predicted by in silico analysis. In addition, randomization of the putative -10 (P mmsA _-10) and -35 (P mmsA _-35) regions decreased the promoter strength by 52% compared with PmmsA_wt. The 5'-end mapping and random mutagenesis support the in silico predictions of the -10 and -35 regions of P mmsA .

[0425] Table 1: In vivo analysis of the mmsR-mmsA intergenic region based on GFP expression after controlled serial mutagenesis in the promoter and operator regions (AU / OD; AU, arbitrary unit).

[0426]

[0427] a. Indicates complementary expression of MmsR on the plasmid

[0428] b. Fluorescence was measured 3 times (n = 3) 4 h after induction, and the average value was taken.

[0429] c. 25 mM 3-HP was added to the growth medium for induction.

[0430] 5'-end mapping and random mutagenesis were also performed to study the position and identity of the operator regions (O1 and O2) in vivo. Since mmsR may be autoregulated (see the examples below), mmsR was constitutively expressed from a plasmid under the control of a weak P c1 promoter (hbdH-1 promoter). As shown in Table 1, BS_wt and BS_Δ1, which contain two operators (O1 and O2), showed high 3-HP induction (up to fold). In contrast, when the first half-site (BS_Δ2) of the putative O1 site was deleted alone or together with the other half-site of O1 or O2 (BS_Δ3 or BS_Δ4), the inducibility was completely lost.

[0431] Promoters with mutations in the O1 and / or O2 regions were also studied. Here, the operator sequences were randomized to disrupt the dyad symmetry, but the -35 region was not changed. As expected, randomization of O1 or both O1 and O2 completely abolished 3-HP inducibility. These results confirmed the position and identity of the O1 operator region, which is thought to be located downstream of -98. However, these results did not clearly define or determine the role of the O2 region. When only the putative O2 region was randomized (BS_ΔO2), the promoter strength was greatly increased in the absence of 3-HP ( fold) compared to the wild-type BS_wt. When 3-HP was present, the strength was reduced by 37% (compared to the wild-type BS_wt), but still remained at a high level. In addition, when both O1 and O2 were mutated (BS_ΔO1O2), the promoter strength was significantly reduced, and the promoter was not inducible. This indicates that the O2 site works closely with the O1 site and plays an important role in regulating the function and strength of PmmsA.

[0432] In general, in vivo studies of the operator region can be summarized as follows: (i) Alone, O1 can activate transcription (by the 3-HP-MmsR complex), although less efficiently than when both O1 and O2 are present; (ii) Binding of MmsR without 3-HP to the O2 operator can inhibit transcription from the PmmsA promoter, and (iii) The high strength of the ΔO2 mutant promoter still involves the presence of the O1 site. It is very likely that the PmmsA promoter is both positively regulated (in the presence of 3-HP) and negatively regulated (in the absence of 3-HP).

[0433] The effect of MmsR binding in the operator region on mmsR expression was also studied using a GFP reporter gene. P mmsR The promoter is negatively regulated by its own protein product, the MmsR protein ( Figure 4B ); GFP expression increased approximately 2-fold when mmsR (P mmsR _wtΔmmsR) was deleted. Mutations in O2 (P mmsR _ΔO2) did not affect GFP expression, while an O1 mutation (P mmsR _ΔO1) slightly increased expression These results suggest that occupancy of the O1 region by MmsR alone can inhibit transcription from the P mmsR promoter. In addition, the presence of 3-HP did not affect the repression by the MmsR protein, indicating that the repression is not 3-HP-dependent. Attempts were also made to identify the -10 and -35 regions of P mmsR by mapping the 5' end of the putative promoter region ( Figure 5A ), but this failed. The strength of the P mmsR promoter was too weak to achieve differences between mutant promoters (p > 0.05).

[0434] LTTR proteins and their cis-acting elements have been studied in several microorganisms. In Escherichia coli and Salmonella typhimurium LT2, the IlvY protein (a LysR-type transcriptional regulator) that controls the expression of the ilvC gene (encoding acetohydroxyacid isomerase; EC 1.1.1.86) has been studied. In these studies, transcription of the ilvC gene is induced by the substrates of acetohydroxyacid isomerase, acetohydroxybutyrate or acetolactate, and this induction is mediated by the IlvY protein.

[0435] Similar to the PmmsA promoter, the promoter regions of the ilvY and ilvC genes have two operators, O1 and O2, each of which consists of a 9-bp long inverted repeat. These repeat sequences have the same homologous sequences, which are AACGTTAC(T)A in Escherichia coli and NG(A)CGTTG(A)TA in Salmonella typhimurium LT2, respectively. In addition, similar to the PmmsA promoter in Pseudomonas denitrificans, the symmetry between the two dyads in these strains is more stringent in O1 (single mismatch) than in O2 (six mismatches). It has been reported that another LysR-type activator, AtzR, present in Pseudomonas sp., shows dissimilarity in the ABS operator region. AtzR activates the expression of the cyanuric acid degradation operon atzDEF, which is transcribed differently. However, ABS contains three motifs, called ABS-1, ABS-2, and ABS-3, respectively, and each sub-site has a different role during activation. In vivo mutagenesis analysis showed that the ABS-1 and ABS-2 sub-sites were involved in the full activation of the P aztDEF promoter. In contrast, when AtzR is located at the ABS-2 and ABS-3 sub-sites, ABS-3 acts as a "subunit trap", resulting in the inactivation of P aztDEF For the P mmsA promoter, like many other LTTR-mediated systems, the O2 region is assumed to completely overlap with the -35 region. Transcriptional activators, IlvY, ClcR, and AtuR, from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa, respectively, also have the same arrangement, where the -35 promoter element is located within the O2 sub-site. This overlapping region controls the upregulation of downstream genes by binding to the dimers of LTTRs. However, the function of suppressing downstream genes in the absence of an inducer molecule has not been reported.

[0436] Example 4. In vitro production of MmsR protein and its binding to operator sites

[0437] For in vitro biochemical characterization, a recombinant MmsR protein labeled with six histidine residues at the C-terminus of the protein was produced and purified from recombinant Escherichia coli. Whether His is present at the C-terminus or N-terminus of MmsR, in the complementation experiments conducted as described in Example 1 (see Figure 6C ), the His-tagged MmsR protein seemed to function as well as the native MmsR protein. Therefore, only the recombinant C-terminal His-tagged MmsR was further studied in vitro. After optimizing the culture conditions (such as temperature, medium, IPTG concentration, harvest time, and 3-HP concentration, as well as co-expression with various molecular chaperones such as GroEL-ES, DnaKJ-GrpE, and trigger factor), the recombinant MmsR was highly expressed in a soluble form in Escherichia coli and purified by affinity chromatography ( Figure 5A - 5E)。The size of the His-tagged MmsR protein was estimated to be 34.4 kDa, which was in good agreement with the size estimated based on the 6-fold his-mmsR gene sequence. According to native PAGE and / or gel filtration analysis, the MmsR protein was monomeric at a low concentration of 65 nM and dimeric at a high concentration of 550 nM( Figure 6B )。

[0438] The binding of MmsR to the mmsR-mmsA intergenic region was studied in vitro by EMSA( Figure 7A )。A 40 nM intact 130 bp DNA fragment was used as a probe (referred to as the F12 fragment; see Figure 3B ), and 0 - 72.7 nM purified MmsR protein was incubated with the DNA probe in the presence and absence of 3-HP as an inducer. A DNA fragment (F1M2M) with randomization in both O1 and O2 was used as a control (see Figure 3B and Table 2). In the electrophoresis, when MmsR was added, a band shift of the intact 130 bp DNA fragment occurred, and the ratio of the shifted DNA to the unshifted DNA increased with the increase in protein concentration during incubation. In contrast, no such decrease in mobility was observed in the control DNA fragment with up to 72.7 nM MmsR (data not shown). This indicates that MmsR has a strong binding affinity for the native P mmsA and forms a binding complex in vitro. At high concentrations of the MmsR protein, multiple bands with shorter migration distances appeared. This was attributed to the formation of various oligomeric complexes between the DNA fragment and the protein molecules. The DNA probe has two binding sites (O1 and O2) for MmsR, and MmsR binds to the DNA in the form of a dimer. Therefore, when the concentrations of DNA and protein are very high, it is very likely to form multiple oligomeric complexes. Figure 7A The effect of 3-HP on the binding affinity between MmsR and the P mmsA promoter was also shown. When 3-HP was added, a band shift occurred at a lower MmsR concentration. Similarly, when 3-HP was present, oligomeric complexes with shorter migration distances appeared earlier at a lower MmsR concentration. This indicates that 3-HP promotes the binding affinity between MmsR and the P mmsA promoter.

[0439] Table 2: EMSA fragments used in this study

[0440]

[0441]

[0442] Bold letters indicate the half-sites of the O1 operator; italic letters indicate the half-sites of the O2 operator; underlined letters indicate the site-directed mutagenesis regions.

[0443] To analyze the MmsR binding region in vitro, DNase I footprinting was performed by capillary electrophoresis ( Figure 7B ). A longer 169 bp DNA fragment containing the complete 130 bp intergenic region (used for the above EMSA experiment) was used as a probe (Table 2). The concentration of DNA was fixed at a specific concentration, while the concentration of MmsR varied between 0 - 1.2 μM. The footprint results clearly showed the presence of two regions protected by MmsR, corresponding to the operator O1 (centered at -81) and O2 (centered at -33), respectively. As the concentration of MmsR increased, the protection became more obvious. However, it was difficult to precisely determine the specific protected regions at the base pair level of the DNA sequence.

[0444] The EMSA experiment was repeated at a low DNA concentration (0.4 nM) to evaluate the dissociation constant KD ( Figure 8 ) between MmsR and the PmmsA promoter. Three DNA fragments were used. One contained both the O1 and O2 operator sites (F12), and the other two were the same length as F12 but had mutations (randomizations) in the palindromic regions of the O1 (F 1M2 ) or O2 (F 12M ) operator sites. Among these three DNA fragments, F 12 had the highest affinity for MmsR, followed by F 12M , and then F 1M2 .

[0445] The dissociation constants of MmsR binding (K D ) to these three DNA fragments were determined from the MmsR protein-DNA isotherm curves, in which the fraction of DNA bound by MmsR was plotted against the free MmsR concentration in the reaction buffer ( Figure 9 ). In the absence of 3-HP, when estimating the monomeric MmsR protein, the K 12 of F D was 10.7 nM, the K 12M of F D was 18.6 nM, and the K 1M2 of F D was 79.8 nM ( Figure 9 ). 3-HP changed the binding affinities of these DNA fragments in different ways. In the presence of 3-HP, the K 12 value (monomeric MmsR concentration) of F D was estimated to be 5.4 nM, the K 12M value of F D was 19.8 nM, and F1M2 The K D value is 316 nM ( Figure 9 ). F 12 has a higher affinity for F 12M or F 1M2 indicating that there is a certain cooperativity in the operator binding. Thus, the binding of MmsR to one operator (possibly O1) stimulates the binding to another operator (possibly O2). In the absence of 3-HP, F 12 The presence of only one DNA-MmsR complex band also supports the concept that the binding between the two operators is cooperative: if the binding of MmsR to O1 does not stimulate the binding to O2, then two DNA-MmsR complex bands should appear (one for O1 binding and the other for O1 and O2 binding).

