Production and isolation of 3-hydroxypropionic acid

Abstract

Methods and apparatus are provided for producing 3-hydroxypropionic acid or a salt thereof, for removing 3-hydroxypropionic acid from aqueous solutions (e.g., aqueous broths), and for using it to make various chemicals. The method includes evaporating a first solvent; condensing the evaporated first solvent; and redirecting the flow of the first solvent to remove 3-hydroxypropionic acid from the aqueous solution, wherein the 3-hydroxypropionic acid is removed from the aqueous solution without using countercurrent liquid flow.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is hereby incorporated by reference in its entirety under 35 U.S.C. §119(e). No. 62 / 577,361, filed October 26, 2017, and U.S. Provisional Application No. 62 / 577,361, filed December 26, 2017, which are incorporated herein by reference. This application claims the benefit of priority from Provisional Application No. 62 / 594,318, filed on the 4th.

[0002] (Technical field) The present disclosure provides methods for its production from glycerol by recombinant strains and in aqueous solutions (e.g., aqueous blue). production of 3-hydroxypropionic acid, including its removal from the soluble fraction (loss), as well as various chemicals and its use for producing a [Background technology]

[0003] (background) 3-Hydroxypropionic acid (3-HP) and 3-hydroxypropanoate (salts of 3-HP) are poly It can be used as a cross-linking agent for marking coatings, a metal lubricant, and an antistatic agent for textiles. It is used in the industrial production of various chemicals, such as acrylic acid. Summary of the Invention

[0004] (overview) The present disclosure provides a method for extracting 3-HP from an aqueous solution (e.g., an aqueous broth) to produce 3-HP or a salt thereof. The present invention provides a method and apparatus for removing and using the same to create various chemicals. Provide.

[0005] In some embodiments, the present disclosure provides a method for the production of Pseudomonas denitrificans (Pseudomonas denitrificans). A genetically engineered strain of Pseudomonas denitrificans was used to produce 3-HP or its salts from glycerol. Provide a way to create

[0006] In certain embodiments, the present disclosure provides a method for producing high potency 3-HP or its derivatives without exogenous supplementation of coenzyme B12. The present invention provides a method for producing a Pseudomonas strain capable of producing a salt of

[0007] In some embodiments, the present disclosure provides expression systems for use in Pseudomonas strains. Such an expression system (e.g., an expression module or an expression construct) is provided. The stem comprises, inter alia, two or more promoters operably linked to regulate the expression of downstream genes. Such promoters may include tandem promoter systems. The promoter can be an inducible promoter or a constitutive promoter. The present system includes at least one inducible promoter. metabolic pathways by producing beneficial proteins / enzymes or minimizing toxic intermediates to produce high titers of a target product, e.g., 3-HP or a salt thereof, and / or to synthesize 3-HP. It can increase the production of vitamin B12, a cofactor in the first reaction of the synthesis pathway. .

[0008] In some embodiments, the present disclosure provides nucleic acids encoding these systems, A recombinant bacterium expressing the system and / or the bacterium is cultured to produce 3-HP or a salt thereof. These bacteria produce a target product level, in this case 3-HP in the medium. or a salt thereof and used to produce the target product, e.g., 3-HP or a salt thereof. In some cases, the vector is modified to contain one or more expression systems that express the gene. Therefore, these bacteria have one or more gene expression pathways that increase the expression of genes used in the production of B12. The bacterium is also modified to contain the present system. The bacterium is cultured to detect 3-HP production (or a salt thereof). Conditions for augmentation are also provided herein.

[0009] In one aspect, the present disclosure provides a first promoter that is inducible by a small molecule, a second promoter that is inducible by a small molecule, and a a promoter encoding a protein involved in the synthesis of 3-HP (or a salt thereof) or B12; The first gene, modified UTR, and native Pseudomonas denitrificans tan containing or consisting of a naturally occurring sequence encoding up to 20 amino acids from the N-terminus of the protein an expression system comprising or consisting essentially of said modified native sequence, operably linked to the 3'-end of the UTR and operably linked to the 5'-end of the first gene and the first and second promoters function in tandem upstream of the modified UTR. An expression system is provided in which the vector is operably linked to the vector.

[0010] In some embodiments of all aspects, the expression system further comprises a third promoter. and a second gene encoding a transcription factor configured to regulate expression of the first gene. comprising, consisting of, or consisting essentially of two genes, wherein the third In some instances, the promoter of The second promoter is inducible by a small molecule. a native sequence operably linked to the 5'-end of the first gene to define a fusion gene; and the second promoter is a promoter encoding a naturally occurring Pseudomonas denitrificans protein. The second promoter is a natural promoter and functions at the 5'-end of the fusion gene. In some embodiments, the first promoter is operably linked to a second promoter. a second promoter operably linked to the 5'-end of the second promoter, the second promoter being a modified the modified UTR is operably linked to the 5'-end of the fusion gene, and the modified UTR is It is functionally linked to the end.

[0011] In one aspect, the disclosure provides a method for producing a nucleotide sequence comprising or consisting of a first and a second promoter. or consisting essentially of, wherein the first promoter is an inducible promoter. and inducible by a small molecule; wherein the first and second promoters are and the first gene is operably linked to a gene encoding 3-hydroxypropionic acid (3-HP) or a salt thereof) or a nucleic acid encoding a protein involved in the synthesis of coenzyme B12. .

[0012] In some embodiments of all aspects, the first gene is 3-hydroxypropiotropic acid. A gene encoding a protein involved in the synthesis of 3-HP (or its salts) and glycerol dehydrogenase (GDH) Glycerol dehydratase, glycerol dehydratase reactivase, and aldehyde dehydrogenase In some cases, the first gene is selected from the group consisting of dhaB1, dhaB2, dhaB3, dhaB4, dhaB5, dhaB6, dhaB7, dhaB8, dhaB9, dhaB10, dhaB11, dhaB12, dhaB13, dhaB14, dhaB15, dhaB16, dhaB17, dha , dhaB2, dhaB3, gdrA, gdrB, and kgsA. In some examples, the first gene includes a dhaB1 gene, a dhaB2 gene, a dhaB 3 genes, the gdrA gene, and the gdrB gene.

[0013] In some embodiments, the first gene is dhaB1 and the sequence is the sequence The present invention relates to a method for producing a compound comprising, consisting of, or derived from a sequence that is at least 95% identical to No. 1. In some cases, the first gene is dhaB2 and its sequence is , comprising, consisting of, or comprising a sequence that is at least 95% identical to SEQ ID NO:2 In some instances, the first gene is dhaB3, and the sequence thereof The sequence comprises, consists of, or is at least 95% identical to SEQ ID NO:3. Consists essentially of an array.

[0014] In some embodiments of all aspects, the first gene is gdrA, and its sequence The sequence comprises, consists of, or 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 is at least 95% identical to SEQ ID NO:5. consists essentially of said sequence.

[0015] In some embodiments of all aspects, the first gene encodes an aldehyde dehydrogenase The enzyme may comprise an aldehyde dehydrogenase gene, may consist of an aldehyde dehydrogenase gene, or may be an aldehyde dehydrogenase gene. In some instances, the aldehyde dehydrogenase gene In some cases, kgsA is a nucleotide sequence that is identical to SEQ ID NO: 6 and at least 9. 5% identical to a sequence comprising, consisting of, or consisting essentially of a sequence.

[0016] In some embodiments of all aspects, the small molecule is an acid or an alcohol. In some cases, the small molecule acid or alcohol is L-lactic acid (LAC), acetic acid (AcOH), propionyl alcohol (PHA), or acetic acid (ACOH). carboxylic acid (PA), 3-hydroxypropionic acid (3-HP), 3-hydroxybutyrate (3-HB), 1,3-propionic acid Pandiol (1,3-PDO), 2,3-butanediol (2,3-BDO), L-valine (L-val), and 3-hydroxybenzoates hydroxyisobutyrate (3-HIB), or salts thereof. In examples, the small molecule acid or alcohol is 3-hydroxypropionic acid (3-HP), 3-hydroxypropionic acid (3-HPO), It consists of 3-hydroxyisobutyrate (3-HB), L-valine (L-val), and 3-hydroxyisobutyrate (3-HIB). In some cases, the small molecule is selected from the group consisting of 3-hydroxypropionic acid (3 -HP) (or a salt thereof).

[0017] In some embodiments of all aspects, the nucleic acid is at least 85% identical to SEQ ID NO:65. In some cases, the sequence may comprise, consist of, or consist essentially of a sequence wherein the nucleic acid comprises, consists of, or is derived from SEQ ID NO:65. Become qualitative.

[0018] In some embodiments of all aspects, the second promoter is a promoter the first gene is operably linked to the 3'-end of the second promoter, In some embodiments of all aspects, the first promoter is functionally downstream of the first gene is operably linked to the 3'-end of the second promoter, and the first gene is It is functionally downstream of the second promoter.

[0019] In some embodiments of all aspects, the second promoter is a constitutive promoter. In some instances, the second promoter is a second inducible promoter. In some cases, the gene between the first promoter and the second promoter The intervening space does not include a terminator sequence. The first gene and the second promoter regulate expression of the first gene.

[0020] In some embodiments of all aspects, the first promoter is P mmsA Promoter - P hbdH-1 Promoter, P hbdH-4 Promoter, or P hpdH It comes from the promoter. In some instances, the first promoter is at least as similar as SEQ ID NO: 14 (PmmsA promoter). It comprises, consists of, or consists essentially of a sequence that is at least 95% identical to the sequence. In some cases, the first promoter is SEQ ID NO: 13 (PhbdH-1 promoter) comprising, consisting of, or consisting essentially of a sequence that is at least 95% identical to the sequence In some examples, the first promoter is SEQ ID NO: 11 (PhbdH-4 promoter). comprising, consisting of, or consisting essentially of a sequence that is at least 95% identical to In some cases, the first promoter is SEQ ID NO: 12 (PhpdH promoter). ) comprising, consisting of, or essentially consisting of a sequence that is at least 95% identical to In some examples, the first promoter is SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; and SEQ ID NO: 14; or It consists essentially of said sequence.

[0021] In some embodiments of all aspects, the first promoter is P mmsA Promoter - or P mmsA promoter or P mmsA consisting essentially of a promoter, and the nucleic acid is P mmsA An operator site operably linked to the 5'-end of the promoter is further provided. In some cases, the nucleic acids described herein are 95% identical to SEQ ID NO: 15. In some examples, the sequence may comprise, consist of, or consist essentially of a sequence In this context, the nucleic acids described herein are 95% identical to SEQ ID NO: 16 (O1 of the MmsA operator). A sequence comprising a sequence that is 95% identical to SEQ ID NO: 17 (O2 of the MmsA operator) or a sequence In some embodiments of all aspects, the sequence consists of or consists essentially of: The nucleic acids described herein are 95% identical to SEQ ID NO: 18 (PmmsA and operator, Figure 3). In some cases, the sequence may comprise, consist of, or consist essentially of a sequence The nucleic acids described herein are SEQ ID NOs: 15, 18, 19 (PmmsA2a), 20 (PmmsA2b), and 21 (PmmsA2ab), or It essentially becomes

[0022] In some embodiments of all aspects, the second promoter is P mmsA Promoter - P hbdH-1 Promoter, P hbdH-4 Promoter, P hpdH Promoter, or P zwf Promo In some cases, the second promoter is derived from SEQ ID NO: 11 (PhbdH -4 promoter) In some instances, the second promoter consists essentially of the sequence SEQ ID NO: 12 (Php dH promoter) or a sequence that is at least 95% identical to the sequence of the In some embodiments of all aspects, the second promoter consists essentially of the sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 14 (PmmsA promoter) or the sequence In some cases, the second promoter comprises or consists essentially of the sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 13 (PhbdH-1 promoter) or In some embodiments of all aspects, the sequence and the second promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 7 (Pzwf). In some cases, the second promoter comprises a sequence selected from the group consisting of, or consisting essentially of, the sequence. The promoter comprises or is at least 95% identical to SEQ ID NO: 8 (Pzwf-1). In some instances, the second promoter consists of, or consists essentially of, the sequence. , comprising or consisting of a sequence (Pzwf-7) that is at least 95% identical to SEQ ID NO: 9; In some embodiments of all aspects, the second promoter consists essentially of the sequence. The target comprises a sequence that is at least 95% identical to SEQ ID NO: 62 (shorter Pzwf-7) or In some cases, the second promoter comprises or consists essentially of the sequence comprises or consists of a sequence that is at least 95% identical to SEQ ID NO: 10 (Pzwf-12). In some instances, the second promoter comprises or consists essentially of the sequence Numbers 7, 8, 9, 10, 11, 12, 13, 14, 52-60, 61, 62, and 63 (e.g., Pzwf promoter) (a) a sequence selected from the group consisting of: become.

[0023] In some embodiments of all aspects, the first promoter is P mmsA Promoter - or P mmsA promoter or P mmsA consisting essentially of a promoter, and the second promoter is P hbdH-4 Contains a promoter or P hbdH-4 From the promoter or P hbdH-4 In some cases, the first promoter The promoter has a sequence that is at least 95% identical to SEQ ID NO: 14 (the PmmsA promoter sequence). the second promoter comprises, consists of, or consists essentially of the sequence, comprising or consisting of a sequence at least 95% identical to SEQ ID NO: 11 (PhbdH-4 promoter); In some examples, the first promoter consists of or consists essentially of the sequence , comprising, consisting of, or containing the PhbdH-1 promoter. and the second promoter comprises a PhpdH promoter or a Php The gene may consist of the dH promoter or consist essentially of the PhpdH promoter. wherein the first promoter is a sequence of SEQ ID NO: 13 (PhbdH-1 promoter sequence) and at least 9 5% identical to a sequence of the first amino acid sequence, or consisting essentially of the sequence of the first amino acid sequence, The second promoter has a sequence that is at least 95% identical to SEQ ID NO: 12 (PhpdH promoter). The nucleic acid sequence of the present invention may comprise, consist of, or consist essentially of the sequence:

[0024] In some embodiments of all aspects, the second promoter is a PhbdH promoter. comprising, consisting of, or consisting essentially of a PhbdH promoter, and the nucleic acid further comprises an operator region operably linked to the N-terminus of the PhbdH promoter. comprising, consisting of, or consisting essentially of an operator site In some cases, the operator site is selected from the group consisting of SEQ ID NOs: RBS-1, RBS-2, ABS-1, ABS-2, ABS-3, ABS-4, ABS-5, ABS-6, ABS-7, ABS-8, ABS-9, ABS-10, ABS-11, ABS-12, ABS-13, ABS-14, ABS-15, ABS-16, ABS-17, ABS-1 ABS-2, and one or more sequences that are at least 95% identical to ABS-3, or In some examples, the operator site consists essentially of the sequence SEQ ID NO: RB comprising, consisting of, or comprising a sequence that is at least 95% identical to the S-1 site and ABS-2; In some cases, the operator site consists essentially of the sequence RBS- The compound may comprise, consist of, or consist essentially of a nucleotide sequence of ABS-1 and ABS-2.

[0025] In some embodiments of all aspects, the nucleic acid further comprises a nucleic acid sequence that modulates expression of the first gene. The gene encoding the transcriptional regulator In some instances, the transcriptional regulator consists essentially of a first or second promoter. In some cases, the transcriptional regulator is a LysR-type transcriptional regulator (LTTR). In some embodiments, the transcriptional regulator is MmsR or HpdR. In this case, the transcriptional regulator is MmsR.

[0026] In some embodiments of all aspects, the transcriptional regulator binds to the first promoter. In some cases, the transcriptional regulator is derived from the mmsR protein and is primarily The promoter of this gene is derived from the PmmsA promoter. The nodal factor may comprise, consist of, or be the mmsR protein. and the first promoter comprises or is a PmmsA promoter. The PmmsA promoter may consist of, or consist essentially of, the PmmsA promoter.

[0027] In some embodiments of all aspects, the nucleic acids described herein further comprise an operable comprising an operator moiety, consisting of an operator moiety, or consisting essentially of an operator moiety where the MmsR protein binds to the operator site. The nucleic acids described herein are at least 95% identical to SEQ ID NO: 19 (PmmsA2a). In some instances, the sequence comprises, consists of, or consists essentially of the sequence. The nucleic acids described herein are selected from the group consisting of SEQ ID NOs: 19 to 21 (PmmsA2a, A2b, A2ab). In some cases, the sequence may comprise, consist of, or consist essentially of a sequence selected from the group consisting of: In this case, the transcriptional regulatory factor binds to the second promoter. In some embodiments, the second promoter is a constitutive promoter. In the present invention, the transcriptional regulator is the HpdR protein and the second promoter is the hpdH protein. In some instances, the second promoter is derived from the P promoter of SEQ ID NO: 12. h pdH comprising, consisting of, or consisting of a sequence that is at least 95% identical to the sequence of It essentially becomes

[0028] In some embodiments of all aspects, the transcriptional regulator binds to the first promoter. and has enhanced binding to the first promoter in the presence of a small molecule. In some cases, the transcription factor binds to the second promoter and reacts to the presence of a small molecule. In some instances, the gene has enhanced binding to the second promoter in the presence of Transcriptional regulators are autoregulatory. In some cases, the nuclear transcription factors described herein The gene further comprises a third promoter operably linked to the gene encoding the first transcriptional regulator. the third promoter, or the third promoter, or Become essential.

[0029] In some embodiments of all aspects, the third promoter is selected from the group consisting of SEQ ID NOS: 7-10 and and 52 to 63, and a sequence that is 95% identical to or consists of the sequence. In some cases, a second constitutive promoter is selected from the group consisting of SEQ ID NOs: 9, 10, 57-60, 62, and 63; the transcriptional regulator is MmsR comprising, consisting of, or consisting essentially of MmsR protein and the first promoter is P mmsA Contains or P mmsA or P mmsA From essentially In some examples, the second constitutive promoter is selected from the group consisting of SEQ ID NOs: 9, 10, 57-60, 6 2, and 63; the transcriptional regulator comprises or is selected from the group consisting of HpdR protein. the first promoter consists of, or consists essentially of, a protein; and the second promoter is hbdH Contains or P hbdH or P hbdH In some cases, the third promoter comprises, consists of, or is SEQ ID NO: 10; In some cases, the third promoter comprises SEQ ID NO: 62. or consisting of, or consisting essentially of, SEQ ID NO:62.

[0030] In some embodiments of all aspects, the first gene comprises a modified 5' end of the gene. a modified 5'UTR of the gene, or a modified 5'UTR of the gene In some examples, the first gene consists essentially of SEQ ID NOS: 22-28 and 64 (UTR 0 to 6), In some cases, the 5'UTR comprises SEQ ID NO: 28 (UTR-6) or SEQ ID NO: 28 (UTR-6), or consisting essentially of SEQ ID NO: 28 (UTR-6). Does the first gene comprise or consist of the gene encoding kgsA? or kgsA, and the 5'UTR comprises SEQ ID NO: 28 (UTR-6). Alternatively, it may consist of, or consist essentially of, SEQ ID NO: 28 (UTR-6).

[0031] In some embodiments of all aspects, at least 10 amino acids at the 5' end of the first gene Codons are optimized for translation in Pseudomonas denitrificans In some cases, up to 10 codons at the 5' end of the first gene are Pseudomonas In some cases, the translation in S. denitrificans has been optimized. , codon-optimized: Encoding native Pseudomonas denitrificans proteins Determining the codon frequency of each amino acid in the gene; The 10 codons at the 5'-end of the first gene were converted to 10 optimized codons using the nucleotide sequence frequency. and wherein: The codon for each amino acid of the ten optimized codons is the codon for the native protein. In some cases, the optimized second 10 codes are present at the same frequency as the The codon frequency of each amino acid in the codon is greater than 0. The frequency of genes encoding natural Pseudomonas denitrificans proteins is It is measured for each amino acid in the 10 codons at the 5' end.

[0032] In some embodiments of all aspects, the nucleic acid is SEQ ID NO: 29, 30, and 31 (Opt1-3 or consisting of a sequence that is 95% identical to a sequence selected from the group consisting of: In some cases, the first gene encodes kgsA. or consisting of the gene encoding kgsA, or consisting of the gene encoding kgsA. and comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 29 to 31. In some instances, the first gene comprises or consists essentially of the sequence. At the 5'-end, a second gene encoding a native Pseudomonas denitrificans protein It is fused to a sequence encoding up to 20 amino acids from the 5' end of the gene, In some cases, the mRNA of the fusion gene is expressed by the first In some instances, the fusion gene has greater stability than the mRNA of the gene alone. Increases gene translation when compared to one gene alone. In embodiments, the mRNA of the fusion gene is more resistant to ribonuclease activity than the mRNA of the first gene alone. In some cases, the fusion gene is the first gene only. Increases gene translation compared to

[0033] In some embodiments of all aspects, the first gene has at its 5'-end a naturally occurring Derived from the 5' end of a second gene encoding a Pseudomonas denitrificans protein fused to a sequence encoding up to 20 (5, 10, 15, or 20) amino acids a second promoter for the second gene, thereby creating a fusion gene; In some cases, the second promoter is derived from the native promoter of P m msA The fusion gene is derived from a promoter and contains at least the N-terminal sequence of the native MmsA protein. each comprising or consisting of a sequence encoding 5, 10, 15, or 20 amino acids; In some instances, the second promoter consists essentially of the sequence P mmsA professional Includes motor or P mmsA promoter or PmmsA Essentially from the promoter and the fusion gene encodes five or more amino acids from the N-terminus of the native MmsA gene. and the fusion gene comprises, consists of, or consists essentially of a sequence The P mmsA operably linked to the 3' end of the promoter. The acid comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 32 to 35; or It consists essentially of said sequence.