[0446] Rhee et al. also studied the binding affinity of the Escherichia coli IlvY protein to DNA fragments containing tandem operators (O1O2) or only the O1 or O2 operator. For the fragment containing the tandem O1O2 operator, the binding affinity was the highest, while for the fragment with only the O2 operator, the binding affinity was the lowest. The Kapp determination for monomeric IlvY was: 4.4 nM for the tandem operator, 35.2 nM for O1, and for O2 was However, Rhee et al. also reported that the inducer does not affect the binding of the IlvY protein to these operators. Here, in the presence of 3-HP, two retarded bands could be resolved at the highest protein concentration. The change in the nature of the DNA-binding protein in the presence of the inducer may affect the formation of the complex. As explained in the above examples, when the concentrations of DNA and MmsR increase above a certain level, they may form multiple complex oligomeric structures with different mobilities.

[0447] Example 5. Specificity of the Small Molecule Inducer for the HpdR Transcriptional Activator

[0448] HpdR is a transcriptional activator protein, similar to MmsR, that can recognize 3-HP or a similar small acid and stimulate the expression of specific genes in Pseudomonas denitrificans. Figure 10AShows the gene arrangement and intergenic region in the HpdR-regulated operon in Pseudomonas denitrificans. The hpdR and hpdH genes are conserved in all sequenced Pseudomonas genomes available in the Pseudomonas genome database, and the organization of this genomic region is the same. The hpdH gene encodes 3-hydroxypropionate dehydrogenase (EC number 1.1.1.59), an enzyme involved in 3-HP catabolism. The hpdR gene encodes an LTTR protein consisting of 304 amino acid residues, which is transcribed differently compared to its structural gene hpdH. Sequence analysis shows that the intergenic region between hpdR and hpdH is 124 nucleotide base pairs long and contains two promoters for the expression of hpdR and hpdH, HpdR dimer binding sites (regulator binding site and activator binding site, RBS and ABS respectively) and ribosome binding sites. The DNA binding site of HpdR may have three putative tandem operator sites ABS-1, -2 and -3 and RBS-1 and -2, and only two T-N11-A motifs are located within this intergenic region, one at RBS-1 and the other overlapping with RBS-2 and ABS-1. The presence of the T-N11-A motif is one of the typical features of the LTTR-mediated expression system (see Figure 10A ). These three operators contain dyad symmetry regions located at positions -52, -36 and -21 respectively upstream of the hpdH transcription start site (TSS; predicted using the NNPP tool). For the expression of the hpdR gene, palindromic repeats (such as RBS-2 and ABS-2) mask the region between the transcription start site and the -35 region, indicating that HpdR binding in these operator regions can completely eliminate the binding of RNA polymerase. Thus, automatically inhibiting the transcription of its own gene hpdR. On the other hand, two 9bp fragments of the RBS and ABS sites are separated by 5, 7 and 6 nucleotides respectively. The nucleotide sequences of the three inverted dyads are not completely symmetrical as there are three mismatches in each sub-site.

[0449] Example 6. Transcriptional activation of hpdH by HpdR

[0450] It has been proposed that HpdR is a transcriptional activator of the hpdH gene in Pseudomonas denitrificans. To test this in vivo, we created several Pseudomonas denitrificans strains, including deletion mutants, namely PdΔhpdR and PdΔmmsR, and a recombinant strain PdΔhpdR carrying pUCPK' / Pc1-hpdR (for complementation studies), and analyzed hpdH transcription together with the WT strain (ATCC13867). The PdΔmmsR strain with a putative deletion of the C4 transcriptional activator was tested to see if there is cross-talk between the C3 and C4 transcriptional activators HpdR and MmsR. The mRNA expression levels were determined and compared in the presence and absence of 3-HP as an inducer. 3-HP led to a 43.0 ± 10.9-fold increase in hpdH expression from the Pseudomonas denitrificans chromosome (WT), while its transcriptional activator HpdR did not show a change induced by 3-HP ( Figure 11A ). In the C4 operon, the expression of mmsA and hpdH-IV was also activated 151.3 ± 18.2 and 149.3 ± 9.6-fold, respectively, by 3-HP, while mmsR was not activated at all ( Figure 11B ). In Pseudomonas denitrificans WT, the genes of the regulatory proteins (i.e., hpdR and mmsR) were expressed at low levels, while the genes regulated by these proteins (hpdH, mmsA, and hpdH-IV) were highly expressed in the presence of 3-HP. In particular, mmsA and hpdH-IV of the C4 operon showed high expression, indicating the presence of a strong promoter. Once hpdR was deleted (ΔhpdR), the transcription of hpdH was not activated (1.20 ± 0.58); when hpdR was complemented with a recombinant plasmid (hpdR-C) expressing HpdR from it, its transcriptional activation was restored to the wild-type level (60.5 ± 6.1). Therefore, it can be concluded that when 3-HP is present as an inducer, hpdH (one of the catabolic genes in the C3 operon of 3-HP) is positively regulated by its transcriptional regulator HpdR. To study the cross-talk between the C3 and C4 operons, the expression of the C4 operon genes (mmsA and hbdH-IV) was observed in the PdΔhpdR and recombinant Pseudomonas denitrificans strains complemented with hdpR. The expression of mmsA and hbdH-IV was not affected by the presence and / or absence of hpdR, indicating that the C3 transcriptional activator is specific to the C3 operon and does regulate the C4 operon ( Figure 11B ). Similarly, the C4 transcriptional activator (MmsR) did not affect the expression of the C3 operon genes (data not shown). Taken together, these results suggest that although HpdR and MmsR are activated by the same inducer molecule (3-HP) and belong to the same LTTR family, they are highly specific in binding to the operator site and the genes whose subsequent transcriptional activation is regulated.

[0451] Example 7. Autoregulation of HpdR

[0452] It is known that LTTR regulatory proteins autoregulate their own promoters by inhibiting transcription initiation in an inducer-independent manner. The regulatory binding site (operator) of HpdR upstream of hpdH is considered a possible autoregulation site. To understand the autoregulation of HpdR in the C3 operon, we constructed several plasmids, including (i) GFP expressed under the control of PhpdR, where HpdR was constitutively expressed using the PC1 or Pzwf promoter, and (ii) GFP constitutively expressed using the P eda and P fbp promoters. GFP fluorescence was expressed using the P hpdR promoter, where HpdR was expressed at two different levels with the P zwf or P C1 (weaker than P zwf ) promoters. The autoregulation of GFP was monitored during a 24-hour culture period. When HpdR was expressed using P zwf or P C1 , GFP reached a saturation level at 10 hours, and there was no further expression despite the constitutive expression of HpdR. However, when regulated by constitutive promoters such as P eda and P fbp , GFP fluorescence was still gradually expressed. The expression level of GFP fluorescence was affected by the strength of its promoter, i.e., P hpdR , P eda and P fbp . This result is consistent with the fact that LTTR proteins autoregulate their own promoters ( Figure 12A - 12D ).

[0453] Example 8. Specificity of the inducer of HpdR in the C3 operon

[0454] Several acids and aldehydes were used to examine the inducer specificity of HpdR. These acids and aldehydes were selected based on their similarity to 3-HP in terms of the number of carbon atoms (C3 or 4), molecular mass, and chemical structure, including 3-HP, L-val, BA, IBA, LAC, AA, IVA, and PA. The relative inducibility was examined based on the transcription of the hpdH and gfp genes and the fluorescence of GFP. Based on the transcription levels of HpdH and gfp and GFP fluorescence, 3-HP was determined to be the best inducer of HpdR. Compared with the control (Ctrl) without an inducer, 3-HP increased the transcription of hpdH by 75 ± 8.3-fold. Compared with the results of other chemicals, this was a statistically significant difference ( Figure 12A)。Following exposure to L-valine, the transcription of hpdH also increased by 52.8 ± 27.4-fold, making L-valine the second strongest inducer in these experiments. However, other acids and propionaldehyde were found to be less effective or unable to induce the HpdR-mediated regulatory C3 gene expression system. The results of 3-HP and L-valine inducibility obtained using the C3 (hpdH and hpdR) regulon were similar to those described for the C4 regulon (mmsA and MmsR) in the above examples.

[0455] Structurally, 3-HP has both a carboxyl group and a β-hydroxy group, which play important roles in the ligand-binding ability to HpdR. L-valine was also tested because hpdH, which encodes 3-HP dehydrogenase, is also involved in the degradation of L-valine, and its metabolites can show inductive ability. Even though L-valine is structurally different from 3-HP, it can be easily converted to 3-hydroxyisobutyric acid (3-HIB), which can induce the Cm operon (manuscript in preparation), and the induction of L-val is due to its conversion to 3-HIB. Overall, the transcription of gfp as a reporter gene was examined. In plasmid pUCPK’ / Pzwf-hpdR-PC3-gfp, the hpdH gene was replaced by gfp, and gfp was heterologously expressed. As described above, the reporter strain PdΔhpdRΔhpdH carrying gfp was treated with several chemicals at 25 mM (pH ), including 3-HP, L-val, BA, IBA, LAC, AA, IVA, and PA. 3-HP could maximally induce the expression of gfp mRNA, resulting in a 3.4-fold higher expression than the control. This was statistically significant compared to the control. The other chemicals tested did not induce mRNA expression ( Figure 12B ). Similarly, GFP fluorescence was induced the most (3- and 3.3-fold) by 3-HP at 6 and 11 hours after induction, respectively ( Figure 12C ), which was consistent with the gfp gene expression results showing a 3-fold induction ratio. Therefore, 3-HP is the best inducer for activating the transcription of target genes when complexed with HpdR in the C3 operon.

[0456] The dose effect of 3-HP on the hpdH-inducible promoter system was also studied by varying the concentration range of 3-HP from 0.1 mM to 25 mM. The fold increase in the induction ratio of GFP fluorescence at 6 and 11 hours after induction changed linearly. However, concentrations above 0.5 mM to 25 mM showed a similar increase in inducibility ( ). Figure 12D)。The C3 operon was characterized by examining inducer specificity at the transcriptional and translational levels, where the transcription of hdpH was regulated by HpdR through 3-HP-mediated induction in vivo and in vitro. The inducer specificity and strength of the hpdH promoter system were investigated. The findings obtained indicated that 3-HP showed the highest specificity when complexed with HpdR to induce this gene expression system, and an overall 3-fold induction ratio of the hpdH promoter for GFP expression was observed in vivo.

[0457] Example 9. In Vivo Analysis of the HpdR Binding Site

[0458] In silico sequence analysis of the 5'-region of PhpdH revealed the presence of five putative operators (RBS-1, RBS-2, ABS-1, ABS-2, and ABS-3). To elucidate the importance of these operators in hpdH transcriptional activation, a study was conducted in which the repeat sequences of each putative operator were mutated. Several recombinant plasmids based on the pUCPK' / Pzwf-hpdR-PC3-gfp plasmid were constructed, in which one of the five operator repeat sequences was replaced with a 9-bp random DNA sequence (ACAGGCGTA, generated by the Random DNA Sequence Generator tool, which neither showed the conserved T-N11-A motif nor sequence conservation with the actual subsites), and then the induction ability of gene expression was evaluated by measuring GFP levels (reporter gene) (data not shown). Mutation of the RBS-1 or ABS-2 site significantly reduced the induction ability of 3-HP (data not shown). In contrast, the mutation had a smaller effect on the ABS-1 or ABS-3 site, while mutation of the RBS-2 site only slightly affected the PC3 promoter. These results suggest that the RBS-1 and ABS-2 sites are key binding sites for HpdR to activate the transcription of hpdH (or gfp), while the importance of other operator sites for HpdR binding is not high.

[0459] According to LTTR studies, the LysR protein dimer binds to two operator sites in a cooperative manner. Generally, once the RBS is occupied for the first time, it helps the ABS recruit a second LTTR dimer. In this case, due to the functional role of the RBS in the efficient recruitment of LTTR dimers for target gene transcriptional activation, the RBS site is more important than the ABS. Among the two repeats in an operator (i.e., RBS-1 and RBS-2), it is unclear which repeat is more important in binding the LTTR protein. Both repeats receive the helix-turn-helix motif of the LysR protein and are thus equally important. However, considering the low promoter strength of PC3, the affinity of HpdR for the operator may be weak, which can be attributed to the smaller contribution of RBS-2 in the recruitment of HpdR protein. This study showed that even when mutations in RBS-2 were examined, the inducibility did not change, indicating that the contribution of this repeat was not high enough. By changing RBS-2 to have more consensus sequences to achieve higher affinity for LysR, a more detailed study of the role of RBS-2 in LTTR binding can be conducted. The smaller decrease in expression after mutation of the ABS site (both repeats of the ABS) may be attributed to the fact that this ABS closely matches the putative promoter site of hpdH and these mutations result in changes in the hpdH promoter, leading to fluctuations in inducibility. Mutations on ABS-2 seem to make PC3 constitutive, resulting in no inducibility of HpdR-mediated transcriptional activation in the presence of 3-HP. In summary, in the case of HpdR complexed with 3-HP, mutations in each operator except RBS-2 result in loss of inducibility, and this change leads to functional modification of the promoter, from inducible to constitutive in nature. The changes in gene expression caused by operator mutations can be attributed to several hypothesized reasons: (i) decreased affinity of HpdR for the DNA binding site that activates hpdH (or gfp) transcription; (ii) inappropriate conformational changes in the HpdR protein-operator DNA complex because DNA bending results in low transcription of the target gene; (iii) significant changes in the hpdH promoter strength resulting in decreased expression. Thus, operator mutations can lead to changes in the intrinsic promoter characteristics.

[0460] Example 10. Binding Site Randomization and Modification of Activator Protein Expression Levels

[0461] Gene expression can be modified at the transcriptional and / or translational levels. Initially, we modified the operator sites (O1 and O2) to attempt to improve P mmsATranscription of the promoter. The operator site present in the intergenic region of the mmsR-mmsA genes bound by the activator protein MmsR plays an important role in determining the affinity of the promoter region for RNA polymerase. The kgsA gene was cloned downstream of the intergenic region of the mmsR-mmsA genes in the multi-copy pUCPK plasmid, and two different parts of the O2 operator sequence (each constituting half of the operator site) were independently randomized (P mmsA2a and P mmsA2b ) or used in combination (P mmsA2ab )( Figure 15A ). The transcriptional activator MmsR was expressed from the bacterial chromosome under the native promoter (P mmsR ). The intact intergenic sequence without mutations was used as a control. Mutations in O 2a or O 2b significantly increased the transcriptional level, 7.3 - 10.6-fold in the absence of 3-HP and 1.3 - 1.7-fold in the presence of 3-HP. Mutations in the first half of the O2 region (O 2b ) significantly increased the transcriptional efficiency compared to mutations in the second half (O 2a ). When both half-sites of the O2 operator were randomized, the basal level of transcription (without 3-HP) was comparable to the basal transcriptional level generated by mutations in O 2a or O 2b ( Figure 15B ). However, the maximal transcriptional level in the presence of 3-HP decreased and this level was even lower than that of the wild-type promoter. The O2 region completely overlaps with the -35 region where the σ-factor of RNA polymerase binds. In response to 3-HP, in terms of upregulation, changes in the sequence around the -35 region may directly affect the affinity / function of MmsR for the P mmsA promoter. In this case, reducing the binding interaction by randomizing half of the O2 region greatly increased the level of transcription in an inducible manner.

[0462] By using GFP as a reporter gene, the strengths of the wild-type and mutant promoters were clearly compared ( Figure 15C ). The results showed that during the induction period with 25 mM 3-HP, the promoter with mutations in the first half of the O2 region (P mmsA2a ) showed approximately 2-fold higher expression of GFP. This result was consistent with the transcriptional level results shown in Figure 15B . This study indicates that the transcriptional level can be modified by altering the operator binding site region. We can both increase the basal level expression of the promoter (substantially) and improve the induction level of the promoter expression (by about 2-fold).

[0463] In bacteria, LysR-type transcriptional regulators (LTTR) proteins effectively upregulate downstream genes by interacting with RNA polymerase. As a result, the amount of LTTR protein affects the transcriptional level of target genes. To study the effect of MmsR expression on the P mmsA promoter strength, we developed a constitutive P zwf promoter library ( Figure 16A ) and expressed MmsR ( zwf ) under the promoters selected from the P Figure 16B library. In constructing this synthetic promoter library, the -35 and -10 consensus sequences of the P zwf promoter were kept constant, and the space around these sequences was randomized and the length was variable.

[0464] Several synthetic promoters in the library were evaluated, and these promoters showed a promoter strength distribution as Figure 16A shown. The constitutive promoter P zwf (P zwf-7 ) was used as a positive control. After normalizing the background signal, the GFP fluorescence levels produced by the library promoters were between 11,500 (P zwf-1 ) and 51,000 (P zwf-12 ) relative fluorescence units (AU / OD600). Thus, the range of promoter expression levels was approximately 5 orders of magnitude relative to the wild-type P zwf-7 (32,200). Three promoters were selected from this library: P zwf-1 and P zwf-11 , corresponding to 0.4, 1, and 1.7-fold differences from the wild-type P zwf , respectively. The transcriptional activator MmsR produced at different levels under the control of each of these derived P zwf promoters, and the transcription of the gene (kgsA) downstream of the P mmsA promoter was affected. A strain (P mmsR ) with chromosomally expressed MmsR under the native promoter P mmsA was used as a control. At low MmsR levels (MmsR-1 strain), when MmsR was under the control of the P zwf-1 promoter, the mRNA expression of kgsA was comparable to the control regardless of the presence of 3-HP. When MmsR was placed under the control of two stronger P zwf-1 and P zwf-11When the amount of MmsR was increased under the control of promoters (MmsR-1 and MmsR-11 respectively), the transcription of kgsA was improved in the presence of 3-HP. However, in the absence of 3-HP, the basal expression level of kgsA was significantly reduced. As described above, this result is understandable due to the repression regulation of apo-MmsR. For inducibility, a higher MmsR expression level can greatly increase the difference between non-induced and induced cases by minimizing the basal level. This indicates that enhancing the activator is a good strategy to prevent pre-induction target gene leakage that usually occurs in the case of strong inducible promoters.

[0465] Example 11. Expression comparison of native mmsA and heterologous kgsA genes under the P mmsA promoter

[0466] Pseudomonas denitrificans can actively degrade 3-HP produced from glycerol. 3-Hydroxyisobutyrate dehydrogenase IV (HbdH-4) and methylmalonic semialdehyde dehydrogenase (MmsA) encoded by hbdH-4 and mmsA respectively are partially responsible for the degradation of 3-HP (the mmsA promoter (PmmsA) is highly induced by 3-HP ((>140-fold)) with the help of the activator protein MmsR). This promoter was used to develop a 3-HP sensor that can sensitively detect 3-HP at around 25 mM when added externally.

[0467] Although P mmsA can be highly induced by 3-HP, the expression level of heterologous genes (such as gfp or kgsA) under the control of P mmsA is much lower than the expression level of mmsA inherently controlled by P mmsA in Pseudomonas denitrificans. Before engineering P mmsA the expression of homologous (mmsA) and heterologous (kgsA) genes cloned under P mmsA was quantitatively compared by RT-PCR. The transcription of the heterologous gene kgsA was significantly lower than that of the native gene (mmsA), about 100-fold when expressed chromosomally, and when using multiple copies ( When the pUCPK' plasmid was expressed in episomal form, it was approximately 40-fold. The crude cell KgsA activity clearly demonstrated the effects of 3-HP induction and gene dosage. When expressed from the chromosome (Int-1), the activities were 0.08 U / mg protein (without 3-HP) and 0.15 U / mg protein (with 3-HP), respectively. When expressed from a multi-copy plasmid, the activities were 1.02 U / mg protein (without 3-HP) and 3.15 U / mg protein (with 3-HP), respectively. We noticed that the KgsA activities measured with or without 3-HP faithfully reflected the gene dosage effect and were almost proportional to the gene copy number. When KgsA was expressed with glycerol dehydratase (DhaB) from a multi-copy plasmid, its crude cell activity was 2.5 U / mg protein, resulting in good 3-HP production. This indicates that when kgsA is integrated into the chromosome, the P mmsA promoter should be engineered to increase its gene expression by approximately 20-fold. Fortunately, under the control of the P mmsA promoter, the transcription of the homologous mmsA gene was approximately 100-fold higher than that of kgsA. Even when the copy number of the kgsA gene was very low (e.g., 1 or 2 copies), improving the expression of kgsA by altering the characteristics of the mmsA regulatory structure to enhance its transcription and / or translation efficiency was possible.