[0034] In some embodiments of all aspects, the first gene has at its 5'-end a naturally occurring Derived from the sequence encoding the N-terminus of the Pseudomonas denitrificans MmsA protein fused to a sequence encoding up to 20 amino acids (5, 10, 15, or 20) In some cases, the nucleic acids described herein are selected from the group consisting of SEQ ID NOS: 32-35 (Pcm-mmsA(5) (10)(15)(20)) It essentially consists of:

[0035] In some embodiments of all aspects, the first gene is involved in the synthesis of vitamin B12. In some cases, the first gene encodes a protein that contributes to the replication of a gene. , cobH, cobG, cobL, cobF, cobK, gst, xre, chlD, chlI, dahp, cobN, cobW, cbtBA, c obE, cobM, btuB, cobO, cob, cobR, cobD, cobC, cobQ, cobU, cobP, bgpM, and cobV? comprising, consisting of, or essentially consisting of a gene selected from the group consisting of: In some instances, the first gene comprises, consists of, or In some cases, the first gene is a P. denitrifcans gene. Pseudomonas aeruginosa (P. aeruginosa); P. entomophila (P. entomophila); P. putida (P. putida); P. schidigera P. syringae; P. fluorescens; P. mendocina and P. stutzeri; do.

[0036] In another aspect, the present disclosure provides a gene encoding a protein involved in the synthesis of coenzyme B12. a first promoter operably linked to a gene; In some cases, a nucleic acid is provided that consists of, or consists essentially of, said first promoter. In some examples, the first promoter is a constitutive promoter. The nucleic acid comprises or consists of a sequence selected from SEQ ID NOs: 7 to 10 and 52 to 63. In some embodiments of all aspects, the first gene The genes are cobJ, cobI, cobH, cobG, cobL, cobF, cobK, gst, xre, chlD, chlI, dahp, co bN, cobW, cbtBA, cobE, cobM, btuB, cobO, cob, cobR, cobD, cobC, cobQ, cobU, cobP , bgpM, and cobV, or In some cases, the first gene is a P. dendrobatidis gene. P. nitrifucans; P. aeruginosa; P. entomophila; P. putida; P. syringae; P. fluorescens Pseudomonas species selected from the group consisting of P. mendocina; P. stutzeri; and P. stutzeri. It is from the past.

[0037] In some embodiments of all aspects, the nucleic acids described herein further comprise a first or a second promoter operably linked to the promoter of In some cases, the second promoter may consist of, or consist essentially of, the second promoter. The first and second promoters regulate expression of the first gene. , the first promoter is not the native promoter of the first gene.

[0038] In another aspect, the present disclosure provides a cobG gene, P edd Promoter, P sucA promoter, and the cobL gene; edd The promoter is the cobG gene and P sucA To the promoter operably linked to said P sucA The promoter is operably linked to the cobL gene. In some cases, P edd The promoter is the sequence of SEQ ID NO: 36. comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO:36; or TsuP sucA The promoter comprises, consists of, or consists of the sequence of SEQ ID NO: 37. In some embodiments, the nucleic acid sequence described herein essentially consists of the sequence of SEQ ID NO: 37. The nucleic acid to be used comprises, consists of or is a sequence of SEQ ID NO: 38. The .DELTA..times ...

[0039] In one aspect, the present disclosure provides a cobG gene, P sp9 Promoter, P zwf promoter, and comprising, consisting of, or consisting essentially of a cobL gene; sp9 The promoter is the cobG gene and P zwf operably linked to a promoter, and zwf A nucleic acid is provided in which a promoter is operably linked to the cobL gene. In P sp9 The promoter comprises or consists of SEQ ID NO: 39 or the sequence Consisting essentially of number 39; and P zwf The promoter comprises SEQ ID NO: 40. In the present specification, the nucleic acid comprises, consists of, or is SEQ ID NO: 41. consists essentially of SEQ ID NO:41.

[0040] In one aspect, the present disclosure provides a cobW gene, P zwf Promoter, P sp9 promoter, and comprising, consisting of, or consisting essentially of the cbtB gene, wherein P zwf P The promoter is the cobW gene and P sp9 operably linked to a promoter, and sp9 P In some cases, a nucleic acid is provided in which a promoter is operably linked to the cbtB gene. wherein the Pzwf promoter comprises SEQ ID NO: 42; and the Psp9 promoter comprises SEQ ID NO: 43 In some examples, the nucleic acids described herein comprise or are SEQ ID NO: 44. consisting of or consisting essentially of SEQ ID NO:44.

[0041] In one aspect, the present disclosure provides a cobW gene, P tkt Promoter, P sp2 promoter, and comprising, consisting of, or consisting essentially of the cbtB gene, wherein P tkt P The promoter is the cobW gene and P sp2 operably linked to a promoter, and sp2 P In some embodiments, a nucleic acid is provided in which a promoter is operably linked to the cbtB gene. P tkt The promoter comprises, consists of, or is SEQ ID NO: 45. 45; and the Psp2 promoter comprises or consists of SEQ ID NO:46. In some cases, the sequences described herein may comprise or consist essentially of SEQ ID NO: 46. The nucleic acid comprises, consists of, or consists essentially of SEQ ID NO:47. do.

[0042] In one aspect, the present disclosure provides a P operably linked to a tonB gene (e.g., a butB gene). z wf containing a promoter or zwf or the P zwf From the promoter In some cases, the Pzwf promoter is provided as a nucleic acid consisting essentially of SEQ ID NO: 48, consisting of, or consisting essentially of SEQ ID NO: 48. In the present specification, the nucleic acid comprises, consists of, or is SEQ ID NO: 49. consists essentially of SEQ ID NO:49.

[0043] In one aspect, the present disclosure provides a P operably linked to a tonB gene (e.g., a butB gene). s p9 containing a promoter or sp9 or the P sp9 From the promoter In some cases, the Psp9 promoter is provided as a nucleic acid consisting essentially of SEQ ID NO: 50, consisting of, or consisting essentially of SEQ ID NO: 50. In the present specification, the nucleic acid comprises, consists of, or is SEQ ID NO:51. consists essentially of SEQ ID NO:51.

[0044] In some embodiments of all aspects, the nucleic acid is selected from the group consisting of Pseudomonas denitrifica comprising, consisting of, or consisting essentially of one or more sequences derived from In some cases, the first or second promoter is selected from the group consisting of Pseudomonas denitrificans and Pseudomonas dendritic cells. In some instances, the nucleic acid is derived from Enterobacter sp. ter), Lactobacillus, Pseudomonas, or Azos comprising or consisting of one or more sequences derived from bacteria of the genus Azospirillum; or consisting essentially of said sequence. In some cases, the first gene is It is derived from bacteria of the genus Lactobacillus, Lactobacillus, Pseudomonas, or Azospirillum.

[0045] In one aspect, the disclosure provides a DhaB expression module that is at least 85% identical to SEQ ID NO: 66 (DhaB expression module). DhaB Expression Modules Comprising, Consisting of, or Consisting Essentially of a Sequence In some cases, the sequence is at least 90% identical to SEQ ID NO: 66. % identical. In some examples, the nucleic acids described herein comprise SEQ ID NO: 66. or consisting of, or consisting essentially of, SEQ ID NO:66.

[0046] In one aspect, the present disclosure provides a nucleic acid sequence at least 85% identical to SEQ ID NO: 67 (kgsA expression module). DhaB Expression Modules Comprising, Consisting of, or Consisting Essentially of a Sequence In some cases, the sequence is at least 90% identical to SEQ ID NO: 67. In some examples, the sequence includes or is % identical to SEQ ID NO:67. or consisting essentially of SEQ ID NO:67.

[0047] In one aspect, the disclosure provides a nucleic acid sequence comprising, consisting of, or comprising SEQ ID NO:68. SEQ ID NO: 68 and comprising or consisting of SEQ ID NO: 69. or consisting of a UTR of the gdrB gene consisting essentially of SEQ ID NO: 69; Alternatively, nucleic acids containing a DhaB expression module consisting essentially of these UTRs are provided. In this case, the nucleic acid has at least one sequence selected from the group consisting of SEQ ID NOs: 70 to 72. 85% identical to a sequence comprising, consisting of, or consisting essentially of a sequence. In some instances, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 70-72, or It consists of or consists essentially of the sequence.

[0048] In one aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising: a fermenter comprising, consisting of, or consisting essentially of a sequence that is 85% identical to the sequence of In some cases, a nucleic acid comprising a nucleic acid sensor as described herein is provided. the nucleic acid comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 73 and 74; or consisting essentially of said sequence.

[0049] In one aspect, the present disclosure provides a first gene involved in the synthesis of coenzyme B12, The gene encoding the promoter comprises or consists of a first promoter induced by the promoter and a second promoter. or a coenzyme B12 expression module consisting essentially of the same. or consisting essentially of said coenzyme B12 expression module, wherein said first and a second promoter of the first gene so as to regulate the expression of the first gene. In some cases, a nucleic acid is provided that is operably linked in tandem to the upstream The nucleic acid is further induced by a second gene, a small molecule involved in the synthesis of coenzyme B12. a third promoter, and a fourth promoter, wherein the third and fourth promoters control expression of the second gene. The second gene is operably linked in tandem upstream of the first gene so as to regulate the second gene. In some similar embodiments, the nucleic acid further comprises a third gene involved in the synthesis of coenzyme B12. a gene, a fifth promoter induced by a small molecule, and a sixth promoter , consisting of, or consisting essentially of, wherein the fifth and sixth promoters The target functions in tandem upstream of the third gene to regulate the expression of the third gene. In some cases, the nucleic acid further comprises a nucleic acid functionally linked to a target molecule involved in the synthesis of coenzyme B12. a fourth gene that controls the expression of a gene encoding a nucleotide sequence, a seventh promoter that is induced by a small molecule, and an eighth promoter that is induced by a small molecule. comprising, consisting of, or consisting essentially of a seventh and an eighth promoter upstream of the fourth gene so as to regulate the expression of the fourth gene; In some examples, the first, second, third, and The fourth gene is cobJ, cobI, cobH, cobG, cobL, cobF, cobK, gst, xre, and chlD, respectively. , chlI, dahp, cobN, cobW, cbtBA, cobE, cobM, btuB, cobO, cob, cobR, cobD, cobC, one or more genes selected from the group consisting of cobQ, cobU, cobP, bgpM, and cobV; In some cases, the first The gene comprises, consists of, or consists essentially of bgpM.

[0050] In some embodiments of all aspects, the nucleic acid is a gene involved in the production of coenzyme B12. In some cases, the nucleic acid does not include any naturally occurring riboswitches that regulate the expression of the gene. Does not contain SEQ ID NO: 75 or 76.

[0051] In another aspect, the disclosure includes a nucleic acid described herein or a nucleic acid sequence described herein. and a recombinant bacterium comprising the nucleic acid described herein, or consisting essentially of the nucleic acid described herein. In some cases, the bacterium is a Pseudomonas species. The bacterium is Pseudomonas denitrificans.

[0052] In some embodiments of all aspects, the nucleic acid is on an expression plasmid. In some cases, the nucleic acid is integrated into the bacterial chromosome. The nucleic acid is on an episome.

[0053] In another aspect, the disclosure provides a method for producing a DhaB expression module comprising or comprising a DhaB expression module. a first nucleic acid consisting essentially of a DhaB expression module and an ALDH expression module; a second comprising, consisting of, or consisting essentially of an ALDH expression module; and providing a recombinant bacterium comprising, consisting of, or consisting essentially of the nucleic acid of In some cases, the DhaB expression module may be any of the nucleic acids described herein. or one of the nucleic acids described herein, or consisting of any one of the nucleic acids described herein. In some instances, the ALDH expression module is a nucleic acid sequence encoding the ALDH expression module. The vector may comprise any one of the nucleic acids described herein or may be a nucleic acid sequence described herein. or consisting essentially of any one of the nucleic acids described herein. do.

[0054] In some embodiments of all aspects, the first and second nucleic acids are on an expression plasmid. In some cases, the first and second nucleic acids are on an episome. In some instances, the first and second nucleic acids are integrated into the bacterial chromosome. In some cases, the first nucleic acid is integrated into the bacterial chromosome and the second nucleic acid is expressed. In some instances, the first nucleic acid is on an expression plasmid. and the second nucleic acid is integrated into the chromosome of the bacterium. In this manner, the first nucleic acid is integrated into the bacterial chromosome and the second nucleic acid is an epitope. In some instances, the first nucleic acid is on an episome and the second nucleic acid is on an episome. The nucleic acid is integrated into the bacterial chromosome.

[0055] In some embodiments of all aspects, the first nucleic acid is located in the bacterial chromosome at the first location. and a second nucleic acid is integrated into the bacterial chromosome at a second location. and the first and second positions are within 2500 kilobase pairs of each other. In some examples, 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. wherein the first and second positions are within 1000 base pairs of each other.

[0056] In some embodiments of all aspects, the first nucleic acid is within 4000 base pairs of the origin of replication. In some cases, the first The location of is within 1000 base pairs of the replication origin.

[0057] In another aspect, the present disclosure provides a first gene involved in the synthesis of coenzyme B12, a first promoter induced by a gene encoding ... or consisting essentially of, wherein the first and second promoters are a coenzyme B12 expression module operably linked in tandem upstream of the gene; consisting of or consisting essentially of said coenzyme B12 expression module. The present invention provides a recombinant bacterium that

[0058] In some embodiments of all aspects, the bacterium is Pseudomonas denitrifica In some cases, the bacterium is a first gene that regulates the expression of the first gene. comprising, consisting of, or consisting of a deletion at the first position of the riboswitch and the first and second promoters are configured to regulate expression of the first gene. In some instances, the bacterium is integrated into the chromosome at the first location. Additionally, a second riboswitch regulates the expression of a second gene involved in the synthesis of coenzyme B12. A second deletion at a second position in the gene, which regulates the expression of a third gene involved in the synthesis of coenzyme B12, A third deletion at the third position of the third riboswitch involved in the synthesis of coenzyme B12. A fourth deletion at a fourth position of a fourth riboswitch that regulates the expression of a fourth gene that In some cases, The recombinant bacterium further comprises a second gene operably linked in tandem to regulate expression of the second gene. a third promoter and a fourth promoter, which are integrated into the chromosome at a second location; a motor; operably linked in tandem to regulate expression of a third gene; and a fifth promoter and a sixth promoter integrated into the chromosome at position three; and a fourth gene, which is functionally linked in tandem to regulate the expression of the fourth gene. the seventh and eighth promoters integrated into the chromosome at positions In some embodiments, the promoter may consist of, or consist essentially of, a promoter. In some embodiments, the bacterium is more abundant than naturally occurring Pseudomonas denitrificans. In some embodiments of all aspects, the enzymes described herein produce a large amount of coenzyme B12. The recombinant bacterium further comprises a coenzyme B12 expression module or The coenzyme B12 expression module may consist of, or consist essentially of, a coenzyme B12 expression module.

[0059] In another aspect, the disclosure provides a recombinant bacterium described herein that is capable of expressing 3-HP or a salt thereof. comprising, consisting of, or consisting of culturing under conditions sufficient to produce In some cases, a method for producing 3-HP or a salt thereof is provided, the method consisting essentially of: The conditions are sufficient to produce at least 85 g / L of 3-HP or a salt thereof. In an example, 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 include at least 95 g / L of 3-HP or its In some cases, the conditions are sufficient to produce at least 100 g / L of a salt. It is sufficient to produce 3-HP or a salt thereof.

[0060] In some embodiments of all aspects, culturing the bacteria comprises culturing the bacteria with exogenous coenzyme B12. In some cases, culturing bacteria involves the addition of external fermentation agents to the culture. Includes the addition of vitamin B12.

[0061] In some cases, culturing involves culturing the bacteria in a medium containing glycerol, glucose, glutamic acid, or ethanol. in a medium containing a carbon source selected from the group consisting of conate, glutamate, and citrate; The method comprises, consists of, or consists essentially of propagating the cells in a medium containing the bacteria. In some instances, culturing involves inducing the bacteria by adding glycerol. and producing 3-HP or a salt thereof. In some cases, glycerol is added at mid-log phase.

[0062] In some embodiments of all aspects, the culturing comprises culturing the bacteria in a solution of yeast extract, co-culture medium, or the like. corn steep liquor powder, and corn steep liquor paste. The method comprises, consists of, or consists of growing the plant in a medium containing a nitrogen source that is It essentially consists of:

[0063] In some embodiments of all aspects, culturing comprises culturing the bacteria at a pH of 6.8 to 7.8. Involving, consisting of, or consisting essentially of multiplying. In some cases, culturing may involve the use of NaOH, KOH, NaHCO3, NH4HCO3, NH4OH, (NH4)2CO3, and Na2CO3. In some examples, the culturing comprises culturing a Na OH, KOH, NaHCO3, NH4HCO3, NH4OH, (NH4)2CO3, and Na2CO3 The present invention relates to a method for preparing a process for the preparation of a medicament ... In some cases, culturing can dilute the base concentration and comprising, consisting of, or consisting essentially of increasing the concentration See Figures 46 and 49).

[0064] In some embodiments of all aspects, culturing comprises culturing the bacteria at a temperature of 28 to 40 degrees Celsius. "Propagation" means the propagation of a plant or animal at a temperature of 100°C. In some cases, culturing involves growing the bacteria at 33 degrees Celsius or Consisting of or consisting essentially of

[0065] In another aspect, the disclosure provides a method for producing 3-HP or a salt thereof, comprising: The recombinant bacteria containing the 3-HP or a salt thereof are provided in a culture medium suitable for the growth of the bacteria and the production of 3-HP or a salt thereof. providing a method comprising, consisting of, or consisting essentially of providing In some cases, the methods described herein involve administering 3-HP or a salt thereof to the bacteria. or comprising, consisting of, or consisting essentially of, separating the culture medium from the Become a target.

[0066] In some instances, the culture medium contains a dissolved oxygen concentration that is about 2-20% dissolved oxygen. , consisting of, or consisting essentially of, the dissolved oxygen concentration. In some embodiments, the dissolved oxygen concentration is 20% or less. The dissolved oxygen concentration is at least 2%.

[0067] Additionally, the present disclosure relates to methods for providing and reacting 3-hydroxypropionic acid. do.

[0068] The present disclosure provides a method for removing 3-hydroxypropionic acid (3-HP) from an aqueous solution. The method can be relatively simple and inexpensive to carry out. The method provides a relatively high yield of 3-HP. can be provided.

[0069] The present disclosure also provides a method for producing acrylic acid. The method is relatively simple to carry out. The process can be carried out with little or no acrylic acid polymerization. The method can provide a relatively high yield of acrylic acid.

[0070] In one aspect, the present disclosure provides a method for removing 3-HP from an aqueous solution without using countercurrent liquid flow. This provides a way to

[0071] In another aspect, the present disclosure provides a method for preparing a liquid crystal display comprising: evaporating a first solvent; condensing and redirecting the flow of the first solvent to remove 3-HP from the aqueous solution. The present invention provides a method comprising:

[0072] In another aspect, the present disclosure provides a method for extracting 3-HP from an aqueous solution using a water-immiscible solvent. The method includes removing the hydroxybenzoate in a 50% yield.

[0073] In another aspect, the present disclosure provides a method for the preparation of 3-hydroxybenzoates using an organic liquid comprising at least two solvents. The present invention provides a method for removing 3-hydroxypropionic acid (3-HP) from an aqueous solution.

[0074] In another embodiment, the present disclosure includes removing 3-HP from an aqueous solution using a liquid, wherein the liquid comprises two different solvents (e.g., a water-miscible solvent and a water-immiscible solvent). , a method is provided.

[0075] In another embodiment, the present disclosure includes removing 3-HP from an aqueous solution, wherein the aqueous solution wherein the pH of the solution is at least about 3.

[0076] In another aspect, the present disclosure provides a method for producing acrylic acid by reacting 3-HP at a pressure of less than about 1 atmosphere. The method includes forming a

[0077] In another aspect, the present disclosure provides a process for the preparation of a compound comprising reacting 3-HP to form acrylic acid in a reaction mixture. and removing gaseous acrylic acid from the reaction mixture. .

[0078] In another aspect, the present disclosure provides a method for the preparation of a method for the preparation of a liquid comprising reacting 3-HP to form acrylic acid. The present invention provides a method comprising:

[0079] In another aspect, the present disclosure provides a method for producing acrylic acid esters by reacting 3-HP with acrylic acid esters in at least about 70% yield. forming an acid.

[0080] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present disclosure. The terms "methods and materials" have the same meaning as commonly understood by one of ordinary skill in the art. , as described herein for use in this disclosure; or other suitable methods known in the art. Methods and materials may also be used. The materials, methods, and examples are illustrative only. The disclosures of any publications, patent applications, patents, patent applications, patent notices, and related documents mentioned herein are not intended to be limiting. All sequences, database entries, and other references are incorporated by reference in their entireties. In the case of conflict, the present disclosure, including definitions, will control.