[0468] Example 12. Development of a tandem promoter system

[0469] Attempts to optimize gene expression levels in bacterial hosts have used rational and combined methods to change transcription and translation levels. When strong promoters and / or ribosome binding sites (RBS) are used, gene expression increases. A synthetic promoter library providing a range of expression levels has been developed. In bacteria such as Escherichia coli and Bacillus subtilis, vegetative and stationary phase promoters have also been found, which are stronger than another in response to a σ factor of RNA polymerase. Although the aforementioned regulatory modules can help to better control the expression of genes at a broad level, they cannot express genes horizontally under dynamic growth conditions. To address this problem, we have developed a new gene expression regulation module by creating a tandem promoter, which incorporates the 3-HP inducible promoter expression module described in the above embodiment. In nature, microorganisms use tandem promoters to maintain the concentration of certain molecules and regulate proteins during dynamic cell growth under different physiological conditions. We combine 3-HP or a small acid-inducible promoter with a series of constitutive or inducible promoters. The promoter is natural or synthetic. Here, we developed a library of 3-HP inducible promoters fused to another promoter that is natural / synthetic and / or can activate genes during exponential / stationary growth. In some cases, the tandem promoter system has at least one inducible promoter and at least one constitutive promoter.

[0470] Once they are bound by an activator protein (similar to LysR), the small acid inducible promoters are activated by 3-HP. After characterizing the activator proteins that regulate the 3-HP (or similar small acid) inducible promoter system, it has been determined that these promoters themselves are not suitable for recombinant protein expression because they express the target protein at low intensity. Therefore, by generating random promoter mutations, a small acid inducible promoter library showing a range of expression intensities was developed. The strength of each mutant promoter was analyzed by cloning the mutant promoter upstream of the reporter gene (green fluorescent protein (GFP)). In vivo analysis was performed by fluorescence measurement of strains carrying 3-HP inducible derivative mutant plasmids. In brief, four 10 bp sequence blocks were randomized starting from the -51 position of the predicted TSS (the beginning of the first repeat sequence) to the -9 region. Although mutations in the first 30 bp (i.e., from -51 to -21) did not result in any differences in fluorescence, mutations between -21 and -12 significantly increased the fluorescence level (up to about 6 times). Therefore, randomization of mutations near the -10 region significantly enhanced the promoter strength ( Figure 13A - 13B ). This result, together with the in silico predictions, strongly supports our predicted location of the promoter element of the 3-HP inducible promoter.

[0471] As described herein, self-inducible promoter systems are valuable for enzyme expression and regulation in biochemical and biofuel-producing strains because metabolic pathways can be enhanced by intermediate or end products of the pathway without the addition of any extra inducer. However, the types of self-inducible promoters are very limited in cells, and these promoters generally produce weak expression levels. Inducible systems can be modified (e.g., by modifying promoter elements or operator regions) to achieve better performance, but this must be done carefully to maintain the inducible nature of the promoter. We attempted to increase the transcriptional strength of inducible promoters while maintaining their inducible characteristics by using expression cassettes comprising tandem promoters.

[0472] We designed a tandem promoter expression cassette that combines an inducible promoter and a constitutive promoter. A tandem promoter expression cassette containing P mmsA and P hbdH-4 promoters that control the kgsA gene was cloned into the pUCPK’ plasmid, while the MmsR activator was produced by its native promoter in the chromosome. Figure 17A The tandem promoter expression cassette, and two mRNA transcripts produced by the promoters, are schematically depicted. By combining two promoters in tandem, as Figure 17A shown, two mRNA transcripts were transcribed, one from the P mmsA promoter and the other from the P hbdH-4 promoter, thus increasing the number of kgsA mRNA transcripts. Figure 17B showed that the tandem promoter system increased the mRNA expression level of kgsA by 1.75-fold compared to the native P mmsA promoter alone. Figure 17C showed that using the tandem promoter system led to a 3-fold increase in KgsA enzyme activity compared to the enzyme activity produced by the native P mmsA promoter alone.

[0473] When considering the translation efficiency of each promoter in the tandem construct, we found that one transcript unit from the P hbdH-4 promoter produced two units of protein / activity (a ratio of 1:2); while one unit of transcript form of the P mmsA promoter produced one unit of protein / activity (a ratio of 1:1) (see Figure 17A - 17D ). This result indicates that the 5' untranslated region (5'UTR) of the P mmsA promoter may lead to a higher translation level compared to the 5'UTR region of the P hbdH-4 promoter. Each transcript produced by the tandem promoter expression cassette inherits the UTR region of P hbdH-4 , compared to each promoter alone (P mmsA or PhbdH-4 )Compared with it, it can significantly improve the translation level, resulting in higher activity of KgsA. Therefore, relative to a single individual promoter alone, due to the P mmsA promoter contribution, the inducibility of the P hbdH-4 promoter is maintained (if slightly reduced), and due to the constitutive promoter, the basal expression level without inducer is increased, which is useful for expressing pathway enzymes at early time points.

[0474] Figure 55A Four different expression constructs constructed with tandem promoters are shown, and the tandem promoters include at least one 3-HP inducible promoter. These combine phpdH with the Pc1 or Pzwf promoter. Figure 55B It shows that the tandem promoters have different strengths based on DhaB activity.

[0475] Example 13. UTR Engineering

[0476] Based on the accessibility of ribosomes to these elements, the 5' untranslated region (5'-UTR) and the 5'-proximal coding sequence play important roles in controlling the translation initiation rate and mRNA stability. We created a mutant library in the 5'UTR and the 5'-proximal coding sequence of the PmAdH-4 tandem promoter expression cassette with the aim of increasing the translation of transcripts produced by genes placed under the control of this system. There are several factors that make mutagenesis of these regions challenging, including the total length of these regions and the ease with which translation efficiency may be reduced. RBS engineering is an easy-to-operate method for randomizing the RBS to regulate the affinity of ribosomes for their binding sites. However, this method only modifies 6-9 nucleotides in the RBS, and other important components of translation (5'-UTR or 5'-coding sequence) should be considered.

[0477] A computer tool, UTR Designer, has been developed to accurately predict the translation initiation level. This tool takes into account the folded structure of the 5' untranslated region (5'UTR) of the mRNA (35bp), the 5'-proximal coding sequence (25bp) of the structural gene, and the affinity between the ribosome and the Shine-Dargarno region to understand the translation control of the target gene, so that modifications can be designed to change the translation level. We used this tool to evaluate the combination of features for improving the transcription and translation of the PhbdH-4 tandem promoter expression cassette, including the constructs we generated with Opt-3 and Hyb-20 and MmsR chromosomal expression (which we call UTR-0). As Figure 19CAs shown, the UTR Designer tool is predicted to increase 12-fold, which is higher than the 1.5- to 2-fold improvement we observed when fully expressing this system under normal conditions (200 rpm, 37 °C, 25 mM 3-HP). (Data not shown). To elucidate the effect of UTR modification on translation initiation, the expression levels of relevant genes (in this case KgsA) were measured. The expression levels of proteins were measured on SDS-PAGE gels. We deliberately avoided adding the inducer to keep the expression at a low level so that the differences between various UTRs could be easily distinguished. This experiment only considered the role of translation initiation. Therefore, the basal expression without inducer should provide us with a better and more sufficient evaluation basis. Similarly, by using a low-speed shaker (150 rpm) to limit the growth conditions, the minimization of protein expression limitation was thus achieved.

[0478] As Figure 19A - 19C shown, UTR engineering successfully increased the levels of the target protein. Figure 19A It is shown that a wide range of translation levels were achieved in a library of mutations made to the 5' UTR and 5' proximal coding sequence of the PhbdH-4 tandem promoter expression cassette, and these were observed when the bacteria were grown under normal induction conditions (200 rpm, 37 °C, 25 mM 3-HP). We found that this efficacy could be significantly increased (by approximately 30-fold) compared to the native PmmsA promoter.

[0479] As determined by SDS-PAGE analysis (analyzed using BioRad Image Lab 2.2 software), the UTR-6 construct exhibited the highest fold change, with levels increased 12.5-fold and 30.8-fold respectively compared to UTR-0 and PmmsA. UTR-6 was selected for integration into the chromosomal location of mmsA in the recombinant Pseudomonas denitrificans strain (the mmsR activation gene system remained intact). Finally, as Figure 18B and Figure 18C shown, expression of the target protein from the chromosome was successfully observed at the expected 2-fold and 1.7-fold levels at the transcriptional level (mRNA) and translational level (enzyme activity), respectively. This result indicates that the UTR Designer is a promising tool that can facilitate a wide range of expression and even achieve the ideal level of the target gene with a small number of variants.

[0480] Example 14. Codon Optimization and Protein Hybridization

[0481] If the translation system does not function properly, then even with an effective transcription system, proteins may not be produced effectively. Therefore, optimizing transcriptional and translational control is commonly used to produce sufficient protein levels.

[0482] Codon bias is related to tRNA composition and controls the rate of translation elongation. Codon bias usually affects protein folding. It is generally recommended to use preferred codons when cloning heterologous genes, but this often leads to misfolded, inactivated proteins and even the formation of inclusion bodies. In such cases, the use of a fusion system (hybrid protein) should be considered to increase protein expression, prevent proteolytic degradation, improve solubility and support purification.

[0483] As described in the above examples, using the P mmsA expression system to drive gene expression results in different expression levels between the homologous mmsA gene and the heterologous kgsA gene. The P mmsA poor performance of the expression system in controlling kgsA may come from differences in the properties of the kgsA and mmsA genes (secondary structure, translation initiation ability, codon bias, solubility). We modified the kgsA gene to reduce these differences.

[0484] Codon optimization of the entire gene is a common way to increase translation. However, this is an expensive and time-consuming process. Therefore, this study focused on optimizing only the first ten codons of the heterologous gene to enhance translation initiation. We used the GenScript bioinformatics tool to generate a codon usage table of the highly expressed native mmsA gene, which shows the codon frequency levels of each amino acid in MmsA. We used this information to modify the first ten codons of KgsA (heterologous protein) according to the following three options: (i) using codons that show a higher frequency level in mmsA, (ii) using codons that show a medium frequency level, and (iii) using codons that show the same frequency level as the first ten codons of MmsA and removing the rare codons (frequency of 0%) of KgsA (Table 3).

[0485] We found that codon optimization according to Option 3 (Opt-3) could increase KgsA activity by 30% in the absence or presence of 3-HP. We found that by keeping the frequency of all the first 10 codons the same as that of the mmsA gene (Opt-3), the translation of kgsA was improved. In contrast, increasing the frequency of certain codons, such as the 1st, 4th, 7th, and 9th codons in Opt-1 and Opt-2 (Table 3), did not increase expression and even decreased slightly.

[0486] Table 3: Frequencies of the first 10 codons of wild-type kgsA and the first 10 codons of kgsA optimized based on codon usage of mmsA in Pseudomonas denitrificans.

[0487]

[0488] aUse codons showing a high frequency level.

[0489] b Use codons showing a medium frequency level.

[0490] c Use codons with the same frequency level as the first 10 codons of mmsA and remove rare codons.

[0491] The results were confirmed by producing the protein on SDS-PAGE ( Figure 21B ).

[0492] Fusion proteins are commonly used in heterologous gene expression to enhance protein production, reduce proteolytic degradation and improve folding and solubility. We fused the N-terminus of MmsA produced by the highly expressed mmsA gene in Pseudomonas denitrificans with the KgsA enzyme. We thought that by adding a part of the 5'-coding region of mmsA downstream of the P mmsA promoter, the translation initiation of the heterologous gene (kgsA) transcript could be made more efficient. Since we were concerned about what effect fusing a part of the MmsA protein to KgsA would have on KgsA activity, the length of MmsA was varied by 5, 10, 15 and 20 amino acids, corresponding to the Hyb-5, Hyb-10, Hyb-15 and Hyb-20 strains, respectively. The results showed that as the length of MmsA fused to MgA increased, KgsA activity increased, although this effect tended to level off once 15 amino acids of MmsA (Hyb-15) were used ( Figure 17A ). Compared with the control KgsA (no MmsA fusion), the production of the Hyb-15 fusion protein increased by about 3-fold in the presence of 3-HP. Further extending the length of MmsA to 20 amino acids (Hyb-20) did not improve the expression when the system was fully induced; however, the basal level of protein production in the Hyb-20 strain increased, and this result was confirmed by the band intensity of KgsA on SDS-PAGE ( Figure 17B ).

[0493] In addition to participating in translation initiation, the 5'-end of the target gene also plays an important role in controlling transcript levels by regulating mRNA concentration. Measure the stability of the kgsA transcript without and with the N-terminal fusion of the first 20 aa of MmsA (mmsA(20)kgsA) Figure 17C)。Check this expression by comparison. The half-lives of kgsA and mmsA(20)kgsA are 2.7 minutes and 5.4 minutes, respectively, indicating that the kgsA mRNA fused with mmsA is more stable than the wild-type kgsA mRNA. The half-life of the mmsA transcript was found to be approximately 6 minutes. Therefore, the increased stability of mmsA(20)kgsA may be due to the protective effect of the 5'-terminal coding portion (20 amino acids) of mmsA fused to the kgsA gene. Interestingly, among the genes we tested, the most stable transcript seems to belong to mmsR, with a half-life of 9.5 minutes( Figure 17D )。

[0494] Example 15. Production of Coenzyme B12

[0495] Coenzyme B12 is an essential cofactor for many enzymes, including glycerol dehydratase, methionine synthase, or methylmalonyl-CoA mutase. Glycerol dehydratase (or diol dehydratase) is involved in the conversion of glycerol to 3-hydroxypropanal (3-HPA) and is an essential enzyme for the production of 3-HP (or its salts) or 1,3-PDO from glycerol. 3-HPA can be further converted to other industrially important chemicals, such as 1,3-PDO or 3-HP (or its salts). To continuously produce these biochemicals from glycerol on a commercial scale, an uninterrupted supply of coenzyme B12 is necessary. Since coenzyme B12 is very expensive, we examined methods for producing 3-HP from glycerol using natural coenzyme B12 producers. It has been reported that several microorganisms can naturally produce coenzyme B12, including Enterobacteriaceae, including Klebsiella spp.; species of Streptococcus, including Streptococcus pneumoniae; species of Pseudomonas, including Pseudomonas denitrificans; species of Rhizhobium; species of Sinorhizobium, including Sinorhizobium meliloti; and species of Rhodobacter, including Rhodobacter capsulatus and Rhodobacter sphaeroides, etc.). When we tested some of these microorganisms as hosts for producing 3-HP from glycerol, the amount of coenzyme B12 produced was insufficient to synthesize 3-HP at a high titer (<30 g / L each).

[0496] It was noted that Pseudomonas denitrificans can Produce 3-HP (or its salt). However, when the strain was supplemented with a saturating amount of coenzyme B12, about 100 g / L of 3-HP could be produced. These results indicate that the coenzyme B12 naturally produced by this microorganism is not sufficient to support high-titer 3-HP production. After careful analysis and elimination of the regulation involved in coenzyme B12 synthesis (secondary structure in mRNA), a slight increase in its yield was found. The improvement of coenzyme B12 has been confirmed by various assay methods mentioned in the previous sections. When this recombinant strain was used as a host to produce 3-HP from glycerol, it showed more than twice the improvement in producing 3-HP from glycerol, which was 38.6 g / L.

[0497] In the next experiment, we examined the strain by supplementing a small amount of coenzyme B12 in the medium, and the results are as Figure 50 shown. We noticed that when a small amount of coenzyme B12 (10 mg / L) was added to the culture, the 3-HP yield increased significantly from 38.6 g / L to 100 g / L. This result indicates that the amount of coenzyme B12 produced by the modified strain (IR1-RS1) was improved, but it was not sufficient to support high-titer 3-HP production.

[0498] In addition, to improve the production of coenzyme B12, we need to enhance the activity of the enzymes involved in the coenzyme B12 synthesis pathway. This can be achieved by improving the enzymatic activity of coenzyme B12 to enhance its synthesis pathway. The enzymatic activity can be improved by enhancing its physical and kinetic properties or by improving its expression and increasing its amount. Since this pathway is related to many enzymes, initially we increased the amount of the enzyme and tried to enhance the synthesis of coenzyme B12. To increase the amount of this enzyme in the cell, the native promoter was replaced by the synthetically engineered promoter mentioned above.

[0499] In this study, we attempted to support the production of biochemicals from glycerol at a scale suitable for commercialization by improving the coenzyme B12 production of microorganisms. To achieve this goal, we examined the cobalamin gene cluster in natural coenzyme B12 microbial producers such as Pseudomonas denitrificans to understand the regulation of various coenzyme B12-riboswitches (B12-riboswitches) on transcription and translation. Initially, we used software to identify five secondary structures that could be regulated by coenzyme B12 (see Figure 23 ). We determined that four of these structures were riboswitches. In vitro characterization showed that only three of these riboswitches were transcriptionally regulated, and they were located upstream of the cobG and cbtB genes ( Figure 23 ). These riboswitches are regulated by coenzyme B12, so the addition of cobalamin B12 (at a concentration as low as ) resulted in a significant decrease in transcription / translation (see Figure 28 ).

[0500] In addition, we examined whether six uncharacterized genes (gst, xre, dahp, gntR, bgpM, cobA) located in cob gene clusters I and II were involved in the synthesis of coenzyme B12 (see Figure 23 ). We found that the bgpM gene product is essential for the biosynthesis of coenzyme B12. The cobA gene, which encodes uroporphyrinogen methyltransferase, is involved in an important branch point in the coenzyme B12 biosynthesis pathway. Therefore, we expected that the deletion of cobA would hinder the production of coenzyme B12. However, we found that the cobA deletion mutant could synthesize a large amount of coenzyme B12. When we analyzed the genome of Pseudomonas denitrificans, we noticed the existence of several CobA isozymes that might substitute for CobA activity.

[0501] In the first part of this study, we described the methods for identifying B12-riboswitches and their regulatory roles in transcription / translation involved in coenzyme B12 synthesis. We also studied the method of enhancing coenzyme B12 production by regulating the riboswitch. We further improved the production of coenzyme B12 by replacing the native promoter with the synthetic expression module (tandem promoter, UTR, regulatory protein, N-terminal region of the highly expressed gene) described in the above examples. These modified strains had enhanced coenzyme B12 production, which contributed to an increase in the production of 3-HP (or its salt) from glycerol without the external addition of coenzyme B12.

[0502] The strain with the modified promoter showed good coenzyme B12 production. Analysis showed that this strain could produce 4 times more coenzyme B12 than the wild-type strain and 2 times more than the riboswitch-modified strain. When this strain was transferred with a plasmid containing the 3-HP synthesis pathway enzymes, it showed a significant increase in 3-HP production without the addition of coenzyme B12. This strain produced 3-HP from glycerol at a level higher than 90 g / L, but it grew slowly compared to the wild-type strain, which might be due to the modification in the coenzyme B12 synthesis pathway. In this study, we successfully developed a mutant strain that could produce a higher amount of coenzyme B12 and could support a more efficient production of 3-HP.

[0503] Example 16. In silico analysis and comparison of B12 genes in various Pseudomonas species

[0504] To understand the gene arrangement and its regulation, we compared the coenzyme B12 gene clusters present in various Pseudomonas strains. So far, up to 31 genes have been identified in the coenzyme B12 biosynthesis gene cluster involved in the aerobic coenzyme B12 biosynthesis pathway. Among them, the coenzyme B12 biosynthesis pathway of Pseudomonas denitrificans has been studied at the enzyme level. Based on the comparative analysis of the organization of coenzyme B12 genes in various Pseudomonas species (Figure 24 ), it was found that the genes in Pseudomonas denitrificans ATCC 13867 and Pseudomonas aeruginosa PAO1 strains were highly similar, with an average sequence identity of 73%. Interestingly, these strains also contained four other uncharacterized genes in the coenzyme B12 gene cluster: gst, xre, dahp, and bgpM. Different from other Pseudomonas strains, the gene encoding magnesium chelatase (chlID) was separately located together with the xre gene in these two strains. Additionally, the genes and gene organization in Pseudomonas entomophila, Pseudomonas putida, and Pseudomonas fluorescens were similar to those in Pseudomonas denitrificans. Furthermore, these strains lacked the ton-dependent B12 transporter (btuB), which is known to encode the coenzyme B12 transport system. Similarly, the cobGHIJ operon in these microorganisms was not controlled by the B12-dependent riboswitch structure( Figure 24 ).

[0505] Nitrogen-fixing bacteria (such as Ensifer / Rhizobium species) have also been reported to have high B12 production capabilities. Interestingly, a comparison of the gene organization between Pseudomonas denitrificans SC510 (and industrial B12 producers), Pseudomonas denitrificans ATCC 13867, and Ensifer meliloti revealed that Pseudomonas denitrificans SC510 and Ensifer meliloti were more closely related and very different from the Pseudomonas denitrificans ATCC13867 strain. Since there was no complete genome sequence of Pseudomonas denitrificans SC510, these differences could not be further studied.

[0506] In addition to understanding the gene organization in Pseudomonas denitrificans, we used the Rfam database / tools to identify the presence of four potential B12-riboswitches. A representative of these riboswitch structures is shown in Figure 25A , and it is believed to control the expression of cobalamin biosynthesis. Each of these B12-riboswitches has a cobalamin-binding consensus domain (regulatory domain and receptor domain), similar to the one present in Escherichia coli and similar to the btuB B12-riboswitch in Salmonella enterica. One riboswitch (RS1) is located between two separately located cobGHIJ and cobLFK operons, which encode the enzymes responsible for forming hydrogenobyrinate from the precorrin-2 intermediate (except for the cobM gene)( Figure 25A ; see Figure 23 ). As Figure 23As shown, based on the importance of the gene at this locus, we hypothesized that RS1 exerted strict control over the intergenic region 1 (IR1) in the flux diversion between coenzyme B12 and siroheme biosynthesis nodes. Two B12-specific riboswitch structures (RS2 and RS3) located between the operons cobWN and cbtBA-cobEM seemed to independently regulate the expression of these two operons. Finally, RS4 was located upstream of the cob operon (btuB-cobOBRDCQUP-bgpM-cobV), which contained 11 genes encoding enzymes responsible for the formation of Ado-Cbl from hydrogenobyrinate.