[0081] Other features and advantages of the present disclosure will become apparent from the following detailed description and drawings, and from the claims. It will become clear from this. [Brief explanation of the drawings]

[0082] DESCRIPTION OF THE DRAWINGS [Figure 1]Figures 1A-B are schematic diagrams and graphs showing gene expression regulation by the LysR-type transcriptional activator protein MmsR in the 3-hydroxypropionate degradation pathway of Pseudomonas denitrificans. (Figure 1A) Hypothetical schematic representation of MmsR-based regulation. hpdH, 3-hydroxypropionate dehydrogenase; mmsA, methylmalonate semialdehyde dehydrogenase; hbdH-4, 3-hydroxyisobutyrate dehydrogenase IV. (Figure 1B) Relative mRNA abundance of mmsA (gray bars) and hbdH-4 (black bars) in the presence of various inducer molecules. [Figure 2] Figures 2A-B are graphs showing transcriptional analysis of the mmsA gene (white bars) and the hbdH-4 gene (black bars) by real-time PCR in wild-type (WT) and mutant P. denitrificans strains. (Figure 2A) mRNA levels in deletion mutants P. denitrificans ΔhpdHΔhbdH4ΔhbdH1 (denoted ΔΔΔ) lacking 3-HP degrading enzyme. (Figure 2B) mRNA levels upon deletion and complementation of the transcriptional activator MmsR. ΔmmsR, P. denitrificans mmsR deletion mutant; and mmsR-C, P. denitrificans ΔmmsR with plasmid-derived mmsR complementation. [Figure 3] Figures 3A-B show a schematic analysis of the mmsR-mmsA intergenic region containing the promoter region and identification of the regulatory protein MmsA binding site. (Figure 3A) DNA sequence of the mmsR-mmsA intergenic region and mutations in the promoter region for its in vivo and in vitro characterization. (Figure 3A) Mapping of the 5'-end of PmmsA. (Figure 3B) Mapping of the MmsA binding sites (O1 and O2). [Figure 4]Figures 4A-B are schematic diagrams and graphs showing in vivo experiments to clarify 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 and / or O2 sites were randomized, and GFP was used as a reporter protein to examine the strength of promoters with mutations in the O1 and / or O2 sites. These mutations were introduced into P. denitrificans ΔhpdHΔhbdH1ΔhbdH4ΔmmsR and used as an expression host. [Figure 5] Figures 5A–E show photographs of SDS-PAGE analysis to examine the solubility of recombinant C-His-tagged MmsR protein in recombinant Escherichia coli (E. coli) BL21 using various chaperone plasmids, including pG-KJE8 (Figure 5A), pGro7 (Figure 5B), pKJE7 (Figure 5C), pG-Tf2 (Figure 5D), and pTf16 (Figure 5E). Cell lysates (T) and cell-free extracts (S) of recombinant strains harboring MmsR and chaperones were analyzed. [Figure 6] Figures 6A-C are photographs of gels showing the expression, purification, and oligomeric form of recombinant MmsR protein. (Figure 6A) SDS-PAGE, (Figure 6B) Blue Native-PAGE, and (Figure 6C) relative mRNA levels of recombinant MmsR bearing a His-tag at either the N- or C-terminus. Lane 1, host E. coli BL21 (cell-free extract); lane 2, recombinant E. coli BL21 grown 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 from recombinant E. coli BL21, respectively; lanes 9, 11, and 13 correspond to 65, 220, and 550 nM purified MmsR protein, respectively. [Figure 7]Figures 7A-B are photographs of in vitro electromobility shift assays (EMSAs) to examine the binding of MmsR protein to the operator site. DNA fragments containing the mmsA promoter (PmmsA) were either intact (F12) or mutated at either 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 contain 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 immobilized at 0.4 nM (Figure 7A). Electropherograms obtained from reactions with increasing amounts of MmsR (Figure 7B). The operator region was protected from DNase I digestion by MmsR. Below is a scale giving the nucleotide positions relative to the MmsR transcription start site. [Figure 8] Figure 8 shows photographs of electrophoretic mobility shift assays at low DNA concentrations to estimate the dissociation constants of the MmsR and PmmsA promoters: (F12) a DNA fragment with both operators O1 and O2 intact, (F12M) a DNA fragment with the O2 operator mutated, and (F1M2) a DNA fragment with the O1 mutated. [Figure 9] 9 is a graph and table showing the MmsR protein-DNA binding isotherm and the dissociation constant of the binding. DNA fragments containing either the intact promoter sequence (F12) or the mutated sequence were used. [Figure 10] 10A-B are schematic diagrams of the analysis of the hpdR-hpdH intergenic region. (FIG. 10A) DNA sequence of the hpdR-hpdH intergenic region showing the putative promoter and operator regions. (FIG. 10B) Consensus of the operator palindromic site. [Figure 11] Figures 11A-B are graphs showing the relative transcription of hpdR and hpdH. (Figure 11A) HpdR is a transcription factor that controls the expression of hpdH. (Figure 11B) Absence of crosstalk between the two 3-HP inducible systems, HpdR and MmsR. [Figure 12]Figures 12A-D are graphs showing the relative induction 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 various time intervals, and (Figure 12D) sensitivity at various 3-HP concentrations. [Figure 13] 13A-B are a schematic diagram showing the location of randomized mutations in the PC3 3-HP inducible promoter and graphs showing normalized GFP levels for various promoters. [Figure 14] Figures 14A-B are a series of graphs showing a comparison of mmsA and kgsA transcription and enzymatic activity at the plasmid and genome level: (Figure 14A) mRNA (transcription) and (Figure 14B) KgsA activity. [Figure 15] Figures 15A-C are a series of schematic diagrams showing how MmsR operator site randomization was applied to KgsA overexpression: (Figure 15A) Schematic of operator mutagenesis, (Figure 15B) kgsA mRNA expression levels, and (Figure 15C) GFP fluorescence with the selected operator mutant PmmsA2a. [Figure 16] Figures 16A-B are a series of graphs showing the effect of MmsR overexpression on KgsA transcription using various constitutive synthetic promoters: (Figure 16A) Pzwf promoter library, (Figure 16B) KgsA transcription. [Figure 17] Figures 17A-D are a series of graphs showing the effect of fusing the native MmsA protein on the expression and enzymatic activity of KgsA. Various lengths of mmsA N-terminal sequences (designated Hyb-5, Hyb-10, Hyb-15, and Hyb-20, respectively) corresponding to 5, 10, 15, and 20 amino acids were ligated to the 5'-terminal DNA sequence of kgsA. (Figure 17A) Enzymatic activity of KgsA. (Figure 17B) SDS-PAGE analysis of crude cell extracts expressing the fusion proteins. Transcript stability was also measured (Figures 17C and 17D). [Figure 18]Figures 18A-C are a series of diagrams and graphs showing the effect of tandem promoters on the expression and enzymatic activity of kgsA mRNA. (Figure 18A) Positioning of the tandem promoter and its transcript; TSS, transcription start site; RBS, ribosome binding site; UTR, 5'-untranslated region; (Figure 18B) mRNA expression; and (Figure 18C) KgsA activity. [Figure 19] Figures 19A-C are a table and graphs showing the effect of 5'UTR on kgsA expression. (Figure 19A) Prediction of 5'UTR strength for kgsA expression system. (Figure 19B) Specific KgsA activity of UTR constructs with various inductions at 0, 0.25, and 25 mM 3-HP. (Figure 19C) Comparison of 5'UTR strength between theoretical prediction and experimental measurement at various 3-HP concentrations. [Figure 20] Figures 20A-B show a schematic diagram of the degradation of 3-HP to malonate semialdehyde by hbdH-4 and further to acetyl-CoA by mmsA (Figure 20A), and a schematic diagram of the gene structure of the mmsR-mmsA intergenic region and its regulation (Figure 20B). [Figure 21] Figures 21A-B are graphs and images showing enzyme activity analysis (Figure 21A) and SDS-PAGE analysis (Figure 21B) of KgsA expression levels in strains in which the first 10 codons of KgsA were optimized. Three constructs were developed, differing in terms of codon frequency levels (high, medium, and low), and designated Opt-1, Opt-2, and Opt-3, respectively. [Figure 22] Figure 22 is a series of SDS-PAGE images showing the effect of UTR design on KgsA expression. UTRs with various strengths predicted in silico by the UTR designer were tested. [Figure 23] FIG. 23 is a schematic diagram of the vitamin B12 (coenzyme B12) gene cluster and riboswitch in P. denitrificans. [Figure 24] FIG. 24 is a schematic diagram showing a comparative analysis of the organization of the coenzyme B12 gene from various Pseudomonas species. [Figure 25]Figures 25A-D are schematic diagrams showing the structures of vitamin B12 riboswitches. Figure 25A shows the structure of RS1, which precedes cobG. Figure 25B shows the structure of RS2, which precedes cobW. Figure 25C shows the structure of RS3, which precedes cbtB. Figure 25D shows the structure of RS4, which precedes btuB. [Figure 26] FIG. 26 is a graph showing transcription of the cob gene at the chromosomal level in Pseudomonas denitrificans. [Figure 27] FIG. 27 is a series of graphs featuring the in vivo characterization of the vitamin B12 intergenic region in P. denitrificans. [Figure 28] FIG. 28 is a graph showing the correlation between B12 concentration and GFP fluorescence of the vitamin B12 ribosensor derived from the intergenic region of the genes cobG and cbtB from P. denitrificans. [Figure 29] Figures 29A-B are a series of graphs showing the following: (Figure 29A) Gene essentiality profiling revealed that only the gene encoding bgpM is essential for coenzyme B12 biosynthesis. To understand the essentiality of several uncharacterized or poorly understood genes within the cob gene cluster, various concentrations of cobalt chloride were used. (Figure 29B) Both the Salmonella typhimurium metE-cbiB- and B12 riboswitch-based sensors were used to quantitate the concentration of coenzyme B12. [Figure 30] Figures 30A-B are a series of schematic diagrams showing the development of the dhaB-gdrAB expression cassette: (Figure 30A) Schematic of expression cassette construction; (Figure 30B) Integration of the kgsA gene into the site of the hbdH-4 gene in the ΔmmsA mutant strain. [Figure 31] Figures 31A-B are schematic diagrams showing the combinations of transcriptional and translational modifications used to develop the DhaB-GdrAB expression system (Figure 31A) and the DhaB-GdrAB (DhaB) expression system for chromosomal integration (Figure 31B). [Figure 32]Figure 32 is a series of cartoons (P2-0, P2-10, P2-20, P2-30; strains without the DhaB plasmid) showing the effect of genomic position on KgsA expression (e.g., the position of the kgsA gene and the activity of KgsA after chromosomal integration). Numbers indicate KgsA activity 4 hours after induction with 25 mM 3-HP. [Figure 33] Figure 33 is a series of cartoons showing the genomic integration of the DhaB expression cassette into the chromosome of strain P2-20 to create P4-1 or into P2-10 to create P4-2. The promoter of the DhaB-GdrAB expression cassette in P4-1 and P4-2 was subsequently attenuated (DhaB reduction) to create strains P4-3 and P4-4. [Figure 34] Figure 34 is a cartoon showing strain development (P4-5) for 3-HPA channeling. P4-5 has one copy of KgsA located near DhaB. [Figure 35] FIG. 35 is a graph showing the effect of various neutralizing bases on the production of 3-HP. [Figure 36] FIG. 36 is a graph showing the modification of recombinant strains in media supplemented with various 3-HP concentrations. [Figure 37] FIG. 37 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. [Figure 38] FIG. 38 is a graph showing the cultivation of the 3-HP modified recombinant strain (80 g / L) in media of various pH values. [Figure 39] FIG. 39 is a graph showing modification of a 3-HP (80 g / L) resistant recombinant strain to improve specific growth rate at higher pH. [Figure 40] FIG. 40 is a graph showing the cultivation of the pH 7.6-adapted 3-HP (80 g / L) resistant recombinant strain at various pH values. [Figure 41] Figure 41 is a schematic diagram of the 3-HP synthesis pathway from glycerol adapted in the recombinant strain. The first enzyme is sensitive to oxygen, while the second reaction is enhanced in the presence of oxygen. [Figure 42] FIG. 42 is a graph showing the effect of various dissolved oxygen concentrations on 3-HP production. [Figure 43] FIG. 43 is a graph showing the effect of various temperatures on 3-HP production. [Figure 44] Figure 44 is a graph showing the effect of various induction OD600 on 3-HP production. [Figure 45] FIG. 45 is a graph showing the effect of various concentrations of NH 4 OH on 3-HP production and specific growth rate. [Figure 46] FIG. 46 is a graph showing the effect of NH 4 OH (%) on specific 3-HP titer (%) and amount (%). [Figure 47] Figure 47 is a schematic diagram showing anaplerotic reactions in central carbon metabolism: PPC, PEP carboxylase; PEPK, PEP carboxykinase; ME, malic enzyme; and PC, pyruvate carboxylase. [Figure 48] FIG. 48 is a graph showing the effect of various neutralizing agents on % production of 3-HP. [Figure 49] FIG. 49 is a graph showing the effect of various concentrations of NH 4 HCO 3 on 3-HP titer and amount (%). [Figure 50] Figure 50 is a graph showing the effect of sequential disruption of the B12 riboswitch, promoter replacement, and finally, the use of a tandem promoter to improve coenzyme B12 production and thereby 3-HP production to commercial levels. [Figure 51] FIG. 51 is a graph showing the effect of substrate channeling on 3-HP production. [Figure 52] Figure 52 is a graph showing the effect of various neutralizing agents, such as NHOH and NaOH, on % production of 3-HP. In one condition, NHOH was used as the neutralizing agent up to 24 hours into fermentation, followed by NaOH for up to 48 hours. [Figure 53] Figure 53. In vitro enzyme activity of glycerol dehydratase (DhaB) and KgsA at various stages of the bioreactor. [Figure 54] Figure 54 is a graph showing the effect of various toxic intermediates on the in vitro enzymatic activity of glycerol dehydratase (DhaB). [Figure 55] Figures 55A-B show the development of a 3-HP inducible tandem promoter library for controlled expression of DhaB at various expression levels. (Figure 55A) Schematic of tandem promoter construction, (Figure 55B) Specific DhaB activity of various tandem promoter library constructs. [Figure 56] FIG. 56 is a schematic diagram showing the sequence of the PC3 promoter region. [Figure 57] Figure 57 is a graph showing the effect of natural and synthetic constitutive promoters in P. denitrificans. [Figure 58] FIG. 58 shows an exemplary system for extracting 3-HP from an aqueous solution using a water-immiscible solvent that is less dense than water. [Figure 59] FIG. 59 shows an exemplary system for extracting 3-HP from an aqueous solution using a water-immiscible solvent that is denser than water. [Figure 60] Figure 60 shows an exemplary system for producing acrylic acid from 3-HP by reactive distillation. [Figure 61] FIG. 61 is an exemplary line plot showing the increase in concentration of 3-HP in the solvent vessel when ethyl acetate is used to extract 3-HP from decellularized fermentation. DETAILED DESCRIPTION OF THE INVENTION

[0083] (Detailed explanation) The present disclosure provides an enhanced expression system for producing 3-HP or a salt thereof from glycerol. The present invention provides industrial microbial strains (including, for example, Pseudomonas strains) that are suitable for the production of 3-HP or salts thereof. The enzymes involved in the growth and their regulatory regions were integrated into the chromosome of the industrial strain. The enzymes involved in the production of 3-HP or its salts were identified, characterized, and operably linked to the enzymes. Expression of the 3-HP enzyme can be driven by a 3-HP inducible promoter, constitutive, inducible, and / or synthetic promoters. By combining it with a motor, thereby creating a tandem promoter system The tandem promoter system can be integrated into UTRs (either natural or synthetic). and other regulatory domains that control or enhance expression to improve expression of the system. Tandem promoter systems (e.g., tandem promoters combined with UTRs) can be used to improve The promoter system is inserted into the first amino acid coding sequence of a highly expressed native gene ( For example, sequences encoding up to 20 amino acids can be fused to these modifications. The decoration is an enzyme, downstream gene, in this case a gene involved in 3-HP production (or the production of its salts). Furthermore, the expression of these genes in the chromosomes of industrial strains can be increased. The positioning of the system was examined and its effect on 3-HP (or its salts) production was evaluated. The positioning design of the current system can avoid the accumulation of toxic 3-HPA and 3-HP production can be increased through channeling.

[0084] Furthermore, the present disclosure provides the ability to produce high titers of 3-HP without exogenous supplementation of coenzyme B12 (B12). To identify and characterize regulatory regions that control the expression of enzymes in the B12 production pathway. Removal of many of these regulatory regions (e.g., containing secondary structures) significantly reduced the expression of the B12 enzyme. The incorporation of the constitutive or 3-HP inducible expression systems described herein improves the expression of naturally occurring This can increase the expression of B12 enzymes and increase B12 production. This allows for the production of 3-HP (or a salt thereof) without the addition of any other enzyme.

[0085] The present disclosure also provides methods for removing 3-HP from aqueous solutions. For example, 3-HP can be removed by solvent extraction. The present disclosure provides methods for removing 3-HP (e.g., its removal from aqueous solutions). Additionally, the present disclosure provides methods for purifying 3-HP from 3-HP (e.g., after removal from an aqueous solution). Also provided is a process for producing acrylic acid (after its removal, and optionally after its purification). The present disclosure provides a method for purifying acrylic acid.

[0086] (Production of 3-hydroxypropionic acid by recombinant bacteria) The compositions and methods described herein provide for the production of 3-hydroxybenzoates from carbon sources using recombinant microorganisms. It is useful in the production of 3-hydroxypropionic acid (3-HP) (or its salts). The carbon source is glycerol, which is converted to gluten by a reaction involving coenzyme B12. 3-hydroxypropionyltransferase (3HTP) is produced by glycerol / diol dehydratases (e.g., DhaB). It is then converted to an aldehyde (3-HPA), which is then converted to NAD(P)(+) in a reaction requiring , which is converted to 3-HP by aldehyde dehydrogenases (ALDHs) (e.g., KgsA, EaldH, KaldH). The conversion of glycerol to 3-HP prevents the production of high titers of 3-HP (or its salts). These factors include several rate-limiting factors that contribute to the synthesis of ATP. The complex nature of the enzyme, NAD+ regeneration, and the role of 3-hydroxypropanaldehyde (3 In this study, 3-HP (or its salts) was administered at high titers, e.g. , and produced 3-HP at titers sufficient for commercialization.

[0087] Loss of enzyme activity during 3-HP production (or its salts) due to accumulation of toxic intermediates is a major cause of high titer The production of 3-HP (or its salts) presents a difficult challenge. 3-HPA is a toxic substance that accumulates during 3-HP production. Studies have shown that when 3-HP pathway enzymes are incubated with 3-HPA, It has been shown that the enzyme activity decreases in a dose-dependent manner. By targeting amino groups (NH3+), sulfhydryl groups (-SH), and imidazole groups, Known to react with amino acid residues such as lysine, cysteine, and histidine Accumulation of 3-HPA is involved in the synthesis of 3-HPA in the pathway from glycerol to 3-HP, for example. Proteins containing glycerol dehydratase or diol dehydratase, which are enzymes that Inactivation of glycerol or diol dehydratase by 3-HPA This results in reduced production and low titer of 3-HP (or its salts). Potential approaches to overcome this include, among others, the development of highly resistant plants to 3-HPA toxicity. This includes developing enzymes or continually synthesizing new enzymes. The compositions and methods described herein are directed to the production of toxic intermediates (e.g., 3-H) during the production of 3-HP from glycerol. PA) accumulation, thereby reducing the risk of 3-HP or can increase the amount of salt.

[0088] In some embodiments, the recombinant microorganisms described herein can produce 3- One or more genes encoding enzymes that catalyze the production of HP or a salt thereof, e.g., glycerol dehydratase, diol dehydratase, and / or aldehyde dehydrogenase In some embodiments, the compound can express one or more genes that encode the compound. The proteins involved in the synthesis of a compound are one or more enzymes that catalyze the production of the compound from a carbon source. Proteins involved in the synthesis of 3-HP or salts thereof can be synthesized, for example, by glycerol synthesis. dehydratase, diol dehydratase, and / or aldehyde dehydrogenase In some embodiments, the carbon source can be one or more proteins that comprise the , glycerol, but other carbon sources, e.g., glucose, glutamate, gluconate The recombinant organism can convert 3-hydroxybenzoates from glycerol to 3-hydroxybenzoates. Glycerol dehydratase enzyme catalyzing the synthesis of 3-hydroxypropanaldehyde (3-HPA) The enzymes that can be expressed include a diol dehydratase enzyme (e.g., DhaB) and a diol dehydratase enzyme. It contains three subunits encoding the glycerol / diol dehydratase (dhaBCD). DhaB is a complex enzyme that binds coenzyme B 12 It forms a complex with During the conversion of 3-HPA to 3-HPA, coenzyme B 12 is damaged, and this damaged coenzyme B 12 is different The enzyme glycerol dehydratase reactivating factor (GdrAB) or diol dehydratase reactivating factor (DDRAB) The activator produces a new active coenzyme B 12 GdrAB will be replaced by two sub-sub ... The recombinant organisms subsequently synthesized 3-HPA from 3-HP. expresses the gene encoding aldehyde dehydrogenase (ALDH), which catalyzes the This catalytic activity requires NAD(P)(+). The expression of the ALDH enzyme can include the expression of the kgsA gene. In embodiments, the recombinant microorganism contains DhaB, which catalyzes the conversion of glycerol to 3-HPA. The enzyme 3-HPA is converted to 3-HP by the ALDH enzyme, which then converts 3-HPA to 3-HP.