[0507] We investigated the mechanism by which these riboswitch structures control the expression of cob operon genes. Briefly, the interaction between the conserved regions J6 / 3 and J11 / 10 of the riboswitch confers specificity to each riboswitch and enables it to bind coenzyme B12 with the adenosine moiety, while the interaction between the loops L5 and L13 of the riboswitch alters the regulatory state of the riboswitch (see Figure 25A - 25D ).

[0508] Example 17. Characterization of coenzyme B12-sensing riboswitch structures and their promoter systems

[0509] Riboswitch structures regulate gene expression at the level of transcriptional elongation or translation initiation. The position of the riboswitch within the intergenic region determines the mode of regulation of these riboswitch structures. We noted that most of the B12 riboswitch structures in Pseudomonas denitrificans overlapped with promoter elements rather than with the untranslated region (UTR), indicating that these riboswitches might be controlled at the transcriptional level. Therefore, we used real-time PCR to analyze the mRNA levels of selected genes (the first gene in each operon) in the cob operon in Pseudomonas denitrificans cultures harvested in the late exponential phase and grown in the presence or absence of coenzyme B12. As expected, in the presence of coenzyme B12, the mRNA levels of four genes (cobG, cobW, cbtB, and btuB) in the cob operon were significantly inhibited ( Figure 26)。The transcription of the cbtBA-cobEM operon under RS3 control was highly repressed, 43.3-fold. We note that most genes in this operon do not encode structural genes of the B12 biosynthetic pathway (cobE and cbtBA serve as putative chaperone protein and cobalt transporter, respectively). Notably, in the absence of B12 (unrepressed form), the transcription levels of the structural genes encoding the operon (cobGHIJ, cobWN, and btuB-cobOBRDCQUP-bgpM-cobV) were at least 3.3-fold lower than those of the cbtBA-cobEM operon. From in silico analysis of the intergenic regions, it was not clear whether RS1 controls the expression of the cobGHIJ / cobLFK operons or both. However, based on mRNA levels, it appears that RS1 controls the expression of the cobGHIJ operon but not the cobLFK operon. We observed a decreasing order of transcriptional repression of various B12-RSs in the cob gene cluster as follows: cbtB-RS3 > cobG-RS1 > cobW-RS2 > btuB-RS4. No repression was found in the cobLFK, chlID-xre, and dahp operons, and these results are consistent with the fact that they lack riboswitch structures upstream of these operons.

[0510] We suspect that these riboswitches may be controlled at the translation initiation level in addition to their regulation at the transcriptional level. Furthermore, we attempted to predict the regulatory strength of these riboswitches by cloning the intergenic region between the promoter and the enzyme-coding region (upstream) of the cob operon into a plasmid. For these plasmids, we added green fluorescent protein as a reporter protein (fluorescent label) downstream of the secondary structure positions selected from the cob operon. Two versions of these plasmid systems were constructed. In one version, the first 35 bp of the corresponding native protein-coding sequence was fused to the gene encoding GFP in the plasmid, and in the other version, this fusion was absent. The fused form of cobG (the first 35 bp of cobG fused to gfp) showed 4.2-fold fluorescence intensity compared to the unfused gfp construct ( Figure 27 , Table 6). However, when the first 35 bp of cobG was fused to gfp, we observed no significant difference in the fluorescence levels produced by other promoter systems (intergenic fragments from other B12 operons), indicating that they are not controlled at the translation initiation level. These results further suggest that PcobG-RS1 strictly controls gene expression at both the transcriptional and translation initiation levels. The strict regulation of transcription and translation indicates the importance of the controlled expression of genes in the cobGHIJ operon.

[0511] When overexpressing any pathway involving several genes (such as the vitamin B12 pathway or a pathway involving several membrane proteins), it is crucial to estimate the strength of the native promoter. Overexpression of pathways with many genes can be harmful to cell growth, and unbalanced expression of pathway genes / operons can lead to the accumulation of toxic pathway intermediates. Analysis and quantification of native promoter strength can help us redesign the operon with synthetic promoters without overexpressing pathway enzymes during engineering. Table 5 shows the estimated promoter strengths (relative to fluorescence units) of various native cob promoters that we observed in the operon. Among this group, the promoter PcbtB that controls the expression of the cobalt transporter gene has the highest strength, while the PcobL promoter has the lowest strength. Genes encoding methyltransferases are usually composed of weak promoter systems.

[0512] Table 6: Characterization of the intergenic regions of cob regulon genes in Pseudomonas denitrificans.

[0513]

[0514] Engineering the coenzyme B12 production pathway

[0515] Rational engineering of the B12 promoter requires quantification of promoter strength and its UTR structure (fused and unfused versions). Therefore, the native promoter strength of the Cob operon encoding coenzyme B12 synthesis pathway enzymes was precisely quantified ( Figure 27 , Tables 6 & 10). In addition, to increase the expression levels of these (cob) genes (3-fold and 5-fold increase relative to their native expression). The expression of the cob operon was modified by replacing it with a suitable constitutive promoter ( Figure 57 ). Constitutive promoters were selected by screening and identifying the strengths of several native Pseudomonas promoters (Table 10). For example, to develop the Pedd-IR13-PsucA recombinant strain with a three-fold increased promoter strength, the constitutive promoters Pedd and PsucA were used to replace PcobG and PcobL respectively (for more information, see Table 8).

[0516] Table 10. B12 promoter replacement strategy

[0517]

[0518] Example 18. Development of a riboswitch-based B12 sensing system

[0519] Vitamin B12 quantification methods generally include microbiological assays and high-performance liquid chromatography (HPLC) with UV detection. The HPLC method can detect high concentrations (above the μM level) of B12, but microbiological assays can detect very low concentrations of B12 (down to the nM level). The microbiological assay is based on B12 acting as a growth factor in certain mutant strains of Salmonella typhimurium metE-cbiB-. Briefly, the growth of these microorganisms is proportional to the concentration of B12. However, B12 quantification based on cell growth is affected by various physiological factors, such as the culture medium and incubation time. Therefore, coenzyme B12 quantification based on cell growth is cumbersome and difficult to replicate.

[0520] In this study, we found that among five putative B12 riboswitch structures, two riboswitch structures found after promoters PcobG' and PcbtB could show differential expression of downstream genes in the presence and absence of coenzyme B12. In addition, the kinetic range of these riboswitches was evaluated using various concentrations of coenzyme B12 (up to 100 nM) to verify their potential as novel B12 sensors. It was noted that there was a linear correlation between B12 concentration and GFP fluorescence, with a breakpoint of approximately 5 nM cobalamin B12( Figure 28 ). In the presence and absence of B12, the differential expression of B12 riboswitches PcobG’ and PcbtB was approximately 2.1- and 1.7-fold. Compared with growth-based B12 detection, the fluorescence-based B12 detection method is reproducible, convenient, and labor-saving.

[0521] Example 19. Coenzyme B12 quantification bioassay:

[0522] Pseudomonas denitrificans was grown in M9 minimal medium with an initial OD600 of 0.1, which contained 1 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4, 10 g / L sodium gluconate, and was supplemented with 0.1 mM 5,6-dimethylbenzimidazole (DMB), 1 g / L betaine, and trace elements H3BO3 0.232 g / L, ZnSO4·7H2O 0.174 g / L, Fe(NH4)2SO4·6H2O 0.116 g / L, CoCl2·6H2O 0.025 g / L, (NH4)6Mo7O 24 ·4H2O 0.022 g / L and CuSO4·5H2O 0.008 g / L, MnSO4·4H2O 0.008 g / L. Cells were harvested in the exponential phase and passed through FastPrep-24 TMLysis was performed using a 5G Homogenizer system (MP Biomedicals, Korea). The cell lysate was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was collected and sterilized through a 0.2 μm membrane filter. The obtained lysate solution was used for bioassays. Coenzyme B12 was quantified by using a Salmonella typhimurium metE-cbiB- mutant strain. Briefly, in Salmonella typhimurium, metE encodes methionine synthase that is independent of B12, while cbiB encodes adenosylcobalamin phosphate synthase, an essential enzyme in the B12 biosynthetic pathway. Mutations in these genes render Salmonella typhimurium auxotrophic for B12 or methionine. The indicator strain Salmonella typhimurium metE-cbiB- was pre-cultured in minimal medium containing 0.5 g / L NaCl; 6 g / L Na2HPO4; 3 g / L KH2PO4; 1 g / L NH4Cl; 4 g / L glucose; 2 mM MgSO4; 0.1 mM CaCl2, and supplemented with 50 mg / L methionine. The overnight culture was centrifuged and washed with water. In minimal medium, the washed cells were inoculated to an initial OD600 of 0.01. The cell lysate required for measuring B12 was added to the culture medium. The growth of the indicator strain reflected the concentration of coenzyme B12 in the sample. The concentration of coenzyme B12 was varied to form a standard curve. The dynamic range of B12 concentration was 0 to 100 pM B12. The concentration of B12 was expressed as nM per 1 OD600.

[0523] Example 20. Identification of bgpM as an essential gene for B12 synthesis

[0524] To comprehensively understand the essentiality of genes in the cob biosynthetic operon, the necessity of several uncharacterized genes (such as gnt, gst, xre, dahp, and bgpM) in the cob gene cluster for B12 biosynthesis was investigated. To elucidate whether these genes are essential for B12 biosynthesis, single knockout mutants of each gene were created. Using the B12-specific riboswitch sensor system described in the above example and the conventional Salmonella typhimurium metE-cbiB--based B12 assay system (see Materials), the B12 biosynthetic ability of these mutant strains was investigated. Since the biosynthesis of coenzyme B12 involves cobalt in its metal center, its biosynthesis depends on the concentration of CoCl2. When these mutant strains were cultured with increasing concentrations of CoCl2 (up to 50 μg L-1), the fluorescence of GFP decreased proportionally, except for the bgpM mutant strain ( Figure 29A)。These results indicate that, except for bgpM, each of these mutant strains can synthesize coenzyme B12 similar to that of the wild-type strain. The gene product of bgpM plays a crucial role in the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. However, its exact role in B12 biosynthesis remains to be elucidated. An essentiality analysis was also performed using Salmonella typhimurium as an indicator strain. Quantify the intracellular concentration of coenzyme B12 synthesized by these mutant strains ( Figure 29B ). Similar to the results in Pseudomonas denitrificans, only the bgpM mutant was unable to synthesize (or only synthesized trace amounts of) coenzyme B12 (about 7-fold lower than the wild-type strain). Based on these results, the genes located in IR2 (see Figure 23 ) are not essential for B12 biosynthesis. Therefore, overexpression of these genes may not effectively enhance the production of coenzyme B12. Unexpectedly, the cobA deletion mutant (encoding uroporphyrinogen methyltransferase), which is an important branch point in the B12 biosynthetic pathway, can still synthesize coenzyme B12 ( Figure 29B ). After further analysis, we identified the existence of several CobA isoenzymes in the Pseudomonas denitrificans genome, which may replace the role of CobA in Pseudomonas denitrificans.

[0525] Example 21. Improvement of coenzyme B12 production by deleting riboswitch structures

[0526] To increase the production of B12, individual deletions were made of the identified negative regulatory domains (P5-L5 region) in all riboswitch structures. The mRNA levels produced by mutants with these riboswitch deletions were quantified in the presence and absence of coenzyme B12 (Table 7). In the presence of coenzyme B12 at greater than 5 nM, cob operon transcription can be inhibited due to riboswitches in the Pseudomonas denitrificans strain, while in the riboswitch mutant strain, the presence of coenzyme B12 can lead to the production of more coenzyme B12. Therefore, by measuring the amount of B12 synthesized by these mutant strains, it will be easier to evaluate the enhancement of coenzyme B12 biosynthesis genes due to riboswitch mutations. Interestingly, there was no significant difference in the production of coenzyme B12 between the RS1 (riboswitch 1 deletion) and RS2 / RS3 (riboswitch 2 and 3 deletion) mutants compared to the wild-type strain. However, there was a significant difference in coenzyme B12 in the RS4 (riboswitch 4 deletion) mutant compared to the wild-type. Coenzyme B12 production was substantially inhibited in the RS4 strain. The above results indicate that deletions in RS1 and RS2 / RS3 do not affect coenzyme B12 production, while the RS4 deletion substantially reduces coenzyme B12 production. One possibility is that the regions in riboswitches 1, 2, and 3 randomly selected for mutation were not sufficient to eliminate function, so we did not find any changes in coenzyme B12 production in the RS1 and RS2 / RS3 mutant strains. On the other hand, since the riboswitch overlaps with the coding region, mutations in RS4 may affect protein expression. To overcome this limitation, instead of developing deletion mutants, we modified the riboswitch region where the secondary structure in the mRNA was mutationally neutralized and at the same time the protein sequence was maintained by codon optimization.

[0527] Table 7: Transcription levels of B12 synthesis genes in the presence and absence of 25 mg / L CoCl2.

[0528]

[0529] We designed sequences using software, chemically synthesized nucleic acids and replaced them in the genome to avoid the formation of secondary structures in cobmRNA (encoding enzymes of the coenzyme B12 synthesis pathway). By varying each riboswitch or combination of riboswitches, we developed seven recombinant strains. Interestingly, we noticed that when riboswitch 1 was deleted (ΔRS1 strain), a slight decline in cell growth and coenzyme B12 production was noticeable. This may be due to the accumulation of certain toxic intermediates. However, deleting riboswitches 2 and 3 (ΔRS2 / ΔRS3) or riboswitch 4 (ΔRS4) did not improve coenzyme B12 production. This may be due to the limitation of carbon flux in the upstream pathway. However, when riboswitch 1 was deleted via riboswitches 2 and 3 (ΔRS1ΔRS2 / RS3) or via riboswitch 4 (ΔRS1ΔRS4), the production of coenzyme B12 increased slightly. Moreover, the mutant strain lacking all riboswitches showed relatively high coenzyme B12 production (Table 8).

[0530] This study demonstrated that the ability of Pseudomonas denitrificans to produce coenzyme B12 could be improved by removing the secondary structure of cob mRNA. When the mutant strain with improved coenzyme B12 production was used as a host to produce 3-HP (or its salt) from glycerol, it produced 3HP < 38.6 g / L (Table 8). The titer of 3-HP produced by this mutant strain was slightly higher than that of the wild-type strain, which produced < 30 g / L. The improvement in 3-HP production from glycerol by the mutant strain compared to the wild-type strain could be attributed to the improvement of the coenzyme B12 pathway. When 10 mg / L of coenzyme B12 was externally supplemented in the culture medium for this mutant strain, the strain could produce 3-HP from glycerol at the level of ( Figure 50 ). These experiments clearly showed that this mutant strain had increased coenzyme B12 production. However, the strain could be further modified to more significantly improve the production of 3-HP with a higher titer.

[0531] To further improve the production of coenzyme B12, the native promoter of the cob operon in the riboswitch mutant strain was replaced with the synthetic expression module (tandem promoter, UTR, regulatory protein, N-terminal region of the highly expressed gene) described in the above embodiments. Analysis of the cob gene cluster revealed that there are five promoters that control the gene expression in the B12 biosynthetic pathway. Several recombinant strains were developed by replacing the native promoter alone or in combination, as shown in Table 8. The promoters selected for promoter replacement are shown in Table 10. Replacement of each promoter showed a slight improvement in the production of coenzyme B12. However, when all the promoters were replaced with the synthetic gene expression module, a substantial improvement in coenzyme B12 was found. When this strain (PhpdH-Pedd-IR15-PsucA-PhpdH:PhpdH-Ptkt-IR45-Psp2-PhpdH) was used as a host to produce 3-HP from glycerol in a bioreactor, the strain was able to produce approximately 100 g / L of 3-HP, similar to the amount of 3-HP produced when coenzyme B12 (up to 50 mg / L) was externally supplemented to wild-type Pseudomonas denitrificans in a bioreactor to produce industrial amounts of 3-HP (see Figure 50 ). Therefore, we successfully developed a recombinant Pseudomonas denitrificans strain that can synthesize a larger amount of coenzyme B12, which in turn supports the commercial-scale production of 3-HP from crude glycerol without external supplementation of coenzyme B12. The production cost of 3-HP from glycerol was significantly reduced because nearly 38% of the production cost of 3-HP was attributed to the cost of externally supplying coenzyme B12 to the culture medium.

[0532] Table 8. Summary of the development and evaluation of various coenzyme B12 overproducing Pseudomonas denitrificans strains.

[0533]

[0534]

[0535]

[0536] Example 22. Development of aldehyde dehydrogenase expression cassette

[0537] Aldehyde dehydrogenase (ALDH) catalyzes the conversion of 3-HPA to 3-HP (the second reaction in the 3-HP synthesis pathway). To minimize the accumulation of 3-HPA, which is toxic, in the cells, the expression of ALDH (e.g., via the kgsA gene) should be maintained at a higher level than that of glycerol dehydratase (DhaB). Additionally, to integrate ALDH into the chromosome, its expression was increased to an even higher level (to track gene expression observed from the plasmid). For this purpose, an expression module / cassette containing the inducible tandem promoter system described in the above examples was used as the basal promoter to drive the gene expressing ALDH (e.g., kgsA), and was further developed. As described in the above examples, the strength of the ALDH expression cassette was altered / improved through various genetic modifications. In summary, the expression module / cassette has the following features: (i) tandem promoters, where one promoter is inducible and the other is constitutive or inducible, and these promoters can be native or synthetic to the host microorganism; (ii) mutation (randomization) of the binding (operator) site of the activator protein in the promoter region; (iii) alteration of the activator protein expression level; (iv) variable-length fusions, where a part of the N-terminus of a highly expressed native protein (e.g., MmsA) is fused to ALDH, and the first 10 codons of ALDH are codon-optimized (according to the codon usage of the host microorganism, e.g., Pseudomonas denitrificans); and (v) 5'-untranslated region (UTR) engineering (for a schematic diagram of the genetic modification of the expression system, see Figure 30A ; for the expression system for integrating the ALDH-expressing gene into the chromosome, see Figure 30B ).

[0538] Example 23. Development of the DhaB-GdrAB Expression Cassette

[0539] The dhaB and gdrAB genes were integrated into the chromosome of Pseudomonas denitrificans, and their expression levels were increased by enhancing transcription and translation efficiency. To avoid 3-HPA accumulation, the expression levels of dhaB and gdrAB should be lower than that of ALDH. To achieve this, a 3-HP inducible promoter with medium strength was used to drive the expression of the dhaB and gdrAB genes relative to the promoter used for generating ALDH. The expression cassette was designed as follows: (i) UTR engineering; (ii) use of tandem promoters, where one promoter is constitutive and the other is inducible; (iii) modification of the constitutive promoter; (iv) modification of the inducible promoter (-10 and -35 boxes) and the binding (operator) site of the activator protein HpdR; (v) variation in the activator protein expression level (for a schematic diagram of the genetic modification of the expression system, see Figure 31A ; for the expression system for integrating DhaB into the chromosome, see Figure 31B ).

[0540] Thus, the aldehyde dehydrogenase expression cassette described in Example 22 and the DhaB-gdrAB expression cassette described herein were used to produce 3-HP from glycerol by recombinant strains.

[0541] Example 24. Expression of ALDH and DhaB from plasmids in Pseudomonas denitrificans

[0542] Pseudomonas denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhIpUCPK’ was transformed with different plasmid combinations to express DhaB and KgsA. The plasmids carried different expression modules with different strengths. Thus, a series of promoters with different strengths driving the expression of DhaB and KgsA were tested in order to find such DhaB and KgsA expression modules that would together produce a large amount of 3-HP. To minimize 3-HPA accumulation while still allowing significant 3-HP production and cell growth, a lower-strength expression module was used for DhaB, GdrAB expression, while a higher-strength expression module was used for KgsA expression.

[0543] Among all the strains generated, one strain designated P1-1 showed a large amount of 3-HP production in bioreactor experiments (see Table 9). However, in this strain, we determined that the ALDH activity was relatively low and the toxic intermediate 3-HPA appeared early in fermentation. Using P1-1 as a reference strain (with reference expression levels of DhaB, GdrAB, and KgsA), we next attempted to reduce 3-HPA accumulation by increasing the plasmid expression level of ALDH. We developed a series of plasmids expressing higher ALDH levels using various gene expression cassettes described in the above examples. These plasmids were introduced into Pseudomonas denitrificans to generate strains P1-1x (x = a, b, c, d,...). Compared with the ALDH activity of 2.5 U / mg protein in the P1-1 strain, the ALDH activity in the crude cell extracts of the newly developed recombinant strains increased to 10 to 19 U / mg protein. The 3-HP production of the recombinant strains was evaluated at the bioreactor scale.

[0544] Table 9. Evaluation of Pseudomonas denitrificans strains expressing KgsA and DhaB from plasmids

[0545]

[0546] a Measured from flask experiments 4 h after induction with 25 mM 3-HP.

[0547] b From bioreactor experiments.

[0548] Example 25. Development of Pseudomonas denitrificans with chromosomally integrated and episomal DhaB expression modules with an ALDH expression module

[0549] Development of ALDH-integrated strains:

[0550] It is known that the integrated chromosomal location affects the expression level of the integrated gene, and careful selection should be made. Generally, due to the gene dosage effect, gene expression is higher when located closer to the origin of replication. First, the kgsA gene was integrated to replace the endogenous mmsA gene located approximately 3,000 Kb from the origin of replication. The mmsA gene expression unit (i.e., the intergenic region and mmsA) on the chromosome was replaced by the kgsA expression cassette (see Figure 30A - 30B ; the expression cassette has a tandem promoter (20 amino acids of mmsA fused to the N-terminus of kgsA) (hybrid protein); codon optimization of the first 10 codons of kgsA; and a modified UTR (UTR-6)) to create the first KgsA-integrated strain (named P2-0); for P2-0, wild-type kgsA was used.