[0089] The recombinant microorganisms described herein can be bacteria or fungi. In an embodiment, the recombinant microorganism is a bacterium. The recombinant bacterium is a bioprocessor. Any method capable of producing 3-HP from a carbon source under culture conditions such as in a bioreactor or a microorganism. In some embodiments, the bacteria can be genetically modified to grow under aerobic conditions. In some embodiments, the bacteria are grown under anaerobic conditions. In some embodiments, the bacterial genome is capable of producing 3-HP from a carbon source such as glycerol. Genes useful for catalyzing the synthesis of glycerol dehydratase or diol dehydratase Encoding a ketase (e.g., DhaB) and / or an aldehyde dehydrogenase (e.g., ksgA) In some embodiments, the bacterial genome naturally contains the glycerol Some strains of Escherichia coli do not naturally contain genes useful for producing 3-HP from carbon sources such as Escherichia coli. In one embodiment, the gene used to produce 3-HP from glycerol is not naturally contained in the Bacteria that do not express 3-HP or 3-HP-1 were converted to express at least one gene used for 3-HP production. Genetic modifications can be made to the enzymes, for example, glycerol dehydratase (DhaB), diol dehydratase (DhaB), and Bacteria that do not express hydratase and / or aldehyde dehydrogenase (ALDH) are designated DhaB. and / or can be genetically modified to express ALDH.

[0090] In some embodiments, the recombinant bacteria are Pseudomonas strains, Klebsiella strains, lebsiella strain, or Escherichia strain. The recombinant bacteria are capable of transforming Pseudomonas denitrificans, Klebsiella pneumoniae, and nia), or a strain of E. coli. In some embodiments, the recombinant bacterium is a strain of Pseudomonas aeruginosa. Pseudomonas denitrificans is a strain of Pseudomonas denitrificans. Coenzyme B12 is a naturally occurring aerobic microorganism that can synthesize coenzyme B12. Furthermore, P. denitrificans is an important cofactor for the production of 3-HP from NA. NAD+ regeneration is crucial for the continuous production of 3-HP. Therefore, Pseudomonas denitrificans is a 3-HP producer (or its salt producer). It is believed that the microorganism is suitable for

[0091] In some embodiments, the recombinant bacterium is an Enterobacter bacterium (e.g., a bacterium of the genus Enterobacter, Bucieella or Salmonella or Citrobacter or Lactobacillus or Propionibacterium or Proteus or Serratia species or Clostridium species At least one glycerol dehydratase and / or diol dehydratase from enzymes, such as Enterobacter (e.g., Klebsiella or Salmonella) or Citrobacter) or Lactobacillus or Propionibacterium or Propionibacterium At least one glycerol from a species of the genus Rhodeus, Serratia, or Clostridium In some embodiments, the recombinant β-glucanase expresses a hydroxylase enzyme (e.g., DhaB). The bacterium may express two or more glycerol dehydratase enzymes (e.g., DhaB) and / or diol dehydratase enzymes (e.g., DhaC). and expressing a dehydratase enzyme (e.g., DhaB), wherein each of the dehydratase enzymes (e.g., DhaB) is a different Enterobacter species (e.g., Klebsiella or Salmonella, Citrobacter) or Lactobacillus or Propionibacterium or Proteus or Serratia or can be from a Clostridium species. The recombinant bacterium may be an Enterobacter bacterium (e.g., Klebsiella or Salmonella spp.). Citrobacter or Lactobacillus or Propionibacterium or Proteus at least one aldehyde dehydrogenase from the genus Serratia or Clostridium species In some embodiments, the cell expresses an ALDH enzyme, e.g., at least one ALDH enzyme. In one embodiment, the recombinant bacterium expresses two or more ALDH enzymes, wherein each of the ALDH enzymes is They can be derived from different Enterobacter or Lactobacillus strains. In some embodiments, the recombinant bacterium is an Enterobacter bacterium (e.g., Klebsiella At least one glycerol derived from bacteria of the genus Lactobacillus or Salmonella diol dehydratases (e.g., DhaB) and / or diol dehydratases, and Enterobacter Bacteria of the genus Klebsiella (e.g., Klebsiella or Salmonella) or Lactobacillus The strain expresses at least one aldehyde dehydrogenase (ALDH) enzyme derived from the strain.

[0092] In some embodiments, the recombinant bacterium is derived from a bacterium, in this case, Klebsiella pneumoniae. At least one glycerol dehydratase and / or diol dehydratase is At least one gene encoding the dhaB1, dhaB2, dhaB3, gdrA, and In some embodiments, the recombinant bacterium expresses Azospirillum spp. and / or gdrB. At least one aldehyde dehydrogenase from Azospirullum brasilense At least one gene encoding an enzyme, e.g., from Azospirillum brasilense In some embodiments, the recombinant bacterium expresses at least one of the K. pneumoniae-derived kgsA gene. at least one gene encoding at least one glycerol dehydratase, e.g., dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB from Klebsiella pneumoniae, or at least one At least one gene encoding diol dehydratase, as well as Azospirillum. At least one gene encoding at least one aldehyde dehydrogenase from Brassirense They also express one gene, for example, kgsA from Azospirillum brasilense. In some embodiments, the recombinant bacterium contains at least one glycerol derivative from Klebsiella pneumoniae. at least one gene encoding a hydratase and / or a diol dehydratase, e.g. For example, dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB, and Azospirillum brasiliensis At least one gene encoding at least one aldehyde dehydrogenase from For example, Pseudomonas densiflora expressing kgsA from Azospirillum brasilense trificans strain.

[0093] In some embodiments, the recombinant bacterium is an Enterobacter bacterium (e.g., a bacterium of the genus Enterobacter, at least one glycerin from bacteria of the genus Lactobacillus or Salmonella A cellol dehydratase gene and / or a diol dehydratase gene (e.g., dhaB1 , dhaB2, dhaB3, gdrA, and / or gdrB genes), e.g., at least one from Klebsiella pneumoniae Glycerol / diol dehydratase genes (e.g., dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB gene). In this case, the recombinant bacterium is an Enterobacter bacterium (e.g., Klebsiella or Salmonella At least two glycerol dehydratase genes from bacteria of the genus Nella or Lactobacillus genes and / or diol dehydratase genes (e.g., dhaB1, dhaB2, dhaB3, gdrA, and and / or two or more of the gdrB gene), for example, at least two glyceroglycoproteins from Klebsiella pneumoniae aldehyde hydratase genes (e.g., dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) two or more of the genes) or at least one nucleic acid having a diol dehydratase gene In some embodiments, the recombinant bacterium comprises a small gene from a bacterium of the genus Azospirillum. At least one aldehyde dehydrogenase gene (kgsA gene), e.g., azospirillum At least one aldehyde dehydrogenase gene (kgsA gene) from M. brasilense strain In some embodiments, the recombinant bacterium comprises at least one nucleic acid having a At least two aldehyde dehydrogenase genes from Azospirillum bacteria (e.g., For example, the kgsA gene), for example, at least two alkanes from the Azospirillum brasilense strain At least one nucleic acid containing a aldehyde dehydrogenase gene (e.g., kgsA gene) In some embodiments, the recombinant bacterium is an Enterobacter bacterium (e.g., At least one strain of bacteria from the genus Klebsiella or Salmonella or the genus Lactobacillus Glycerol dehydratase gene and / or diol dehydratase gene (e.g., dh aB1, dhaB2, dhaB3, gdrA, and / or gdrB genes), e.g., at least one gene from Klebsiella pneumoniae Three glycerol dehydratase genes (e.g., dhaB, dhaB1, dhaB2, dhaB3, gdrA, and and / or gdrB gene), and at least one nucleic acid derived from a bacterium of the genus Azospirillum At least one aldehyde dehydrogenase gene (kgsA gene), e.g., Azospirillum sativum At least one aldehyde dehydrogenase gene (kgsA gene) from a strain of Rhodobacterium brasilense The nucleic acid sequence comprises at least one nucleic acid having a gene. [ka] TIFF0007813656000002.tif223170TIFF0007813656000003.tif223170TIFF0007813656000004.tif224170TIFF0007813656000005.tif52170

[0094] In some embodiments, the diol dehydratase gene is derived from a Klebsiella bacterium. For example, from Klebsiella pneumoniae. In some embodiments, the diol dehydratase The enzyme is derived from Klebsiella pneumoniae subsp. pneumoniae MGH78478. wherein the diol dehydratase gene is selected from the group consisting of pduC, pduD, pduE, pduG, and / or pduH. In some embodiments, the recombinant bacterium can be one or more of: For example, at least one diol dehydrogenase, including PduC, PduD, PduE, PduG, and / or PduH. It expresses the enzyme drogenase.

[0095] In some embodiments, the recombinant bacterium comprises a sequence selected from SEQ ID NOs: 1-6 or SEQ ID NOs: 83-87. In some embodiments, the nucleic acid has at least one sequence selected from the group consisting of: In the method, the recombinant bacterium comprises at least one gene having a sequence selected from SEQ ID NOs: 1 to 5. In some embodiments, the recombinant bacterium comprises one or more nucleic acids selected from SEQ ID NOs: 83-87. In some embodiments, the nucleic acid has at least one sequence selected from the group consisting of: In some embodiments, the recombinant bacterium comprises at least one nucleic acid having SEQ ID NO:6. In this embodiment, the recombinant bacterium is selected from SEQ ID NOs: 1-5 and / or SEQ ID NOs: 83-87. and at least one nucleic acid having at least one sequence as defined above, as well as another having SEQ ID NO: 6. It has nucleic acid.

[0096] In some embodiments, the recombinant bacterium comprises any of SEQ ID NOs: 1-6 and 83-87. and at least one nucleic acid having at least one sequence that is at least 80% identical to the In some embodiments, the recombinant bacteria comprises the sequences of SEQ ID NOs: 1-6 and SEQ ID NOs: 83-87. Either and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical In some embodiments, the recombinant vector comprises at least one nucleic acid having a sequence similar to that of the recombinant vector. The bacterium is a strain selected from the group consisting of a nucleic acid sequence of any one of SEQ ID NOs: 1 to 6 and SEQ ID NOs: 83 to 87 and 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 It has at least one nucleic acid having the sequence

[0097] In some embodiments, the recombinant bacterium comprises a sequence selected from SEQ ID NOs: 77-82 and 88-92. In some embodiments, the vector expresses at least one protein having an amino acid sequence wherein the recombinant bacterium has a sequence at least 80% identical to any one of SEQ ID NOs: 77 to 82 and 88 to 92. In some embodiments, the vector expresses at least one protein having an amino acid sequence In the recombinant bacterium, the sequence of SEQ ID NOs: 77 to 82 and 88 to 92 is at least 80%, 81%, or 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, or 99% identical amino acid sequence to at least one of the In some embodiments, the recombinant bacteria expresses a protein selected from SEQ ID NOs: 77-82 and 88. ~92 amino acids and 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 Express at least one protein whose amino acid sequence differs by many amino acids .

[0098] (promoter) As used herein, the term "promoter" refers to a promoter that is operably linked to a gene. The core promoter refers to a DNA sequence that contains regulatory nucleic acid sequences used in the expression of a gene. Essential nucleotide sequences for promoter function, including the A box and transcription initiation site According to this definition, a core promoter contains sequences that enhance activity or enhance organization, for example. It may or may not have detectable activity in the absence of specific sequences that may confer specific activity. The promoter can be either constitutive or inducible. Inducible promoters control gene transcription at a constant rate for the life of the cell. The activity of the motor depends on the presence (or absence) of specific inducers, e.g., extracellular or environmental The 3-HP inducible promoter varies as determined by the presence (or absence) of factors. " indicates that when cells carrying such a promoter are exposed to 3-HP, e.g., 3-HP Cells carrying the promoter were transfected with 3-HP present in the cell culture medium or bioreactor. a promoter that, when exposed to a gene, increases the expression of the gene or genes to which it is operably linked In some cases, the promoter contains one or more operator sites. include.

[0099] The recombinant microorganisms described herein contain a glycoprotein under the control of at least one promoter. A strain expressing at least one gene used to produce 3-HP (or a salt thereof) from cellulose. In some embodiments, the expression system is responsive to a stimulus (i.e., an inducer). at least one inducible promoter that increases expression of the gene in response to a steroid hormone (e.g., 3-HP); Once a stimulus contacts the inducible promoter, the promoter activates the gene In some embodiments, the inducible promoter turns on or upregulates expression of the gene. The promoter is induced by 3-HP. At least one inducible promoter in the expression system The stimulus or inducer that causes upregulation of gene expression by 3-HP is 3-HP. In some embodiments, the expression system is driven by at least one inducible promoter. The stimuli or inducers that result in upregulation of gene expression include 3-HP and chemical and / or In some embodiments, at least one of the expression systems Stimuli or inducers that cause upregulation of gene expression by an inducible promoter The salt is a small molecule, e.g., a small organic molecule such as an acid or an alcohol. In this manner, the small molecule is 3-hydroxypropionic acid (3-HP) or 3-hydroxypropaneal. It is structurally similar to 3-HPA, a hydroxypropyl ester of 3-HPA. 3-HP has the chemical structure C3H6O3 and a molecular weight of 90.08. In some embodiments, the L-valine degradation pathway and central carbon metabolism A variety of small acids structurally similar to 3-HP or its intermediates found in In embodiments, the small molecule acid or alcohol is L-lactic acid (LAC), acetic acid (AcOH), propionic acid ( PA), 3-hydroxybutyrate (3-HB), 1,3-propanediol (1,3-PDO), 2,3-butanediol 2,3-BDO, L-valine (L-val), and 3-hydroxyisobutyrate (3-HIB) In some embodiments, the inducible promoter can be, but is not limited to, A promoter can be induced by more than one inducer.

[0100] Presented herein are studies that demonstrate that 3-HP-induced C3 platform chemical The present disclosure provides novel promoters that can reported the mechanism by which 3-HP activates / initiat- es gene expression. We have found that when Pseudomonas strains are provided with 3-HP as the sole carbon source, the strains We observed that 3-HP was actively consumed by the bacteria, indicating growth. putative 3-hydroxyisobutyrate dehydrogenase IV (HbdH-4), 3-hydroxypro Pionate dehydrogenase (HpdH), and / or methylmalonate semialdehyde dehydrogenase We identified an enzyme (MmsA) involved in 3-HP degradation. However, transcription of the genes encoding these enzymes is not high, and 3-HP or similar small We found that these 3-HP degradation genes were significantly upregulated only in the presence of hydroxybenzoates. Analysis of the gene arrangement revealed putative transcriptional regulator proteins corresponding to mmsA, hbdH-4, or hpdH. These transcriptional regulator proteins are complexed by 3-HP. When the body is formed, it activates the transcription of mmsA, hpdH, and others.

[0101] The inducible nature of the promoters described herein and their high induction efficiency allow for the The promoter is useful for the expression of 3-HP synthesis pathway enzymes, the development of 3-HP-responsive biosensors, and / or has been useful for the expression of pathway enzymes for coenzyme B12 production. We have elucidated the HP-induced gene regulation system. At the cellular level, we have identified the L-valine degradation pathway and its intermediates. We used various acids that are structurally similar to 3-HP or its intermediates found in high-carbon metabolism. The specificity and / or pharmacological spectrum of small molecule inducers was investigated.

[0102] These promoters are used to determine whether the promoter is inducible by small acids, and A library of promoters showing various expression levels was constructed. Tandem promoter systems have been developed using these promoters in combination with natural or synthetic promoters. Here, we have developed a small acid-inducible system and natural / synthetic promoters. By combining libraries, tandem promoter systems can be stimulated to target We attempted to express target genes, such as genes involved in the 3-HP synthesis pathway.

[0103] In some embodiments, the inducible promoter is induced by 3-HP. In some embodiments, the inducible promoter is a native promoter, e.g., a bacterial promoter. For example, promoters that drive gene expression in Pseudomonas bacteria. In one embodiment, the inducible promoter is a promoter found in bacteria, e.g., Pseudomonas bacteria, e.g., For example, the natural 3-HP inducible promoter present in Pseudomonas denitrificans In Pseudomonas bacteria, such as P. denitrificans, 3-hydroxyisothiazolinone is synthesized. 3-hydroxyisobutyrate dehydrogenase I (HbdH-1), 3-hydroxyisobutyrate dehydrogenase IV (HbdH-4) , 3-hydroxypropionate dehydrogenase (HpdH), and methylmalonate semialdehyde A promoter driving the expression of 3-HP dehydrogenase (MmsA) was used to express the 3-HP degradation enzyme Several natural promoters driving protein expression are sequentially upregulated by 3-HP. In some embodiments, the recombinant bacteria described herein are P. denito. the mmsA gene (PmmsA promoter), the hpdH gene (PhpdH promoter) of S. refificans, or hbdH-4 gene (PhbdH-1 promoter) under the control of at least one native promoter. It expresses at least one gene used to produce 3-HP from glycerol. In some embodiments, the inducible promoter is a synthetic promoter. [ka]

[0104] In some embodiments, the recombinant bacterium is represented by any of SEQ ID NOs: 11-14. and producing 3-HP (or a salt thereof) from glycerol under the control of at least one promoter. In some embodiments, the at least one gene is expressed. The recombinant bacterium is regulated by at least one promoter represented by SEQ ID NOs: 11 to 14. at least one from any of dhaB1, dhaB2, dhaB3, gdrA, gdrB, and / or kgsA below; expresses the gene.

[0105] In some embodiments, the recombinant bacterium comprises at least one of SEQ ID NOs: 11-14. 3 from glycerol under the control of at least one promoter with a sequence that is 80% identical -expressing at least one gene used to produce HP (or a salt thereof). In some embodiments, the recombinant bacterium has at least 80%, 81%, or 90% identity with any of SEQ ID NOs: 11-14. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% identical sequence to at least one promoter. At least one of the following is used to produce 3-HP or a salt thereof from glycerol under the control of a In some embodiments, the recombinant bacterium expresses one gene selected from the group consisting of SEQ ID NOs: 11-14. Any of the following and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleosides Three-phase synthesis from glycerol under the control of at least one promoter with a sequence that differs by only one sequence. -expressing at least one gene used to produce HP or a salt thereof.

[0106] In some embodiments, the expression systems described herein are A small molecule that controls at least one gene involved in the production of 3-HP or its salts from any carbon source. Tandem promoter systems contain at least two promoters in tandem. These are observed in microorganisms that regulate the expression of downstream genes under various physiological or environmental conditions. The system aids in the controlled regulation of target gene expression during various stages of cell growth. A microorganism promoter is a promoter that is native to a microorganism, such as a promoter naturally found in a bacterial strain. Alternatively, it may be endogenous or synthetic. In some embodiments, one or more of the promoters is a native promoter. In some embodiments, one or more of the promoters can be The promoter may be a synthetic promoter.

[0107] The tandem promoters of the expression systems described herein may contain one or more inducible promoters. In some cases, the gene may include one or more constitutive promoters and / or one or more constitutive promoters. Thus, the tandem promoter comprises two or more inducible promoters. Tandem promoters containing motors can have two identical inducible promoters. i.e., an inducible promoter can be induced by the same inducer or stimulus. It can be induced by one gene or have two different inducible promoters, i.e., Either the inducible promoter is induced by a different inducer or stimulus In some embodiments, the tandem promoter comprises at least one The tandem promoter comprises an inducible promoter and at least one constitutive promoter. The promoter is a combination of a 3-HP inducible promoter and a constitutive promoter. It is possible to regulate the expression of genes involved in the development of thyroid cancer.

[0108] In some embodiments, each of the promoters is located downstream of the tandem promoter. The terminator sequence is incorporated into the expression system so that the expression of the gene to be placed can be regulated. between tandem promoters of the same gene, e.g., between any two or more promoters. stomach.

[0109] In some embodiments, the tandem promoter comprises at least two 3-HP-inducible In some embodiments, the tandem promoter comprises a 3-HP inducible promoter. The first promoter is an inducible promoter and the second promoter is a 3-HP inducible promoter. In some embodiments, the first 3-HP inducible promoter and The two 3-HP inducible promoters are P mmsA Promoter (SEQ ID NO: 14), P hbdH-1 Promo promoter (SEQ ID NO: 13), PhbdH-4 promoter (SEQ ID NO: 11), or P hpdH Promoter (sequence It can be number 12).

[0110] In some embodiments, the tandem promoter is a 3-HP inducible promoter. a first promoter that is a constitutive promoter and a second promoter that is a constitutive promoter, the first 3-HP inducible promoter is located 5' or upstream of the second constitutive promoter In some embodiments, the first 3-HP derivative is P mmsA promoter( SEQ ID NO: 14), P hbdH-1 Promoter (SEQ ID NO: 13), P hbdH-4 promoter (SEQ ID NO: 11), or P hpdH In some embodiments, the second construct is a promoter (SEQ ID NO: 12). The constitutive promoter is P zwf (SEQ ID NO: 7) is a 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 as set forth in SEQ ID NO:7. Both are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, and 92%. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. and the sequence of the second constitutive promoter is 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 In some embodiments, the sequence differs from SEQ ID NO: 7 by one or more nucleotides. The sequences of the second constitutive promoter are 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: The number is 63. [ka] TIFF0007813656000008.tif56170

[0111] In some embodiments, the sequence of the tandem promoter in the expression cassette is SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In embodiments, the sequence of the tandem promoter in the expression cassette is SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In an embodiment, the sequence of the tandem promoter in the expression cassette is SEQ ID NO: 65, the sequence SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72 at least 80%, 81%, 82%, 83%, 84%, 85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, In some embodiments, the expression cassette may be 99% identical or more than 99% identical. The sequence of the dem promoter is a sequence selected from any one of SEQ ID NOs: 70 to 72 and 1, 2, 3, 4, 5, 6, 7, and 8. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 The sequences may differ by 29, 30, or more nucleotides. [ka]

[0112] (transcriptional regulator) The regulatory region of the 3-HP degradation gene encodes a natural LysR family transcription regulator (LTTR) protein. Located near the coding sequence. When complexed with 3-HP, the LysR family -Transcriptional regulator proteins activate transcription. In bacteria, LTTR proteins are involved in the transcription of RNA. By interacting with LTT polymerase, it upregulates downstream genes. The amount of R protein affects the transcription level of downstream target genes.