[0551] By changing the integration location, more integrated strains (e.g., P2-0, P2-10, P2-20, and P2-30) can be developed (see Figure 32 ). For these strains, a fused kgsA gene (e.g., 20 amino acids of mmsA fused to the N-terminus of the kgsA gene) was used. Many strains with kgsA at different chromosomal positions were studied, such as far from the origin of replication These strains do not have a plasmid expressing DhaB. The strains were induced with 25 mM 3-HP for 4 hours, and KgsA activity was measured. Figure 32 It was shown that P2-10, in which the mutant kgsA is located closest to the origin of replication, produced the highest KgsA1 activity (24 U / mg). The P2-30 strain, in which the mutant kgsA was inserted at the same chromosomal position as the P2-0 strain (wild-type kgsA), produced more protein than the P2-0 strain (15 U / mg and 10.8 U / mg, respectively). The results showed that: (i) when tested after integration at the same chromosomal position, the activity of mutant KgsA1 was higher than that of KgsA, and (ii) ALDH activity varied significantly with its chromosomal integration position ( Figure 32 ).

[0552] Changing DhaB expression from a plasmid and KgsA expression from chromosomal integration:

[0553] The ratio of DhaB and ALDH can be altered by modulating the expression of episomal DhaB in the plasmid and / or the expression of chromosomally integrated ALDH. Only the recombinant P2-0 and P2-10 strains were modified with plasmids containing the DhaB expression cassette (dhaBgdrAB in the pUCPK backbone) (Table 11). The P2 strains described in Table 11 differed in the promoter system used to express the DhaB expression cassette. The promoters for P2-1 to P2-5 were as follows: Pc3, Pc3 with UTR design (i.e., the modified UTR as described herein), Pc3-Pc1 with UTR design, Pc1-Pc3 with UTR design, and Pc3-Pzwf with UTR design. The promoter strengths of these promoter systems varied in ascending order.

[0554] Table 11. Evaluation of Pseudomonas denitrificans strains with chromosomal ALDH and episomal DhaB

[0555]

[0556] a Measured from flask experiments 4 h after induction with 25 mM 3-HP.

[0557] b From bioreactor experiments.

[0558] Example 26. Development of recombinant Pseudomonas denitrificans with chromosomally integrated ALDH and DhaB

[0559] Optimizing the DhaB / ALDH ratio:

[0560] Recombinant Pseudomonas denitrificans strains with DhaB-GdrAB and KgsA expression cassettes integrated into the chromosome were developed. As described in the above examples, the ALDH activity varied with the chromosomal location of the kgsA gene insertion ( Figure 32 ), and we developed several ALDH integrants showing different activities (P2-10, P2-20, P2-30). We integrated the DhaB-GdrAB expression cassette into the P2-10 and P2-20 recombinant strains described in Example 25. The location of the chromosomal integration site of the DhaB-GdrAB expression cassette differed relative to the KgsA expression cassette in the recombinant strains. For example, by integrating the DhaB-GdrAB expression cassette (see Figure 31B ) into the chromosome of the host P2-20 (see Figure 32 and 37) to develop the P4-1 integrated strain. In the P4-1 strain, the DhaB-GdrAB cassette was integrated at a position approximately 4,150 kb from the replication origin. The P4-1 strain highly expressed DhaB and KgsA from the chromosome (Table 12). By altering the expression of KgsA, DhaB, or both KgsA and DhaB, other strains were developed to optimize the ratio between DhaB and KgsA activities. We also employed the P2-10 strain and integrated t...

Claims

1. An expression system comprising: A first promoter that can be induced by a small molecule, which comprises the PmmsA promoter, i.e., SEQ ID NO: 14, or the PhbdH-1 promoter, i.e., SEQ ID NO: 13, A constitutive or small molecule-induced second promoter, which comprises the PhbdH-4 promoter, i.e., SEQ ID NO: 11, or the PhpdH promoter, i.e., SEQ ID NO: 12, A first gene encoding a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP) or a salt of 3-HP, said first gene comprising at least one gene selected from dhaB1, dhaB2, dhaB3, gdrA, gdrB, and kgsA, A modified untranslated region (UTR) and a native sequence encoding 5-20 amino acids at the N-terminus of a protein from Pseudomonas denitrificans, Wherein: The first promoter comprises the PmmsA promoter, the second promoter comprises the PhbdH-4 promoter, and the first gene comprises kgsA; or the first promoter comprises the PhbdH-1 promoter, the second promoter comprises the PhpdH promoter, and the first gene comprises the dhaB1 gene, the dhaB2 gene, the dhaB3 gene, the gdrA gene, and the gdrB gene; The Pseudomonas denitrificans protein is MmsA; The native sequence is operably linked to the 3'-end of the modified UTR and operably linked to the 5'-end of the first gene; The first promoter and the second promoter are operably linked in series upstream of the modified UTR; and Wherein the modified UTR comprises a sequence selected from SEQ ID NO: 22-28.

2. The expression system according to claim 1, further comprising a third promoter and a second gene encoding a transcriptional regulator configured to regulate the expression of the first gene, wherein the third promoter is operably linked to the second gene, and wherein the third promoter comprises a sequence selected from SEQ ID NO: 7-10 and 52-63.

3. The expression system according to claim 1, wherein the first promoter is operably linked to the 5'-end of the second promoter, the second promoter is operably linked to the 5'-end of the modified UTR, and the modified UTR is operably linked to the 5'-end of the first gene.

4. The expression system according to claim 1, wherein the small molecule is selected from L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB), or a salt thereof.

5. The expression system according to claim 1, wherein the small molecule is selected from 1,3-propanediol (1,3-PDO) and 2,3-butanediol (2,3-BDO).

6. A nucleic acid comprising a first promoter containing the PmmsA promoter, i.e., SEQ ID NO: 14, or the PhbdH-1 promoter, i.e., SEQ ID NO: 13, and a second promoter containing the PhbdH-4 promoter, i.e., SEQ ID NO: 11, or the PhpdH promoter, SEQ ID NO: 12, wherein the first promoter is an inducible promoter and can be induced by a small molecule; the first promoter and the second promoter are operably linked in tandem upstream of a first gene; and the first gene encodes a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP) or a salt of 3-HP, and the first gene comprises at least one gene selected from dhaB1, dhaB2, dhaB3, gdrA, gdrB, and kgsA, wherein the first gene is fused at its 5'-end with a sequence of 5-20 amino acids encoding the 5'-end of a second gene encoding a protein derived from the native Pseudomonas denitrificans protein, thereby generating a fusion gene; the first promoter comprises the PmmsA promoter, the second promoter comprises the PhbdH-4 promoter, and the first gene comprises kgsA; or the first promoter comprises the PhbdH-1 promoter, the second promoter comprises the PhpdH promoter, and the first gene comprises the dhaB1 gene, the dhaB2 gene, the dhaB3 gene, the gdrA gene, and the gdrB gene; wherein the native Pseudomonas denitrificans protein is MmsA; and wherein the nucleic acid comprises a sequence selected from SEQ ID NO: 22-28.

7. The nucleic acid according to claim 6, wherein the first gene comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

8. The nucleic acid according to claim 6, wherein the small molecule is selected from L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB), or a salt thereof.

9. The nucleic acid according to claim 8, wherein the small molecule is selected from 1,3-propanediol (1,3-PDO) and 2,3-butanediol (2,3-BDO).

10. The nucleic acid according to claim 6, wherein the nucleic acid further comprises a gene encoding a transcriptional regulator that regulates the expression of the first gene, and the transcriptional regulator binds to the first promoter or the second promoter; optionally wherein the transcriptional regulator is a LysR-type transcriptional regulator (LTTR), an MmsR regulator, or an HpdR regulator.

11. The nucleic acid according to claim 6, wherein the first gene is fused at its 5'-end to a sequence of 5 to 20 amino acids at the 5'-end of a second gene encoding a protein derived from a native Pseudomonas denitrificans protein, thereby generating a fusion gene, and the second promoter is derived from the native promoter of the second gene, and the fusion gene comprises a sequence encoding at least 5 amino acids at the N-terminus of the native protein.

12. The nucleic acid according to claim 11, wherein the fusion gene comprises a sequence encoding at least 10 amino acids at the N-terminus of the native protein.

13. The nucleic acid according to claim 11, wherein the fusion gene comprises a sequence encoding at least 15 amino acids at the N-terminus of the native protein.

14. The nucleic acid according to claim 11, wherein the fusion gene comprises a sequence encoding at least 20 amino acids at the N-terminus of the native protein.

15. The nucleic acid according to claim 11, wherein, compared to the first gene alone, the mRNA of the fusion gene has higher stability and increased gene translation.

16. A nucleic acid comprising: a first promoter and a second promoter, wherein the first promoter is an inducible promoter capable of being induced by a small molecule and comprises the PmmsA promoter, i.e., SEQ ID NO: 14, or the PhbdH-1 promoter, i.e., SEQ ID NO: 13; the second promoter comprises the PhbdH-4 promoter, i.e., SEQ ID NO: 11, or the PhpdH promoter, i.e., SEQ ID NO: 12; and the first promoter and the second promoter are operably linked in tandem upstream of a first gene; the first gene encodes a protein involved in the synthesis of 3-hydroxypropionic acid (3-HP) or a salt of 3-HP, and the first gene comprises at least one gene selected from dhaB1, dhaB2, dhaB3, gdrA, gdrB, and kgsA, wherein at least 10 codons at the 5'-end of the first gene are optimized for translation in Pseudomonas denitrificans, and wherein codon optimization comprises: measuring the codon frequency of each amino acid in the gene encoding the native Pseudomonas denitrificans protein; replacing the 10 codons at the 5'-end of the first gene with 10 optimized codons using the codon frequency of the native protein, wherein the codons of each amino acid of the 10 optimized codons are present at the same frequency as the codon frequency of the native protein; wherein the gene encoding the native Pseudomonas denitrificans protein is mmsA, and wherein the nucleic acid comprises a sequence selected from SEQ ID NO: 29, 30, and 31.

17. The nucleic acid according to claim 16, wherein the small molecule is selected from L-lactic acid (LAC), acetic acid (AcOH), propionic acid (PA), 3-hydroxypropionic acid (3-HP), 3-hydroxybutyric acid (3-HB), L-valine (L-val), and 3-hydroxyisobutyric acid (3-HIB), or a salt thereof.

18. The nucleic acid according to claim 17, wherein the small molecule is selected from 1,3-propanediol (1,3-PDO) and 2,3-butanediol (2,3-BDO).

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