[0113] In some embodiments, at least one transcriptional regulator protein is a recombinant microorganism, such as a bacterium, in combination with at least one 3-HP producing gene described in In some cases, at least one transcriptional regulator protein is expressed in The LTTR protein is under the control of at least one constitutive or inducible promoter. In some cases, transcriptional regulator proteins are involved in the expression of at least one 3-HP-producing gene. It binds to the site of flux and regulates the expression of at least one gene involved in 3-HP synthesis.

[0114] In some embodiments, the recombinant bacteria described herein contain at least one L At least one gene used to produce the TTR protein and 3-HP or a salt thereof, e.g. , at least one of glycerol dehydratase, diol dehydratase, and / or a In some embodiments, the LTTR protein expresses aldehyde dehydrogenase. , MmsR. In some embodiments, the MmsR promoter is a constitutive promoter In some embodiments, the gene is expressed in a recombinant bacterium under the control of a constitutive promoter. is P zwf promoter (SEQ ID NO: 7). In some embodiments, the constitutive promoter The promoter was mutated to increase or decrease expression of MmsR relative to the Pzwf promoter. In some embodiments, the constitutive promoter is Pzwf (SEQ ID NO: 7). In some embodiments, the sequence of the constitutive promoter is the sequence At least 80% identical to number 7. In some embodiments, a constitutive promoter The sequence of SEQ ID NO: 7 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical In some embodiments, the sequence of the constitutive promoter is SEQ ID NO: 7 and SEQ ID NO: 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, They differ by 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 the sequence In some embodiments, the mmsR gene is located on a chromosome, e.g., The mmsR gene, under the control of a constitutive promoter, is expressed from the chromosome within the recombinant bacteria.

[0115] (5'UTR) In some embodiments, the expression systems described herein express the gene of interest ( For example, the 5'UTR can be operably linked to a gene involved in 3-HP synthesis, The 5'UTR is a 5'UTR that is expressed in the 3-HP gene of bacteria that naturally produce 3-HP, such as P. denitrificans. It is derived from a native gene controlled by an inducible promoter. In embodiments, the expression described herein is achieved by activating the mmsA gene (sequence number 101111111) of P. denitrificans. It has a 5'UTR of number 64).

[0116] In some cases, mutations in the 5'UTR improved the rate of translation initiation of mRNA transcripts. and / or can improve the stability of the mRNA transcripts produced by the expression system. In some embodiments, the expression systems described herein comprise SEQ ID NO: 64. In some embodiments, the 5′ UTR has a sequence that is at least 80% identical. In this manner, the expression systems described herein may be at least 80%, 81% identical to SEQ ID NO: 64. ,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%, It can have a 5'UTR with a sequence that is 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the expression systems described herein comprise SEQ ID NO: 64 and 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 3 or more nucleotides In some embodiments, the 5' UTR may have a sequence as described herein. The expression system includes a 5'UTR having any sequence selected from SEQ ID NOs: 22 to 28. It is possible.

[0117] In non-limiting examples, the PmAdH-4 tandem promoter, Opt-3, Hyb-20, and chromosomal Mm The expression system with sR has the 5'UTR of the mmsA gene. , the mmsA 5'UTR can be mutated. [ka]

[0118] (Genetics in P. denitrificans by fusion of the first few amino acids of a native protein) Optimization of gene expression A Novel Approach to Optimize Heterologous Protein Expression in P. denitrificans The heterologous protein was synthesized by cloning the first few amino acids (5-20 AA) of a highly expressed native enzyme. at the N-terminus of the target protein (e.g., 5-20 amino acids at the N-terminus of the native protein). amino acids were fused to the N-terminus of the heterologous protein). For example, various lengths of mmsA (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length; , Hyb-5, Hyb-6, Hyb-7, Hyb-8, Hyb-9, Hyb-10, Hyb-11, Hyb-12, Hyb-13, Hyb-14, Hyb The N-termini of the 2000-kDa nucleotide sequences (Hyb-15, Hyb-16, Hyb-17, Hyb-18, Hyb-19, and Hyb-20) were ligated to the full-length heterologous kgsA gene. The vector was transfected into the ribosomal DNA and expressed from a multicopy plasmid (Figure 17). The highest activity was observed in Hyb-20 (+3-HP, 9. 3 U / mg protein; -3-HP, 3.0 U / mg protein). The results showed no change in expression or its enzymatic activity (Fig. 17A). SDS-PAGE analysis confirmed that kgsA was N-terminally cleaved. When fused to the terminal mmsA fragment (adding more than five amino acids), protein production Improved expression in wild-type and Hyb-20 kgsA recombinant strains was observed (Fig. 17B). To understand the mechanisms of expression and mRNA stability, the transcripts of mmsA and mmsR were measured and compared. The stability of the transcript was also determined (Figures 17C and 17D). The half-life of the wild-type kgsA transcript (2.7 min) was significantly longer than that of mmsA. However, fusing the first 20 aa (20) of mmsA resulted in a half-life of 2.3 times shorter than that of the 20 aa of mmsA (6.1 min). As a result, the half-life of the fusion protein mRNA is equivalent to that of the native mmsA mRNA. This was significantly improved to 7 minutes, because the fusion protein (mRNA transcript) was able to bind to endogenous nuclei. This suggests that the cells are less susceptible to nucleotide attacks as determined by RT-PCR. The transcription level of the hybrid mmsA(20) kgsA gene was also 2 times higher than that of the wild-type kgsA gene. The present inventors concluded that the higher mRNA stability, along with improved transcription, This should have contributed to the improvement of the KgsA activity of the hybrid enzyme. Among the several genes examined, mmsA showed the highest mRNA stability, with a half-life of 9.5 min. (data not shown).

[0119] (Recombinant bacterial coenzyme B 12 (production of The production of 3-HP from glycerol by microorganisms that produce 3-HP or its salts requires coenzyme B12. Coenzyme B12 is used in the first reaction of the 3-HP synthesis pathway. , an essential cofactor for glycerol / diol dehydratase enzyme activity, Glycerol is converted into 3-hydroxypropanealdehyde by catalytic action. Therefore, a continuous supply of coenzyme B12 is essential for the synthesis of glycerophosphate by microorganisms such as bacteria. Coenzyme B12 is essential for the continuous production of 3-HP or its salts from ethanol. It can be naturally synthesized under aerobic or anaerobic conditions by various organisms (anaerobic synthesis of coenzyme B12). Producers include, for example, Klebsiella, Streptococcus, Salmonella, Aerobic producers of coenzyme B12 include, for example, Pseudomonas sp., Rhizobium sp. However, some bacteria may be resistant to the bacteria, such as Rhizobium, Rhodobacter species. Microorganisms being evaluated as potential hosts for the production of 3-HP or its salts from glycerol It appears that these organisms do not produce sufficient amounts of coenzyme B12 to produce high titers of 3-HP. Further analysis has shown that coenzyme B12 production is transcriptionally and translationally regulated. These processes can be genetically modified in recombinant microorganisms, e.g., bacteria, to increase coenzyme B12 production. This increases the potency of 3-HP (or its salts) without supplementing the bacteria with exogenous coenzyme B12. It can be increased.

[0120] As shown in Figure 23, two gene clusters (cluster I and cluster II) , which produces proteins used in the production of coenzyme B12 in P. denitrificans. Riboswitches and other secondary structures are involved in the nucleotide sequences present in clusters I and II. These riboswitches have been identified in the promoter region of B1 2 biosynthetic operon and can repress gene expression. In some embodiments described, one or more riboswitches or other secondary structures are present in P. One or more operons of cluster I and / or cluster II in the genome of B. denitrificans In some embodiments, the promoter region may be removed. riboswitches 1, 2, 3, and / or 4, or portions thereof, as shown in Figures 1A-D. are deleted in the genome of P. denitrificans. and all or part of the DNA sequence of riboswitch 1 (SEQ ID NO: 75) or riboswitch 2 (SEQ ID NO: 76). In some embodiments, the portion is removed from the genome of P. denitrificans. , all or part of the DNA sequences of riboswitch 1 (SEQ ID NO: 75) and riboswitch 2 (SEQ ID NO: 76) has been removed from the genome of P. denitrificans.

[0121] Any of the expression systems described herein can be used to express a gene involved in the production of coenzyme B12. It is possible to increase gene expression and / or increase the production of coenzyme B12. The 3-HP inducible promoter system described herein can be used to express B12 production genes (e.g., The present disclosure provides a method for the replacement of coenzyme B12 (a transcription factor) with a regulatory region of the coenzyme B12. Increase the production of coenzyme B12 so that supplementation is not used by the organism to produce 3-HP. Provided herein are methods for regulating and / or upregulating genes for the production of 3-HP. Expression systems for regulation and / or upregulation of genes for the production of coenzyme B12 The recombinant organism may comprise an expression system for the [ka]

[0122] (Manifestation System Configuration for Channeling) The expression systems described herein can be expressed in host cells using any method known in the art. The gene can be introduced into a microorganism, e.g., a bacterium. The system can be introduced into a microorganism using a plasmid, artificial chromosome, or other vector. In some embodiments, the expression system can be integrated into the genome of a microorganism. For example, it can be integrated into a chromosome.

[0123] Multicopy expression systems can be introduced into microorganisms, e.g., bacteria. The expression system of is on at least one plasmid, artificial chromosome, or other vector, For example, a multicopy expression system can be introduced into a microorganism, such as a bacterium. on one plasmid, or on two or more different plasmids (i.e., different sequences, selection markers, etc.). It can be introduced into bacteria on a plasmid carrying a multicopy expression system. In some embodiments, the multi-copy gene can be integrated into the genome of the microorganism. The expression systems can be integrated at the same location in the genome, for example at the same chromosomal location. In some embodiments, the multicopy expression system is expressed in different locations, e.g., The integration can occur at different chromosomal locations. The present system was used to identify different chromosomal regions of Pseudomonas bacteria, e.g., P. denitrificans. In some embodiments, the promoter is involved in the production of 3-HP from a carbon source. different genes for, for example, one or more glycerol dehydratase or diol dehydratase Two or more expression systems regulating the aldehyde dehydrogenase gene and the aldehyde dehydrogenase gene In some embodiments, the two or more The above expression systems are integrated at the same location in the genome. The two or more expression systems are integrated at different chromosomal locations.

[0124] In some embodiments, one or more of the expression systems described herein are In some embodiments, the vector is introduced into a bacterium, such as a Domonas denitrificans bacterium. and the glycerol dehydratase gene and / or the diol dehydratase gene (e.g. , dhaB1, dhaB2, dhaB3, gdrA, and / or gdrB genes) and an expression system regulating one or more genes encoding aldehyde dehydrogenase (e.g., The expression system for regulating one or more genes encoding the P. dendrobatidis gene (at least one ksgA gene) is nitrificans bacteria and optionally integrated into the bacterial genome. In one embodiment, glycerol dehydratase (and / or diol dehydratase) and the expression systems regulating the aldehyde dehydrogenase gene are located at different chromosomal locations. It is incorporated in.

[0125] In some embodiments, glycerol dehydratase (and / or diol dehydratase) The expression systems regulating the aldehyde dehydrogenase and aldehyde dehydrogenase genes are different. The specific chromosomal integration site is determined by the location of the expression system or module. can significantly affect the level of expression, resulting in reduced gene expression at a given integration site. The level of gene expression at different integration sites will differ. The positioning of the chromosomal insertion of specific genes within the pathway balances the activity of synthetic pathway enzymes. This avoids the accumulation of toxic intermediates such as 3-hydroxypropanaldehyde. For example, in many prokaryotes, the genes encoding the enzymes of a pathway are clustered together. Clustered. This clustered arrangement is produced by intracellular pathways. This is observed in bacteria, where it is thought to avoid the accumulation of intermediates. In prokaryotes, transcription and translation are synchronized. Sometimes, the product of the first enzyme, often encoded by the first gene, initiates the pathway. The gene is a substrate for a second enzyme encoded by a second gene in the If located in the pathway produced by the enzyme encoded by the first gene, The substrate of the second enzyme, encoded by the second gene, is localized near the second enzyme. This type of mechanism increases the efficiency of the pathway, ensuring that the intermediate is quickly consumed by the next enzyme in the pathway. By allowing the ATP to be consumed, the accumulation of potentially toxic intermediates is avoided. This type of clustering of genes in close proximity is known as channeling.

[0126] Therefore, glycerol dehydratase and aldehyde dehydrogenase in the chromosome The positioning of the enzymes, for example, results in the rapid conversion of 3-HPA to 3-HP. Therefore, glycerol dehydratase (and / or diol) is added to produce a channeling effect. The expression systems that regulate aldehyde dehydrogenase and aldehyde hydratase are different. By integrating at a chromosomal location, the levels of these enzymes can be regulated, resulting in recombination. It is possible to regulate the levels of 3-HPA and 3-HP in bacteria. regulates diol dehydratase (and / or diol dehydratase) and aldehyde dehydrogenase The channeling effect created by the positioning of separate expression systems is beneficial for cells. It can prevent the accumulation of 3-HPA in toxic bacterial cells, allowing the cells to survive longer. This allows the bacteria to survive and grow quickly and produce higher titers of 3-HP or a salt thereof.

[0127] In some cases, the second integration site of the second expression system The first integration site of an expression system affects the production level of 3-HP or a salt thereof. In some cases, the first expression system is a glycerol dehydratase or dinucleotide an expression system that expresses an aldehyde hydratase enzyme (e.g., one or more of the dhaB and / or gdrAB genes); In some cases, the second expression system can be an aldehyde stem. a dehydrogenase enzyme (e.g., one or more ALDH genes, e.g., kgsA, ealdH, and / or In some cases, the expression system may be an expression system that expresses the 2 This system incorporates two expression systems in close proximity to each other, and is useful for the production of toxic intermediates (e.g., 3 -HPA) accumulation and reduced exposure of other enzymes to these toxic intermediates, which This can increase the production of 3-HP or a salt thereof.

[0128] In some embodiments, at least one glycerol derivative described herein is Expression systems encoding hydratase (and / or one or more diol dehydratase genes) The gene or module is expressed in the chromosome of a recombinant bacterium, e.g., P. denitrificans. The expression system encoding at least one aldehyde dehydrogenase as described herein Stem or module length of about 500 to 2,500,000 nucleotide base pairs (e.g., about 500 base pairs) ~2,500 kilobase pairs) (e.g., nucleotides or base pairs) or less (e.g., Within about 500 to 2,500,000 base pairs of each other. and at least one glycerol dehydratase (or diol dehydratase) enzyme and and at least one aldehyde dehydrogenase enzyme. The expression systems are approximately 2,500 kb, 1,500 kb, and 500 kb of each other. Nucleotides, 250 kilonucleotides, 150 kilonucleotides, 50,000 nucleotides, 25,0 00 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 nucleotide, 3,200 nucleotides, 3,000 nucleotides, 2,800 nucleotides, 2,600 nucleotides Tide, 2,400 nucleotides, 2,200 nucleotides, 2,000 nucleotides, 1,800 nucleotides , 1,600 nucleotides, 1,400 nucleotides, 1,200 nucleotides, 1,000 nucleotides, 90 0 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 50 nucleotides In some embodiments, the gene Alternatively, the distance between integration sites of the expression system is about 4000 base pairs. wherein the integration site is at least about 1500 base pairs (e.g., about 1500 base pairs; 2000 base pairs; 2500 bp; 3000 bp; 3500 bp; 4000 bp; 4500 bp; 5000 bp; 5500 bp base pairs; 6000 base pairs; 6500 base pairs; 7000 base pairs; 7500 base pairs; 8000 base pairs; 8500 base pairs; 90 00 base pairs; 9500 base pairs; 10,000 base pairs; 20,000 base pairs; 50,000 base pairs; 100,000 base pairs; 2 In some embodiments, the sequences are separated by at least 500,000 base pairs; or 500,000 base pairs). Encodes another glycerol dehydratase enzyme or a diol dehydratase , an expression system as described herein, and at least one aldehyde dehydrogenase The expression systems described herein, which encode enzymes, are expressed in the bacterial chromosome. located within about 500 nucleotides of the The distance between the target sites is approximately 500 base pairs.

[0129] In some embodiments, the integration site of the expression system into the bacterial chromosome is In some cases, the closer the replication origin, the better. In some embodiments, the expression system has a higher expression level. The stem is located approximately 500 to 4000 nucleotides (e.g., nucleotides or base pairs) away from the origin of replication. In some embodiments, the expression system is integrated into the expression system at about 4000 nucleotides from the origin of replication. nucleotides apart, ~3000 nucleotides apart, ~2000 nucleotides apart, ~1000 nucleotides apart nucleotides apart, ~750 nucleotides apart, or ~500 nucleotides apart 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 The fragment is located at least 500 nucleotides (e.g., about 500 nucleotides; 600 nucleotides) from the origin of replication. 700 base nucleotide pairs; 800 nucleotides; 900 nucleotides; 1000 nucleotides; 1100 nucleotides; 1200 nucleotides; 1300 nucleotides; 1400 nucleotides; 1500 nucleotides Nucleotides; 2000 nucleotides; 2500 nucleotides; 4000 nucleotides; or 5000 nucleotides In some embodiments, the expression system is about 500 nm from the origin of replication. nucleotides apart.

[0130] As described above, 3-HPA is channeled via DhaB and ALDH enzymes to promote 3-HP production and cell They created a system for improving survival that is linked to other enzymes and cellular components. It reduces exposure to toxic 3-HPA and increases production of 3-HP or a salt thereof. In this case, the dhaB, gdrAB genes, and kgsA were placed adjacent to each other. The location of this gene was more than 2000 bp away from the DhaB and GdrAB coding genes. The location of a gene's integration in relation to the replication origin and to the target site affects its expression and activity. .

[0131] kgsA was cloned at various positions (near DhaB and GdrAB (P4-20 strain) and 2000 bp away from DhaB and GdrAB). When the P4-10 and P4-20 strains were placed in the same culture medium, the inventors noticed a difference in enzyme activity. To investigate the channeling effect, enzyme activity was measured in the strain (by gene expression, such as promoter and UTR). These modifications synchronized both The strains (with channeling effect (P4-20) and without channeling effect (P4-10)) had similar KgsA and The DhaB activity was observed between these two strains (with and without channeling). The only difference between the two was the position of the kgsA gene relative to the dhaB and gdrAB genes (Figure 51). ).

[0132] In these same strains (P4-10 and P4-20), the production of 3-HP was compared. The dhaB and gdrAB genes are channeled (e.g., located close to each other). The P4-20 strain has unchanneled genes (kgsA gene, dhaB and gdrAB genes) ( For example, higher 3-HP production was observed when compared to strain P4-10 (located 2000 bp apart on the chromosome). The P4-20 strain showed improved viability and 3-HP titer. The level of 3-HPA accumulation was significantly higher than that of the P4-10 strain. In some cases, this was due to the 3-HP enzyme being activated by the nearby KgsA enzyme. In the P4-20 strain, the DhaB enzyme (which produces 3-HPA) ) is located near the KgsA enzyme, and therefore 3-HPA is involved in the accumulation and / or transport of K It is rapidly consumed by the gsA enzyme before it can affect it.

[0133] (Bio-production) The optimal physiological parameters, such as medium, aeration, temperature, pH, and induction time of the target product, are determined by: Individually or in combination, they have a significant effect on cell proliferation and target molecule production.

[0134] The culture medium plays a vital role in maintaining cell viability and improving the titer of the target product. It is an important parameter in bioprocesses that play a key role in the efficient 3-HP synthesis. It must be properly formulated for production. Analytical grade chemicals are used to Bioprocesses that produce this salt are not commercially available. Therefore, careful selection of medium components and Among the ingredients, coenzyme B12 for DhaB enzyme activity, carbon source, and The nitrogen source is an expensive component that is supplemented in the medium for 3-HP production, and therefore, a cheaper To identify and investigate the important medium components and formulate a suitable medium for 3-HP production and commercialization. Both are extremely important.

[0135] Apart from the formulation of industrial media, bioprocessing conditions such as temperature, pH, aeration, etc. The physiological parameters were investigated and optimized under controlled conditions. By converting the 3-HP into The important role of the recombinant strain in improving the acid tolerance and enzyme efficiency of the 3-HP production pathway was investigated. The effect of pH on 3-HP production was investigated. The optimization of aeration, temperature, and induction time was investigated. Improved performance of the recombinant strain producing 3-HP from ethanol was achieved.

[0136] As described in the Examples, for example, glucose, glutamate, gluconate The recombinant strain grew to higher cell densities and produced high titers of 3-HP. Various carbon sources were investigated that could support the production of β-glucan. The pH of the growth conditions was also investigated, and in some cases In this case, a neutralizing base was used in the bioreactor to maintain the pH. Also provided herein are bacterial strains that can grow at / or higher 3-HP concentrations. In some cases, the level of bicarbonate in the growth culture is controlled.

[0137] Nitrogen is another important component in the medium. As shown in the examples below, e.g. 3-HP production using various nitrogen sources, including corn steep liquor, yeast extract, etc. I looked it up.

[0138] Aeration is an important characteristic for cell growth. Aeration of the culture conditions used for cell growth However, excessive aeration can reduce the level of 3-HP production. This shows adequate aeration for bioproduction.

[0139] The present disclosure also provides for the identification of optimal temperatures for cell growth and target molecule production by recombinant microorganisms. This provides a balance between the optimal temperature for cell growth and the optimal temperature for target molecule production. Includes.

[0140] The timing of induction for 3-HP production can be adjusted based on the culture conditions and recombinant bacteria. For example, induction by addition of glycerol (as a source of 3-HP) at mid-logarithmic phase The introduction of the recombinant strain resulted in high levels of 3-HP production. Other culture conditions and / or recombinant strains were High levels of 3-HP can be produced by inducing the cells in early or late logarithmic phase. do.

[0141] (Removal of 3-HP from aqueous solution) In some embodiments, the method for extracting 3-HP from an aqueous solution comprises: To evaporate; To condense evaporated water-immiscible solvent to the liquid state; Water-immiscible liquid Extract 3-HP from the aqueous phase containing 3-HP using a water-immiscible solvent and dissolve 3-HP in a water-immiscible solvent. providing a solution of 3-HP in a water-immiscible solvent; and separating the solution of 3-HP in the water-immiscible solvent from the aqueous phase. Typically, the aqueous phase containing 3-HP is further treated with a water-miscible solvent prior to contact with the liquid water-immiscible solvent. Contains an organic solvent.

[0142] Generally, any suitable water-miscible or water-immiscible solvent, or any combination thereof, can be used as a water-soluble Examples of these solvents and their combinations are: Typical embodiments are the corresponding "water-immiscible solvents," "water-miscible solvents," and "solvent combinations" of the present disclosure. This is described in the "Matching" section.

[0143] Generally, any suitable process conditions may be used in the method for removing 3-HP from an aqueous solution. Illustrative example process parameters are referred to below as "process parameters." is described in the section entitled:

[0144] In general, 3-HP is a polar compound that has relatively high solubility in water and relatively low solubility in organic solvents. 3-HP is a commercially available product. For example, 3-HP is available as a 30 wt.% aqueous solution (catalog no. 3-HP can be purchased from Sigma-Aldrich as 792659, while 3-HP with 5-6 wt.% ethyl acetate can be purchased from Sigma-Aldrich as 792659. Although not wishing to be bound by theory, water-miscible solvents are difficult to obtain. When a solvent (e.g., methanol) is added to an aqueous solution of 3-HP, the solubility of 3-HP in the resulting solution The reaction temperature decreases, thereby reducing the removal of 3-HP from aqueous solutions with water-immiscible solvents (e.g., ethyl acetate). It is thought to make removal easier.

[0145] (aqueous solution) As used herein, the term "aqueous solution" refers to a solution containing one or more solvents (e.g., a mixture of solvents). refers to a solution of at least one solute in a liquid containing at least one solvent is water, and the weight percentage of water in the solvent or mixture of solvents 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.

[0146] 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., about 20g / L~Approx. 140g / L, Approx. 30g / L~Approx. 130g / L, Approx. 40g / L~Approx. 120g / L, Approx. 50g / L~Approx. 110g / L, Approx. 50g / L The concentration is about 60g / L to about 100g / L, about 60g / L to about 100g / L, about 60g / L to about 80g / L, or about 80g / L to about 120g / L). The concentration of 3-HP in a solution is the mass (titer) of 3-HP in a solution containing a specified volume of 3-HP, The amount of 3-HP in a given volume of solvent (molarity) or the amount of 3-HP in a given volume of solution (molarity) Additionally or alternatively, any of the following may be used to express the concentration of a solute in a solution: Other methods may be used to describe the concentration of 3-HP in aqueous solution. For example, some In an embodiment, the potency of 3-HP in the aqueous solution is at least about 40 g / L (e.g., about 50 g / L, about 6 0 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, or about 120 g / L).

[0147] In some embodiments, the aqueous solution is a fermentation broth obtained after decellularization. Any method for removing whole cells from tissue can be used for decellularization. The fermentation broth can then be decellularized using centrifugation. In the above, the aqueous solution is a fermentation broth (e.g., In some cases, the aqueous solution is decellularized. In an embodiment, the broth containing 3-HP is prepared by culturing a microorganism that produces 3-HP. Illustrative examples of such microorganisms include gram-negative bacteria, e.g., Escherichia coli, Oligotropha carboxidovorans, pneumonia Bacillus or Pseudomonas species; and Gram-positive bacteria, e.g., Bacillus subtilis lis), Lactobacillus sp, or Lactococcus spp. Microorganisms that can produce 3-HP by fermentation include Clostridium spp. Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas Domonas, Bacillus, Lactobacillus, Enterococcus, Al Alcaligenes, Klebsiella, Paenibacillus, Al Arthrobacter, Corynebacterium, Brevibacter Brevibacterium, Pichia, Candida, Hansenula Hansenula, or any member of the genus Saccharomyces. In some embodiments, the 3-HP-producing microorganisms are selected from the group consisting of the compounds described herein. Some examples of recombinant bacteria that produce 3-HP include Pseudomonas sp. strains, Creutzfeldt-Jakob strains, and others. Among the microorganisms that can produce 3-HP by fermentation are Bacillus subtilis and Escherichia sp. strains. Some examples include Alcaligenes eutrophus (Capria Cupriavidus necator, Bacillus licheniformis eniformis, Paenibacillus macerans, Rhodococcus Rhodococcus erythropolis, Pseudomonas putida a), Lactobacillus plantarum, Enterococcus fumigatus Enterococcus faecium, Enterococcus gallinarium inarium, Enterococcus faecalis, Bacillus subtilis, and Examples include Saccharomyces cerevisiae.

[0148] The fermentation medium in broth contains various nutrients and components normally available in bacterial or fungal growth media. Examples of such nutrients and components include: Illustrative, non-limiting examples are described herein. Examples of carbon sources include, for example, glucose. sugar, glutamate, gluconate, fructose, arabinose, or galactose sugars such as glucose, citric acid, or citric acid cycle intermediates such as pyruvate or cocoa Another example of a carbon source is glycerol. Suitable nitrogen sources include ammonium salts, corn steep liquor, yeast extract, and nitrates. Illustrative examples of additional components useful in fermentation media include serum proteins. , vitamins, nucleic acids, and amino acids.

[0149] In some embodiments, the decellularized broth containing 3-HP after fermentation is about 0.5 wt.% % to about 5 wt. % (e.g., about 1 wt. % to about 3 wt. %) of a carbon source, such as glycerol. do.

[0150] In some embodiments, the decellularized fermentation broth is cultured in a microorganism to produce 3-HP. Therefore, the combined amount of about 0.5 wt.% to about 20 wt.% (for example, about 1 wt.% to about 10 wt.%) is used. The composition contains components (e.g., as described herein) comprising:

[0151] In some embodiments, the broth after fermentation and decellularization is about 7 to about 8 (e.g., about 7 Such broths have a pH of about 7.5 or less. In some embodiments, an acid is added to the decellularized fermentation broth. Any suitable 3-HP may be added to increase the pH and obtain the free acid form of 3-HP in the broth. The pH of the fermentation broth can be adjusted using inorganic or organic acids. Exemplary inorganic acids include: Exemplary organic acids include formic acid, These include 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, about 40 g / L to about 50 g / L (for example, , approximately 45 g / L).

[0152] In some embodiments, the pH of the aqueous solution containing 3-HP is about 3 to about 7 (e.g., about 4 to about 7). About 7, about 4 to about 5, about 4 to about 6, about 4.1 to about 4.9, about 4.2 to about 4.7, about 4.2 to about 4.8, about 4.3 to about 4.6 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, It is about 4.5, about 4.6, or about 4.7.

[0153] In some embodiments, the pH of the aqueous solution typically increases upon removal of 3-HP from the aqueous solution. In such embodiments, the pH of the aqueous solution is adjusted by adding one or more acids to the aqueous solution sequentially or sequentially. By adding discontinuously, the amount of oxidized cellulose can be adjusted (continuously or discontinuously) during the removal process. Any gradual increase in pH due to 3-HP removal can be accommodated as desired.

[0154] In some embodiments, 3-HP is 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%, or less In either case, the yield of water is 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 solution (e.g., by HPLC). or by LCMS) so that only trace amounts of 3-HP can be detected in aqueous solution. In some embodiments, 3-HP can be completely removed from the solution. (i.e., the yield of the removal process is 100%).

[0155] As used herein, the term "yield" refers to the amount of the process used to obtain a product. The total amount of product obtained based on the amount of starting material used in the process and the amount of product obtained by the process It refers to the ratio (expressed as a percentage) of the theoretical amount of product that can be produced.

[0156] In some embodiments, 3-HP is removed from the aqueous solution without using countercurrent liquid flow. As used herein, the term "countercurrent liquid flow" means It refers to the bidirectional flow of two immiscible liquids after contact and phase separation. In other embodiments, the flow rate of one liquid is equal to the flow rate of the other liquid. Countercurrent extraction of a solute from an aqueous solvent by a water-immiscible solvent is a countercurrent liquid flow This is just one example of a method where 3-HP is removed from an aqueous solution without using countercurrent flow. In embodiments, 3-HP is used in, for example, techniques and devices for countercurrent flow of aqueous and organic solvents. It is possible to extract the aqueous solution with an organic solvent without using any of the countercurrent flow techniques. Methods and apparatus are described, for example, in PCT publications WO 2005 / 003074, WO 2013 / 192450, and WO 2013 / 1 It is described in No. 92451.

[0157] In some embodiments, 3-HP is prepared using an organic solvent or a combination of two or more organic solvents. In some embodiments, the aqueous solution can be removed by using The organic solvent for the removal of 3-HP from solution is prepared by (1) evaporating the solvent; and (2) dissolving the evaporated solvent. After condensation, it can be used. In some embodiments, the condensed solvent can be used. The flow is then turned so that, for example, 3-HP is removed from the aqueous phase by the solvent. By directing the flow into the aqueous solution to achieve effective mixing of the solvent and aqueous phase, 3-HP can be removed from

[0158] As used herein, the term "evaporation" refers to the conversion of a liquid into the gas phase (vapor). Evaporation of a liquid occurs when the vapor pressure is equal to the pressure exerted on the liquid by the surrounding atmosphere. In some embodiments, evaporation occurs when a liquid is heated. Typically, boiling of a liquid occurs when the liquid is heated above its boiling point. When a liquid containing a solvent is heated, the two solvents form an azeotrope and evaporate simultaneously. Unless you do this, the solvent with the lower boiling point will evaporate first, followed by the solvent with the higher boiling point. It is understood that the vaporizes.

[0159] As used herein, the term "condensation" refers to the process of conversion of a vapor to a liquid. Typically, condensation of a vapor occurs when the vapor is cooled below the boiling point of the liquid.

[0160] (Water-immiscible solvent) In some embodiments, the water-immiscible solvent (e.g., the water-immiscible organic solvent) is It can be used in processes to remove 3-HP from solutions, i.e., solutions of 3-HP that are water-immiscible. The aqueous solution can be combined with a water-immiscible solvent so as to form in a miscible solvent. The solution of 3-HP in the water-immiscible solvent can then be separated from the aqueous phase.

[0161] As used herein, the term "water-immiscible solvent" refers to a solvent that can be mixed with water to form a homogeneous It refers to a solvent that cannot form a liquid. For example, the solubility of a water-immiscible solvent in water is 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., For example, less than about 2 g, or less than about 1 g) of the water-immiscible solvent dissolves in 100 mL of water.

[0162] In some embodiments, the water-immiscible solvent has a density of less than about 1 g / mL. In such embodiments, the water-immiscible solvent is less dense than water and, when combined with water, In some embodiments, the water-immiscible solvent forms an organic phase above the aqueous phase. The solvent may be 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).

[0163] Illustrative examples of water-immiscible solvents that are less dense than water include C 5-10 Alkane, C 5-8 Shik C alkyl alkane, aromatic hydrocarbon solvent 1-6 Alkyl acetate, C 4-6 Alcohol, and C 1-6 Combinations of such water-immiscible solvents can be used. Cut.

[0164] Exemplary C 5-10 Alkanes include n-pentane, n-hexane, n-heptane, and n-octane. , and isooctane.

[0165] Exemplary C 5-8 Cycloalkanes include cyclopentane and cyclohexane. do.

[0166] Exemplary aromatic hydrocarbon solvents include benzene, toluene, o-xylene, and m-xylene. , p-xylene, and cumene.

[0167] Exemplary C 1-6 Alkyl acetates include methyl acetate, ethyl acetate, and n-propyl acetate. , isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, tert-butyl acetate Examples include n-pentyl acetate, n-hexyl acetate, and n-pentyl acetate.

[0168] Exemplary C 4-6 Alcohols include n-butyl alcohol, isobutyl alcohol, tert n-butyl alcohol, n-pentyl alcohol, and n-hexyl alcohol.

[0169] Exemplary C 1-6 Alkyl ethers include diethyl ether, dipropyl ether, methyl propyl ether, methyl tert-butyl ether (MTBE), and methyl hexyl ether Examples include:

[0170] In some embodiments, the water-immiscible solvent has a density greater than about 1 g / mL. In such embodiments, the water-immiscible solvent is denser than water and when combined with water In some embodiments, the water-immiscible solvent forms an organic phase below the aqueous phase. The solvent may be about 1 g / mL to about 1.5 g / mL (e.g., about 1.1 g / mL, about 1.15 g / mL, about 1.2 g / mL, about 1.25 g / mL, It has a density of about 1.3 g / mL, or about 1.4 g / mL.

[0171] In some embodiments, the water-immiscible solvent that is denser than water is C 1-4 Alkanes, C 1-4 Alkene, C 4-6 Cycloalkane, C 4-6 Cycloalkene, aromatic hydrocarbon solvent, C 1-6 a C 2-6 Alcohol, or C 1-6 Optionally, such alkyl ethers Such solvents may contain one or more (e.g., 1, 2, 3, 4, or 5) independently selected halogen atoms, e.g., For example, one or more Cl atoms, one or more Br atoms, one or more F atoms, or a combination thereof may be substituted. Exemplary embodiments of such water-immiscible solvents include C 1-4 Haloalkanes, C 1-4 Haloalkene, C 2-6 Haloalcohols and halogenated aromatic hydrocarbon solvents are included. Combinations of the water-immiscible solvents described in this paragraph may be used.

[0172] C 1-6 Illustrative examples of haloalkanes include chloroform, bromoform, chlorofluorocarbons, Carbon dioxide, methylene chloride, carbon tetrachloride, 1,1-dichloro-1-fluoroethane, 1,1,1-trichloroethane chloroethane, and perfluorodecalin.

[0173] C 1-4 Illustrative examples of haloalkenes include 1,2-dichloroethene, 1,1-dichloroethene, and trichloroethylene.

[0174] Illustrative examples of halogenated aromatic hydrocarbon solvents include chlorobenzene, 1,2-difluoromethane, Examples include trichlorobenzene, 1,2,4-trichlorobenzene, and trifluorotoluene.

[0175] C 2-6 Illustrative examples of haloalcohols include hexafluoro-2-propanol, 2,2,2- trifluoroethanol, and trichloro-2-methyl-2-propanol.

[0176] When using a water-immiscible solvent to remove 3-HP from an aqueous solution, the conditions are The concentration of 3-HP in the solvent is about 1 g / L to about 300 g / L (e.g., about 5 g / L to about 250 g / L, about 10 g / L to about 200 g / L). / L, about 20g / L to about 150g / L, about 30g / L to about 100g / L, about 40g / L to about 90g / L, or about 50g / L to about 80g / L ) can be selected.

[0177] In general, the amount of water-immiscible solvent relative to the amount of aqueous solution can be selected as needed. In some embodiments, the amount of water-immiscible solvent is based on the amount of aqueous solution. About 50 v / v% to about 150 v / v% (for example, about 80 v / v% to about 120 v / v%, or about 90 v / v% to about 110 v / v%) For example, the amount of the water-immiscible solvent is about 50 v / v%, about 60 v / v, based on the amount of the aqueous solution. / v%, approx. 70v / v%, approx. 80v / v%, approx. 90v / v%, approx. 100v / v%, approx. 105v / v%, approx. 110v / v%, approx. 120 v / v%, about 125 v / v%, about 130 v / v%, or about 140 v / v%.

[0178] (Water-miscible solvent) In some embodiments, a water-miscible solvent (e.g., a water-miscible organic solvent) is added from an aqueous solution. As used herein, "water-mixed" refers to a process for removing 3-HP from a water-mixed cellulose. The term "compatible" refers to a solvent that can be mixed with water in any proportion to form a homogeneous liquid. .

[0179] Generally, in embodiments where a water-miscible solvent is used in the process for removing 3-HP from an aqueous solution, In one embodiment, the water-miscible solvent is combined with the aqueous solution to form a homogeneous aqueous solution. A water-miscible solvent can be poured into an aqueous solution to form a homogeneous liquid. Then, a water-miscible solvent is added to the aqueous solution to: (1) evaporate the water-miscible solvent; (2) remove the evaporated water-miscible solvent; (3) directing a flow of the condensed water-miscible solvent into an aqueous solution, followed by forming a homogeneous solution. It can form a body.

[0180] In embodiments where a water-miscible solvent is used in the process of removing 3-HP from an aqueous solution, A single water-miscible solvent can be used, or a combination of water-miscible solvents can be used. Cut.

[0181] In some embodiments, the water-miscible solvent is C 1-3 It is an alcohol. C 1-3 Alcor Illustrative examples of alcohols include methanol, ethanol, n-propanol, and isopropanol. Examples include:

[0182] In some embodiments, the water-miscible solvent is a polar aprotic solvent. Illustrative examples of protic solvents include tetrahydrofuran (THF), dimethylsulfoxide (DMSO), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HMPT), dimethylformamide (DMF), acetonitrile Nitriles, dioxane, and acetone are included.

[0183] Generally, the amount of the water-miscible solvent relative to the amount of the aqueous solution can be selected as needed. In some embodiments, the amount of water-miscible solvent is about 1 v / v based on the amount of aqueous solution. % to about 50v / v% (e.g., about 1v / v% to about 40v / v%, about 1v / v% to about 30v / v%, about 5v / v% to about 50v / v%, about 10 v / v% to about 40 v / v%, about 15 v / v% to about 35 v / v%, or about 20 v / v% to about 30 v / v%) For example, the amount of the water-miscible solvent may be about 5 v / v%, about 10 v / v%, or about 1 It may be 5% v / v, about 20% v / v, about 25% v / v, about 30% v / v, or about 40% v / v.

[0184] (Combination of water-immiscible and water-miscible solvents) In some embodiments, both the water-miscible solvent and the water-immiscible solvent are converted from aqueous solution to 3- In such an embodiment, a water-immiscible solvent is generally used in the process of removing HP. The volume ratio of the solvent to the water-miscible solvent can be selected as needed. In the above, the volume ratio of the water-immiscible solvent to the water-miscible solvent is about 10:1 to about 1:1 (for example, about 9:1 to about 2:1, about 8:1 to about 2:1, about 7:1 to about 2:1, about 6:1 to about 2:1, or about 5:1 to about 3:1). , 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, It is about 8:1, or about 10:1.

[0185] In general, the ratio of 1) the combined volume of the water-miscible and water-immiscible solvents to 2) the volume of the aqueous solution must be In some embodiments, the water-miscible solvent and the water-miscible solvent may be selected as desired. The ratio of the combined volume of the immiscible solvents to the volume of the aqueous solution is about 1:1 to about 2:1 (e.g., about 1:1 to about 1. 5:1), for example, about 1:1, about 1.2:1, about 1.3:1, about 1.4:1, or about 1.5:1. do.

[0186] Generally, the relative boiling points of the water-miscible and water-immiscible solvents can be selected as desired. In some embodiments, the boiling point of the water-miscible solvent is higher than the boiling point of the water-immiscible solvent. In some embodiments, the boiling point of the water-miscible solvent is lower than the boiling point of the water-immiscible solvent. Higher than.

[0187] Generally, the water-immiscible and water-miscible solvents and their respective amounts may be selected as needed. You can choose.

[0188] In some embodiments, the water-miscible solvent is methanol, ethanol, acetone, Acetonitrile, THF, dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (HM The water-immiscible solvent is selected from methyl acetate, acetic acid, acetic acid, acetic acid methyl ester ... Ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, se acetate c-butyl, tert-butyl acetate, benzene, chlorobenzene, toluene, o-xylene, m-xylene xylene, p-xylene, n-butyl alcohol, isobutyl alcohol, tert-butyl alcohol Alcohol, diethyl ether, methyl tert-butyl ether (MTBE), methyl hexyl ether The solvent is selected from ethylenediamine, chloroform, methylene chloride, and carbon tetrachloride. In embodiments, the amounts of water-miscible and water-immiscible solvents can be selected as needed. By way of example, in such an embodiment, the amount of water-miscible solvent may be adjusted based on the amount of aqueous solution. The ratio of the water-miscible solvent to the water-immiscible solvent can be about 5 v / v % to about 50 v / v %. The volume ratio can be about 1:10 to about 1:2, and / or 1) a water-miscible solvent and a water-immiscible solvent. The ratio of the combined volume of the solvent to the volume of the aqueous solution 2) can be from about 1:1 to about 2:1.

[0189] In some embodiments, the water-miscible solvent is C 1-3 Alcohol (e.g., methanol) , ethanol, or isopropanol), and the water-immiscible solvent is an aromatic hydrocarbon solvent ( For example, benzene, toluene, o-xylene, m-xylene, p-xylene, or chlorobenzene In such an embodiment, the amounts of water-miscible and water-immiscible solvents are By way of example, in such an embodiment, a water-miscible The amount of the solvent can be about 5 v / v % to about 50 v / v % based on the amount of the aqueous solution. The volume ratio of the miscible solvent to the water-immiscible solvent can be about 1:10 to about 1:2, and / or 1) water The ratio of the combined volume of the miscible solvent and the water-immiscible solvent to the volume of the aqueous solution is about 1:1 to about 2:1. It is possible.

[0190] In some embodiments, the water-miscible solvent is C 1-3 Alcohol (e.g., methanol) , ethanol, or isopropanol), and the water-immiscible solvent is C 1-6 Alkyl acetate acetate (e.g., methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate) butyl acetate, isobutyl acetate, sec-butyl acetate, or tert-butyl acetate). In embodiments, the amounts of water-miscible and water-immiscible solvents can be selected as needed. By way of example, in such an embodiment, the amount of water-miscible solvent may be adjusted based on the amount of aqueous solution. The ratio of the water-miscible solvent to the water-immiscible solvent can be about 5 v / v % to about 50 v / v %. The volume ratio can be about 1:10 to about 1:2, and / or 1) a water-miscible solvent and a water-immiscible solvent. The ratio of the combined volume of the solvent to the volume of the aqueous solution 2) can be from about 1:1 to about 2:1.

[0191] In some embodiments, the water-miscible solvent is C1-3 Alcohol (e.g., methanol) , ethanol, or isopropanol), and the water-immiscible solvent is C 1-6 Alkyl ether diethyl ether, methyl tert-butyl ether (MTBE), or methylhexyl In such embodiments, the amount of water-miscible and water-immiscible solvents is can be selected as needed. By way of example, in such an embodiment, water The amount of miscible solvent can be about 5 v / v % to about 50 v / v % based on the amount of the aqueous solution. 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 combined volume of the water-miscible solvent and the water-immiscible solvent to 2) the volume of the aqueous solution is about 1:1 to about 2: :1.

[0192] In some embodiments, the water-miscible solvent is C 1-3 Alcohol (e.g., methanol) , ethanol, or isopropanol), and the water-immiscible solvent is C 1-6 Haloalkanes (e.g. For example, chloroform, methylene chloride, or carbon tetrachloride. In this case, the amounts of the water-miscible solvent and the water-immiscible solvent can be selected as needed. By way of example, in such an embodiment, the amount of water-miscible solvent is based on the amount of aqueous solution. The volume ratio of the water-miscible solvent to the water-immiscible solvent can be about 5 v / v% to about 50 v / v%. 1) a combination of a water-miscible solvent and a water-immiscible solvent; The ratio of the volume of the solution to the volume of the aqueous solution 2) can be about 1:1 to about 2:1.

[0193] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such an embodiment, the amounts of methanol and butyl acetate are: This can be selected as needed. The amount of methanol is about 5 v / v% to about 50 v / v% of the aqueous solution, and the volume ratio of methanol to butyl acetate is , and / or 1) the combined volume of methanol and butyl acetate. and 2) the aqueous solution, the volume ratio can be about 1:1 to about 2:1.

[0194] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such embodiments, the amounts of methanol and MTBE are adjusted as needed. By way of example, in such an embodiment, the amount of methanol can be selected from: The aqueous solution may be about 5 v / v% to about 50 v / v%, and the volume ratio of methanol to MTBE is about 1:10. and / or the ratio of 1) the combined volume of methanol and MTBE to 2) the volume of the aqueous solution The ratio of the amounts can be from about 1:1 to about 2:1.

[0195] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such an embodiment, methanol may be used. The amount of isobutyl alcohol can be selected as needed. 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 isobutyl alcohol is about 1:10 to about 1:2. and / or 1) the combined volume of methanol and isobutyl alcohol and 2) the volume of the aqueous solution. The ratio can be from about 1:1 to about 2:1.

[0196] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is and tert-butyl alcohol. The amount of ethyl alcohol can be selected as needed. In 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-butyl alcohol can be from about 1:10 to about 1:2; and and / or the ratio of 1) the combined volume of methanol and tert-butyl alcohol to 2) the volume of the aqueous solution is , can be about 1:1 to about 2:1.

[0197] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such an embodiment, the amounts of methanol and benzene are As an example, in such an embodiment, methanol The amount of methanol and benzene can be about 5% to about 50% v / v of the aqueous solution. The ratio can be from about 1:10 to about 1:2, and / or the combined volume of methanol and benzene and 2) the aqueous solution, the volume ratio can be about 1:1 to about 2:1.

[0198] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such an embodiment, the amounts of methanol and toluene are As an example, in such an embodiment, methanol The amount of can be about 5 v / v% to about 50 v / v% of the aqueous solution, and the volume of methanol and toluene The ratio can be from about 1:10 to about 1:2, and / or 1) the combined volume of methanol and toluene The ratio of the amount of 2) to the volume of the aqueous solution can be about 1:1 to about 2:1.

[0199] In some embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is , chloroform. In such an embodiment, the amounts of methanol and chloroform can be selected as needed. The amount of ethanol can be about 5 v / v% to about 50 v / v% of the aqueous solution, and the amount of methanol and chloroform can be about 5 v / v% to about 50 v / v% of the aqueous solution. The volume ratio of 1) methanol and chlorine can be from about 1:10 to about 1:2, and / or The ratio of the combined volume of chloroform to the volume of 2) aqueous solution can be from about 1:1 to about 2:1.

[0200] In certain exemplary embodiments, the water-miscible solvent is methanol and the water-immiscible solvent is In such an embodiment, the amount of methanol and ethyl acetate is can be selected as needed. The amount of ethanol can be about 15 v / v% to about 35 v / v% of the aqueous solution, and the amount of methanol and ethanol can be about 15 v / v% to about 35 v / v% of the aqueous solution. The volume ratio of the chill is about 1:9 to about 3:7, and / or 1) a combination of methanol and ethyl acetate. The ratio of the volume of the solution to the volume of the aqueous solution of 2) can be about 1:1 to about 2:1. In an embodiment, the concentration of 3-HP in the aqueous solution can be from about 50 g / L to about 80 g / L, and the aqueous solution 3. The pH can be about 4 to about 5, and the water content in the process of removing 3-HP from the aqueous solution The temperature of the solution is about 15°C to about 40°C.

[0201] In some embodiments, the method for extracting 3-HP from an aqueous solution comprises: (1) extracting 3-HP-containing extractant from the aqueous solution; An extraction vessel containing the cellular fermentation broth, approximately 90 v / v based on the amount of broth in the extraction vessel. providing a solvent container containing ethyl acetate in an amount of v / v% to about 110%; and a condenser; (2) evaporating ethyl acetate from the solvent container; (3) transferring the evaporated ethyl acetate to the condensate. (4) condensing the 3-HP in an ethyl acetate solution to a liquid state in the extraction vessel; (5) directing a flow of ethyl acetate from the condenser to the extraction vessel so that the ethyl acetate is (6) separating the ethyl acetate solution of 3-HP from the ethyl acetate solution of 3-HP in the extraction vessel; and In some embodiments, the method further comprises directing a flow of solvent solution from the extraction vessel to the solvent vessel. In this case, the extraction vessel contains about 20 v / v% to about 30 v / v% of methanol based on the amount of broth. In such an embodiment, the methanol is completely miscible with the broth and the extract The amount of ethyl acetate in such a system and the amount of methyl ethyl acetate remaining in the solvent vessel during the extraction process The ratio of the amount of ethanol is about 5:1 to about 3:1.

[0202] (process parameters) Generally, in the process of removing 3-HP from an aqueous solution, any suitable flow rate can be used to For example, in some embodiments, aqueous solutions can be used. The solvent flow in the process of removing 3-HP from the HCl 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).

[0203] Generally, any suitable temperature may be used in the process of removing 3-HP from an aqueous solution. For example, in some embodiments, removing 3-HP from an aqueous solution can be performed by about The reaction is carried out at an aqueous solution temperature of 15°C to about 50°C (e.g., about 15°C to about 40°C, or about 20°C to about 30°C). In some embodiments, removing 3-HP from the aqueous solution is carried out at temperatures up to about 50°C (e.g., For example, the temperature of the aqueous solution is about 40°C, or about 30°C. Optionally, the 3-HP is removed from the aqueous solution. The reaction is carried out at room temperature.

[0204] In some embodiments, 3- Heating HP results in decomposition of 3-HP as shown in Scheme 1: (Scheme 1) [ka]

[0205] Regarding Scheme 1, the hydrogen atom of the hydroxyl group at the 3-position is bonded to the carbonyl group of 3-HP. A polar protic solvent can form a bond, thereby forming a six-membered ring. Promotes the formation of the ring and the decomposition of 3-HP to form ethylene, carbon dioxide, and water. In one example, decomposition can be achieved by adding 50 mL of a solution of 3-HP in a polar protic solvent (e.g., water). This may occur when heated to temperatures above 100°C. Although not required, in some embodiments, the temperature is adjusted when removing 3-HP from the aqueous solution. Maintaining the temperature below 50°C reduces the formation of undesirable ethylene and / or carbon dioxide. It can be lowered.

[0206] In some embodiments, the temperature of the solution of 3-HP in the water-immiscible solvent is greater than the boiling point of the water-immiscible solvent. As an example, when ethyl acetate is used in the process, the acetic acid The temperature of the ethyl acetate solution may be about 78° C. to about 80° C. In another example, isobutanol may be used as a protease inhibitor. When used in the process, the temperature of the 3-HP isobutanol solution is about 105°C to about 110°C. Advantageously, the temperature is adjusted during the removal of the compound from the aqueous solution, and during the removal of the compound from the aqueous solution. When the solution is heated, it is chosen so that there is little or no decomposition of 3-HP to undesired products. It is selected.

[0207] In some embodiments, the process for removing 3-HP from an aqueous solution comprises, for example, at least at least about 70% (e.g., at least about 80%, at least about 90%, at least about 95%, or at least This results in a relatively high yield of recovered 3-HP, such as at least about 99%.

[0208] Exemplary Systems and Methods for Removing 3-HP from Aqueous Solutions Figure 58 shows the method for removing 3-HP from aqueous solution using a water-immiscible solvent that is less dense than water. 58, the system includes a solvent container 100 and a bypass tube 110. and a condenser 110 connected to the solvent container 100 via a hot water circulation system. The solvent vessel 10 is also connected to the extraction vessel 116 via a siphon tube 112 in a hot water circulation system. 00 includes a stir bar 102, a thermometer 124, and a heating element 104. The extraction vessel 116 includes a stir bar 120, a pH The pump includes a meter 122 and an inlet tube 114. The inlet tube 114 is connected to a peristaltic pump 126. A pump 126 is provided to add acid from an acid storage vessel 130 via an inlet line 114 to the extraction vessel 116. It is composed of:

[0209] In some embodiments, both the water-miscible solvent and the water-immiscible solvent are contained in the solvent container 10. Typically, an aqueous solution of 3-HP (e.g., a decellularized fermentation broth containing 3-HP) is placed in After the water-miscible and water-immiscible solvents are placed in the solvent vessel 100, they are then placed in the extraction vessel 116. Optionally, the aqueous solution of 3-HP may be placed in the solvent container 100 before the water-miscible solvent is placed in the solvent container 100. or simultaneously with, and / or before or after the water-immiscible solvent is placed in the solvent container 100. can be simultaneously placed in the extraction vessel 116. The amount of aqueous 3-HP solution added is determined so that no aqueous 3-HP solution is carried over into the solvent container 100. The solvent container 100 is selected so that the aqueous solution does not reach the side arm 108. The temperature of the liquid contained in the vessel 100, as determined by the temperature sensor 124, is The boiling point of a solvent (either water-immiscible or water-miscible) with a lower boiling point than that of the solvent The water-miscible solvent is heated using the heating element 104 until the temperature reaches a certain temperature. The water-miscible solvent in the solvent vessel 100 has a lower boiling point than the water-immiscible solvent in the solvent vessel 100. The water-miscible solvent vaporizes (without evaporation of the water-miscible solvent), and the vapor of the water-miscible solvent is released from the solvent container 100 through the side tube 108. The condenser 110 is heated to a temperature below the boiling point of the water-miscible solvent (e.g., For example, use the coolant at about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C. The evaporated water-miscible solvent condenses to a liquid state in the condenser 110, forming a liquid water-miscible solvent. The solvent (eg, at about room temperature) flows via siphon tube 112 to the bottom of extraction vessel 116 . The condensed water-miscible solvent combines with the aqueous solution of 3-HP to form an aqueous phase in the extraction vessel 116. A water-miscible solvent is initially placed in the solvent vessel 100 and then transferred to the extraction vessel 116. This amount is determined so that the aqueous phase in vessel 116 is side-amounted so that the aqueous phase is not carried over into solvent vessel 100. The solvent container 100 is then selected so that it does not extend to the room 108. The temperature of the liquid reaches the boiling point of the water-immiscible solvent, evaporating the water-immiscible solvent in the solvent container 100. The water-immiscible solvent is heated to a higher temperature using the heating element 104 until the The vapor flows from the solvent vessel 100 through a bypass 108 to a condenser 110. The condenser 110 is a A temperature below the boiling point of the immiscible solvent (e.g., about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C) Therefore, the evaporated water-immiscible solvent is The liquid water-immiscible solvent (e.g., at about room temperature) is condensed into a liquid state and the liquid water-immiscible solvent (e.g., at about room temperature) is removed from the siphon tube. The condensed water-immiscible solvent flows via 112 to the bottom of the extraction vessel 116. Extracting 3-HP from the aqueous phase, thereby forming a solution of 3-HP in a water-immiscible solvent (organic phase). The extraction vessel 116 is then filled with the aqueous phase and the organic phase. Since water-immiscible solvents are less dense than water, the organic phase will be in contact with the aqueous phase. The aqueous solution of 3-HP, the water-miscible solvent, and the water-immiscible solvent are initially placed in the system. The combined amount of solvent is selected so 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 to the solvent vessel 100. In this way, 3-HP is transferred from the extraction vessel 116 to the solvent vessel 100. A decrease in the amount of aqueous phase in extraction vessel 116, as detected, for example, by pH meter 122, In response to this increase in pH, the peristaltic pump 126 pumps acid into the acid storage volume. The water is then pumped from the pump 130 through the injection pipe 114 into the extraction vessel 116, thereby The aqueous phase in the extraction vessel 116 is stirred (e.g., continuously) with a stirrer 120 to adjust the pH of the aqueous phase. The aqueous phase in extraction vessel 116 and the aqueous phase in extraction vessel 116 may be stirred (by stirring) to enhance pH uniformity of the aqueous phase. The temperature of the organic phase can be about the same, typically about 15°C to about 40°C (e.g., Typically, the pH of the aqueous phase in extraction vessel 116 is adjusted to within the ranges discussed above. 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).

[0210] In one embodiment, the water-miscible solvent is first placed in the extraction vessel 116, and the water-immiscible solvent is The reactive solvent is first placed in a solvent container 100. Typically, an aqueous solution of 3-HP (e.g., a solution containing 3-HP) is The decellularized fermentation broth is placed in an extraction vessel 116 with a water-miscible solvent and a water-immiscible solvent. is placed in the solvent vessel 100 and then placed in the extraction vessel 116, optionally containing an aqueous solution of 3-HP. The liquid may be added before or at the same time as the water-miscible solvent is placed in the extraction vessel 116 and / or after the water-miscible solvent is placed in the extraction vessel 116. The immiscible solvent is placed in the extraction vessel 116 before or at the same time as it is placed in the solvent vessel 100. The aqueous solution of 3-HP and a water-miscible solvent in extraction vessel 116 can be combined. to form an aqueous phase in extraction vessel 116. The combined amounts of 3-HP and the aqueous solution of the water-miscible solvent are To prevent the aqueous phase from being carried over into the solvent vessel 100, the aqueous phase must not extend to the side arm 108. The solvent container 100 is selected to have a solvent volume as determined, for example, by a thermometer 124. Heating element 104 is used to heat the water-immiscible solvent contained in vessel 100 until the temperature reaches its boiling point. The water-immiscible solvent in the solvent container 100 is evaporated, and the water-immiscible solvent vapor is The water flows from the vessel 100 through a side tube 108 to a condenser 110. The condenser 110 contains a water-immiscible solvent. Coolant at a temperature below the boiling point (for example, about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C) Therefore, the evaporated water-immiscible solvent is evaporated to a liquid state in the condenser 110. The condensed liquid water-immiscible solvent (e.g., at about room temperature) is then released via siphon tube 112. The condensed water-immiscible solvent flows to the bottom of the extraction vessel 116. The water-immiscible solvent is then removed from the aqueous phase to form 3-H The extraction is carried out so as to extract P, thereby forming a solution of 3-HP in a water-immiscible solvent (organic phase). The aqueous phase in the extraction vessel 116 is mixed with the organic phase. The water-immiscible solvent is less dense than water, so the organic phase is on top of the aqueous phase. Aqueous solutions of 3-HP, water-miscible solvents, and water-immiscible solvents are initially placed in the stem. The amount is selected so that the organic phase in the extraction vessel 116 reaches the side arm 108. The organic phase flows from the extraction vessel 116 through the side arm 108 to the solvent vessel 100. 3-HP is then transferred from extraction vessel 116 to solvent vessel 100. As the amount of 3-HP in extraction vessel 116 decreases, This increases the pH of the aqueous phase in the extraction vessel 116, as detected, for example, by pH meter 122. In response to this increase in pH, peristaltic pump 126 pumps acid from acid storage container 130. The aqueous phase is then fed via pipe 114 into extraction vessel 116, thereby adjusting the pH of the aqueous phase in extraction vessel 116. The aqueous phase in the extraction vessel 116 is stirred (e.g., continuously stirred) with a stir bar 120 to separate the aqueous phase. The temperature of the aqueous phase in extraction vessel 116 and the organic phase in extraction vessel 116 may be increased. can be approximately the same, and is typically about 15°C to about 40°C (for example, about room temperature). Typically, the pH of the aqueous phase in extraction vessel 116 is adjusted to within the ranges discussed above. 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, about 4.3 to about 4.4, about 4.3, or about 4.4).

[0211] Figure 59 shows the method for removing 3-HP from aqueous solution using a water-immiscible solvent that is denser than water. 3 shows an exemplary system including a solvent reservoir 300, a condenser 310, and The solvent vessel 300 includes an extraction vessel 316. The solvent vessel 300 is hydronicly connected to a condenser 310 through a side pipe 308. The solvent vessel 300 is also connected to an extraction vessel 316 through a side pipe 312 in a hot water circulation system. The condenser 310 is hydronicly connected to the extraction vessel 316 through a connecting tube 334. The solvent vessel 300 includes a stir bar 302, a heating element 304, and a thermometer 332. The extraction vessel 316 is The system includes a meter 320 and an inlet tube 324. The inlet tube 324 is connected to a peristaltic pump 326, which Pump 326 is configured to add acid from acid storage vessel 330 via inlet line 324 to extraction vessel 316. It has been completed.

[0212] In some embodiments, both the water-miscible solvent and the water-immiscible solvent are present in the extraction vessel 300. Typically, a water-miscible solvent and a water-immiscible solvent are placed in the solvent container 300. Prior to this, an aqueous solution of 3-HP (e.g., a decellularized fermentation broth containing 3-HP) was placed in extraction vessel 316. However, optionally, the aqueous solution of 3-HP may be placed in a water-miscible solvent in the solvent container 300. and / or after the water-immiscible solvent is placed in the solvent container 300. After or simultaneously with the extraction, the extractor 316 may be placed in the extraction vessel 316. The volume of the aqueous solution of 3-HP placed in the solution container 300 is determined by the volume of the solution that flows into the solution container 300 via the side tube 312. The solvent vessel 300 is selected to be sufficiently large that it does not overshoot the temperature range. When determined by the above, the temperature of the liquid contained in the solvent container 300 is higher than that of the liquid contained in the solvent container 300. Until the boiling point of the solvent with the lower boiling point (either water-immiscible or water-miscible) is reached , and heated using heating element 304. Generally, water-miscible solvents have a lower viscosity than water-immiscible solvents. The water-miscible solvent in the solvent vessel 300 has a low boiling point (compared to the vaporization of the water-immiscible solvent in the solvent vessel 300). The water-miscible solvent vapors (without evaporation) from the solvent container 300 through the side pipe 308 to the condenser. The condenser 310 is configured to have a temperature below the boiling point of the water-miscible solvent (e.g., about 5°C to Use the coolant at approximately 25°C, approximately 5°C, approximately 10°C, or approximately 15°C. Therefore, the evaporated water The miscible solvent condenses to a liquid state in the condenser 310, leaving a liquid water-miscible solvent (e.g., The condensed water-miscible solvent (which is at about room temperature) flows into the extraction vessel 316 via the connecting tube 334. , combined with an aqueous solution of 3-HP to form an aqueous phase in extraction vessel 316. The amount of water-miscible solvent initially placed and then transferred to extraction vessel 316 is determined by the amount of aqueous is selected so that the phase is not large enough to flow into the solution container 300 via the side tube 312. The solvent container 300 is then heated to a temperature above the boiling point of the water-immiscible solvent. 300 is heated using the heating element 304 until the temperature reaches 100° C. and the water-immiscible solvent in the solvent container 300 is evaporated. The water-immiscible solvent vapor is heated to a high temperature. The condenser 310 is heated to a temperature below the boiling point of the water-immiscible solvent (e.g., The coolant is used at a temperature of about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C. The evaporated water-immiscible solvent condenses to a liquid state in the condenser 310, forming a liquid water-immiscible solvent. The condensed solvent (e.g., at about room temperature) flows into the extraction vessel 316 via connecting tube 334. The water-immiscible solvent extracts 3-HP from the aqueous phase, thereby The organic phase is mixed with the aqueous phase in the extraction vessel 316 to form a solution of 3-HP in the solvent (organic phase). Therefore, extraction vessel 316 contains an aqueous phase and an organic phase. The organic phase is below the aqueous phase because it is denser than the 3-HP that is initially placed in the system. The combined amounts of the aqueous solution, water-miscible solvent, and water-immiscible solvent are determined so that the organic phase in extraction vessel 316 is: Enough to allow the organic phase to pass from extraction vessel 316 via side conduit 312 to solvent vessel 300. In this way, 3-HP is transferred from extraction vessel 316 to solvent vessel 300. A decrease in the amount of 3-HP in the extraction vessel 316, as detected, for example, by a pH meter 320, The pH of the aqueous phase in the extraction vessel 316 tends to increase. In response to this increase in pH, the peristaltic pump Pump 326 pumps acid from acid storage vessel 330 through injection tube 324 into extraction vessel 316, thereby The pH of the aqueous phase in the extraction vessel 316 is adjusted by the addition of the HCl solution. The temperature of the organic phase can be about the same, typically about 15°C to about 40°C (e.g., about Typically, the pH of the aqueous phase in extraction vessel 316 is adjusted to within the ranges discussed above. 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).

[0213] In one embodiment, a water-miscible solvent is placed in extraction vessel 316 and a water-immiscible solvent is placed in extraction vessel 300. This is usually preceded by an aqueous solution of 3-HP (e.g., The decellularized fermentation broth (produced by the method described above) is placed in extraction vessel 316. However, optionally, 3-HP The aqueous solution may be added after or at the same time as the water-miscible solvent is placed in the extraction vessel 316, and / or After or simultaneously with the water-immiscible solvent being placed in the solvent vessel 300, The aqueous solution of 3-HP and the water-miscible solvent in the extraction vessel 316 can be combined. This forms an aqueous phase in extraction vessel 316. The aqueous solution of 3-HP in extraction vessel 316 and the water-miscible The combined volume of solvent is determined by the amount of aqueous phase in extraction vessel 316 flowing into solution vessel 300 via side tube 312. The solvent container 300 is selected so that it is not large enough to cause a The temperature of the liquid reaches the boiling point of the water-immiscible solvent, causing the water-immiscible solvent in the solvent container 300 to evaporate. The water-immiscible solvent vapor is heated to a certain temperature using heating element 304 until the vapor is The solvent flows from the solvent container 300 through a side pipe 308 to a condenser 310. The condenser 310 contains a water-immiscible solvent. The reaction mixture is cooled to a temperature below the boiling point of the solvent (for example, about 5°C to about 25°C, about 5°C, about 10°C, or about 15°C). Therefore, the evaporated water-immiscible solvent is in a liquid state in the condenser 310. The liquid water-immiscible solvent (e.g., at about room temperature) is then passed through connecting tube 334. The condensed water-immiscible solvent flows from the aqueous phase to the extraction vessel 316. to form a solution of 3-HP in a water-immiscible solvent (organic phase). The aqueous phase in the extraction vessel 316 is mixed with the organic phase. Since the water-immiscible solvent is denser than water, the organic phase is below the aqueous phase. The combined amount of the aqueous solution of 3-HP, the water-miscible solvent, and the water-immiscible solvent initially placed in the system The organic phase in the extraction vessel 316 is transferred from the extraction vessel 316 to the solvent vessel 300 via the side pipe 312. The amount of 3-HP is selected so that it is large enough to transfer to the extraction vessel. 316 to the solvent vessel 300. As the amount of 3-HP in the extraction vessel 316 decreases, e.g., a pH As detected by the detector 320, the pH of the aqueous phase in the extraction vessel 316 tends to increase. In response to the increase in pH, peristaltic pump 326 extracts acid from acid storage vessel 330 via inlet tube 324. The aqueous phase is then pumped into extraction vessel 316, thereby adjusting the pH of the aqueous phase in extraction vessel 316. The temperatures of the aqueous phase in extraction vessel 316 and the organic phase in extraction vessel 316 can be about the same, typically The pH of the aqueous phase in extraction vessel 316 is typically about 15° C. to about 40° C. (e.g., about room temperature). The pH of the aqueous phase is adjusted to within the ranges discussed above. For example, in some embodiments, the pH of the aqueous phase is , about 4 to about 7 (for example, 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) .

[0214] (Purification of 3-HP) In some embodiments, 3-HP can be purified after removal from the aqueous solution. As an example, crude 3-HP (3-HP after removal from aqueous solution but before purification) was treated with alkali. The salt is then washed with an organic solvent to give a pure alkaline gold salt of 3-HP. The pure alkali metal salts of 3-HP can be converted to the pure 3-HP free acid. In such an embodiment, the alkali metal salt of 3-HP can be obtained by hydroxylation of crude 3-HP. formed by treatment with an alkali hydroxide such as sodium hydroxide or potassium hydroxide To carry out the reaction, a solution of crude 3-HP in an organic solvent can be formed, The resulting solution can be treated with an alkali metal hydroxide. 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 about 4 to about 5 (e.g., In some embodiments, the alkali metal hydroxide is The reaction mixture is added until the pH of the reaction mixture is about 7. 3-HP precipitates from the reaction mixture. The alkali metal salt can be recovered by filtration and further washed with an organic solvent such as those described above. can.

[0215] To obtain pure 3-HP free acid, a salt of 3-HP, e.g., the sodium salt, can be dissolved in water. and treating the resulting aqueous solution with any one of the acids described herein. For example, an aqueous solution of 3-HP salt can be treated with hydrochloric acid or oxalic acid until the pH reaches about 4 to about 5. Pure 3-HP free acid can be obtained by any of the methods described herein. It can be removed from the aqueous solution.

[0216] (Production of acrylic acid) In some embodiments, the method for producing acrylic acid from 3-HP comprises reacting 3-HP. The method includes reducing the acrylic acid content by at least about 50% (e.g., at least 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% yield of acrylic acid. In this case, the process provides a quantitative yield of acrylic acid (i.e., 100% yield).

[0217] In some embodiments, the method includes reacting 3-HP in a liquid state. In such embodiments, 3-HP may be undiluted or in an aqueous solution or in one or more The solvent may be in a solution of the above organic solvents. Exemplary organic solvents include DMSO, DMF, and water.

[0218] Generally, the process for producing acrylic acid can be carried out at any suitable temperature. In some embodiments, the method for producing acrylic acid includes heating 3-HP to at least about 50°C (e.g., For example, heating to a temperature of about 60°C, about 70°C, or about 80°C. In the reaction, 3-HP is reacted at a temperature of up to about 200°C (e.g., about 190°C, or about 180°C). In some embodiments, 3-HP is reacted to produce acrylic acid. This is carried out at a temperature of about 50°C to about 200°C (for example, about 60°C to about 190°C, or about 80°C to about 180°C). It can be done.

[0219] Generally, the reaction of 3-HP to produce acrylic acid can be carried out at any suitable pressure. In some embodiments, the pressure adjacent to the reaction mixture containing 3-HP is greater than atmospheric pressure. When the temperature is lower than the reaction temperature, acrylic acid evaporates from the reaction mixture. The acid can be removed from the reaction mixture in gas form. Reacting HP to form acrylic acid can be accomplished, for example, by adding 3-HP to a reaction mixture containing 3-HP. In some embodiments, the reaction is carried out at low pressure, such as at a pressure of less than about 1 atmosphere. The pressure adjacent to the containing reaction mixture 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 less than r. is 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 In some embodiments, the reaction mixture containing 3-HP is The adjacent pressures are about 70 mbar, about 74 mbar, about 75 mbar, about 80 mbar, about 90 mbar, about 100 mbar, or is about 120 mbar.

[0220] In some embodiments, the reaction mixture catalyzes the conversion of 3-HP to acrylic acid. Typically, an acid catalyst is used to catalyze the conversion of 3-HP to acrylic acid. It can be mediated. (Scheme 2) [ka]

[0221] With reference to Scheme 2, when 3-HP is contacted with an acid, the hydroxyl group at the 3-position is protonated, Then, an elimination reaction occurs to produce acrylic acid and water. , any one of the organic and inorganic acids described herein. For example, Hydrochloric acid, sulfuric acid, polyphosphoric acid, oxalic acid, or acetic acid are used to catalyze the reaction of 3-HP to form acrylic acid. In some embodiments, the zeolite, silica, or Sea sand may be used as a catalyst for the reaction. Suitable examples of zeolites include 3A molecular sieves, Examples of molecular sieves include 4A molecular sieves and 5A molecular sieves.

[0222] In general, the amount of catalyst used can be selected as needed. In embodiments, the reaction mixture comprises from about 1 wt.% to about 25 wt.% 3-HP based on the amount of 3-HP in the reaction mixture. wt.% (for example, about 1wt.% to about 20wt.%, about 2wt.% to about 20wt.%, about 1wt.% to about 10wt.%, about 1 The catalyst may be present in an amount of from about 2 wt.% to about 4 wt.%. In an embodiment, the amount of catalyst in the reaction mixture is based on the amount of 3-HP in the reaction mixture. , about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 10 wt.%, about 20 wt.%, or about It is 25wt.%.

[0223] The acrylic acid formed from 3-HP in the reaction mixture polymerizes to form polyacrylic acid. The method for producing acrylic acid described herein facilitates the formation of polyacrylic acid. The desired product is advantageously isolated, e.g., at least 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 It provides high yields, such as approximately 99%.

[0224] To reduce undesired polymerization, in some embodiments, 3-HP is used as a polymerization inhibitor. Illustrative examples of polymerization inhibitors include: Phenothiazine, hydroquinone, 4-tert-butylpyrocatechol, tert-butylhydroquinone 1,4-benzoquinone, 6-tert-butyl-2,4-xylenol, 2,6-di-tert-butyl-p-cre Examples of suitable phenols include phenol, 2,6-di-tert-butylphenol, and 4-methoxyphenol.

[0225] Generally, the amount of polymerization inhibitor used can be selected as needed. In one embodiment, the polymerization inhibitor is about 1 wt.% based on the amount of 3-HP in the reaction mixture. ~ 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, the amount of polymerization inhibitor is present in the reaction mixture in an amount of 0.1 to 1.0% by weight. Based on the amount of 3-HP in the reaction mixture, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 5 wt.%, about 10 ... %, or about 15 wt.%.

[0226] In some embodiments, the acrylic acid formed from 3-HP is removed from the reaction mixture. The removal of acrylic acid may be carried out continuously throughout the course of the reaction, optionally until all of the 3-HP has been removed. The reaction can be carried out until acrylic acid is formed, or the removal of acrylic acid can be carried out during the reaction period. Optionally, discontinuously throughout the reaction until all of the 3-HP has reacted to form acrylic acid. can be done.

[0227] As mentioned above, acrylic acid can be removed from the reaction mixture as gaseous acrylic acid. In such an embodiment, the gaseous acrylic acid removed from the reaction mixture can be converted into liquid acrylic acid. It can be condensed to a state where it can be recovered and used for its intended purpose. In an embodiment, a coolant is used to condense gaseous acrylic acid. The temperature of the coolant used to condense acrylic acid at pressures lower than to about 10°C (for example, about 0°C to about 5°C).

[0228] Exemplary Systems and Methods for Producing Acrylic Acid To react 3-HP to form acrylic acid and distill the acrylic acid from the reaction mixture An exemplary system is shown in Figure 60. The system includes a reaction vessel 200, a solvent liquid, and a A still head 214 connected to the reaction vessel 200 via a connection 212, a solvent trap 224, a still head A condenser 226 on the still head 214 and a distilled gas stream connected to the still head 214 via a valve 236 The reaction vessel 200 includes a stirrer 206, a heating element 202, a collection vessel 238 for collecting the acrylic acid. A thermometer 208 for controlling the temperature inside the reaction vessel 200, as well as 3-HP (and optionally, a solvent and catalyst) into the reaction vessel 200. The still head 214 includes a cooling jack having a coolant inlet pipe 220 and a coolant outlet pipe 222. The collection vessel 238 includes a container 218. Additional components, such as polymerization inhibitors, may be added to the collection vessel 238. The recovery vessel 238 includes an inlet tube 242 that can remove acrylic acid from the system. The inlet tube 242 is used to connect the collection vessel 238 to a further vacuum line. The condenser 226 is connected to the coolant inlet pipe 230 and the coolant outlet pipe 232. The condenser 226 provides the system with a cooling jacket 228 with tubes 232. It also includes a junction 234 that can be connected to the jack.

[0229] Prior to the removal process, the reaction vessel 200 is charged with 3-HP and, optionally, a catalyst such as, for example, 4A molecular sieves. Then, apply a vacuum to the system to create a pressure of approximately 70-100 mbar in the system. The reaction mixture is then heated using a heating mantle 202 until the temperature is at a constant temperature, as determined by a thermometer 208. Heat to a temperature of about 80°C. At this temperature, the 3-HP begins to react to form reaction products, acrylic acid and At a pressure of about 70-100 mbar, the acrylic acid and water formed during the reaction The acrylic acid and water are evaporated and the vapor flows through the solvent line 212 to the still head 214. A portion of the contained vapor condenses in the still head 214, and the liquid contained in the still head 214 The remaining vapor travels through trap 224 to condenser 226, where it The remaining gaseous acrylic acid and water condense and flow through trap 224 to stillhead 214. Once a sufficient amount of liquid product mixture has been collected in the still head 214, the liquid is transferred to the open The gas flows through valve 236 into collection vessel 238. Valve 236 is closed to allow the gas to flow through the still head 214. Undesired flow of contained liquid into collection vessel 238 may be prevented.

[0230] In some embodiments, the present disclosure provides a process for the preparation of acrylic acid by reacting 3-HP to form acrylic acid. The method includes: (1) providing a reaction vessel containing 3-HP, a collection vessel, and a condenser; (2) reacting 3-HP to form acrylic acid in a reaction vessel; (3) reacting the acrylic acid with the (3) evaporating the evaporated acrylic acid in the condenser to a liquid state; (4) directing the flow of acrylic acid from the condenser to the collection vessel; In some embodiments, the reaction vessel also includes changing the catalyst. In some embodiments, the reaction vessel also contains a polymerization inhibitor. Exemplary embodiments of polymerization inhibitors, catalyst and polymerization inhibitor amounts, and reaction conditions are described in the " The method is described in the section entitled "Production of Acrylic Acid."

[0231] (Acrylic acid purification) In some embodiments, the method described in the "Production of Acrylic Acid" section of this disclosure is The acrylic acid prepared by any of the methods may be further purified to remove any polymerization products and unreacted products. Acrylic acid free from 3-HP can be obtained by the conventional method for purifying acrylic acid. The method involves the polymerization of acrylic acid at atmospheric pressure (of which acrylic acid is 2-methyl-2-propanol) in the presence of a polymerization inhibitor such as 4-methoxyphenol (MEHQ). The boiling point of the acrylic acid is about 141°C at about 760 mmHg. When heated to near 1000 K, acrylic acid polymerizes rapidly, even in the presence of polymerization inhibitors. Therefore, the yield of the conventional process is only moderate (for example, 40-60%). The disclosed method advantageously avoids polymerization and provides, based on the amount of crude acrylic acid before purification, It is possible to purify acrylic acid with a yield of at least about 50% (e.g., about 75% to about 95%). In some embodiments, the pure acrylic acid is obtained in the form of an aqueous mixture. The concentration of acrylic acid in such a mixture is 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.%.

[0232] In some embodiments, the present disclosure provides a process for the preparation of acrylic acid by distilling it at low pressure. In one embodiment, the acrylic acid is purified by distillation. The pressure exerted by the atmosphere around the acid is typically between about 70 mbar and about 100 mbar (e.g. , about 75 mbar to about 105 mbar, or about 80 mbar to about 100 mbar), and the temperature is typically about 80 °C to about 120 °C (for example, about 90 °C to about 110 °C, or about 80 °C to about 100 °C). 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 an embodiment, the distillation is carried out in the presence of a polymerization inhibitor, and the amount of polymerization inhibitor is optionally The polymerization inhibitor can be selected depending on the amount of the polymerizable compound (for example, about 1 wt.% to about 20 wt.%). The polymerization inhibitor may be any one of the polymerization inhibitors described in, or a combination thereof.

[0233] In some embodiments, the present disclosure provides a process for purging a vessel containing crude acrylic acid with a gas. In some embodiments, a method for purifying acrylic acid is provided, comprising: The gas may be air, nitrogen, or argon. In some embodiments, the temperature of the gas is , below the boiling point of acrylic acid. In such an embodiment, 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, purging is In this process, acrylic acid is heated to a temperature of about 1000 K at about atmospheric pressure. It slowly evaporates (without boiling) and the flow of warm gas releases pure water from the container. In some embodiments, the acrylic acid vapor is purged with a gas. The system for cooling includes a condenser, and the temperature of the coolant in the condenser is about The temperature is between 5°C and approximately 25°C. When the warm gas containing acrylic acid vapor passes through the condenser, , the acrylic acid condenses to a liquid state while the carrier gas is not retained in the condenser; Leave the system. Pure acrylic acid is recovered from the condenser and used for its intended purpose. It can be used. [Example]

[0234] (Example) The present disclosure will be further illustrated by the following examples, which are provided in accordance with the claims. This does not limit the scope of the present disclosure, which is set forth in the ranges.

[0235] Example 1. Specificity of small molecule inducers for the MmsR transcriptional activator protein A gene expression system inducible by 3-HP or similar small acids was developed in P. denitrifican. This is a novel expression system identified in the literature. Analysis of the gene arrangement around the 3-HP decomposition gene in the P. denitrificans genome revealed that This revealed the presence of a putative LysR family transcriptional regulator, MmsR. When complexed with 3-HP, the protein activates the transcription of mmsA, hpdH, and other genes. The putative transcriptional activator protein MmsR has an N-terminal tail for DNA binding. Rix-turn-helix domain, C-terminus for inducer binding in response to 3-HP It consists of a terminal domain, a linker connecting these two domains, and induces MmsR. Studies were conducted to determine the therapeutic range of molecules that can be used to treat L-lactic acid (LAC), acetic acid ( AcOH), propionic acid (PA), 3-hydroxybutyrate (3-HB), 1,3-propanediol (1,3- PDO), 2,3-butanediol (2,3-BDO), L-valine (L-val) and its degradation intermediates Various acids and alcohols, including 3-hydroxyisobutyrate (3-HIB), were used to induce MmsR. Most of the acids and alcohols tested were found to be soluble in water, primarily with 3-HP. It was selected for its similarity in size and / or structure. However, its transcription is not regulated by MmsR. The prepared mmsA and hbdH-4 enzymes are involved in L-val degradation. Malonylsemialdehyde dehydrogenase and 3-hydroxyisobutyl dehydrogenase Therefore, L-val and 3-HIB encode methylmalonyl semialdehyde dehydrogenase. and was selected based on its similarity to 3-hydroxyisobutyl dehydrogenase.

[0236] P. denitrificans was cultured in a minimum of 100 ml of water with and without each compound to be tested. The strains were cultured in medium, and the transcription of mmsA and hbdH-4 was measured by quantitative RT-PCR (Fig. 2A). The housekeeping gene rpoD, which encodes 70, was used as a reference. Two genes (mmsA and The transcription of the genes (mmsA, 154-fold; hbdH-4, 146-fold) was observed in P. denitrificans, which was induced by 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), as well as 3-HP (mmsA , 134-fold; hbdH-4, 128-fold). In contrast, LAC, AcOH, When exposed to PA, 1,3-PDO, and 2,3-BDO, limited induction was observed or induction was not observed. Since 3-HP, 3-HB, and 3-HIB are all β-hydroxy acids, the structures Both the carboxyl group and the β-hydroxyl group are similar in 3-HP, 3-HB, and 3-HIB. These three compounds appear to be essential for the ability of L-val to bind to MmsR. The structural difference is large and it is converted to 3-HIB. The induction by L-val is not due to L-val itself, but to , due to 3-HIB derived from L-val.

[0237] Malonic semialdehyde (MSA) and malonic acid derived from 3-HP, but not 3-HP and / or 3-HIB Methylmalonic acid semialdehyde (MMSA) derived from L-val and 3-HIB is a physiological inductive We also investigated the possibility that aldehydes are toxic and act as catalysts (see Figures 2A and 2B). Aldehyde degradation genes are often upregulated when aldehydes accumulate in cells. 3-HP is degraded by enzymes encoded by hpdH, hbdH-4, and hbdH-1, and the missing The mutant P. denitrificans ΔhpdHΔhbdH-4ΔhbdH-1 expresses the ΔhpdH gene at an appreciable rate. In this triple mutant, which does not produce MSA from 3-HP, the translocation of mmsA is The transcription factor was still upregulated by 3-HP (Fig. 3). This suggests that 3-HP is a key player in the conversion of MSA to MSA. It was shown that the MmsR protein can be activated without the need for a pre-existing MmsA protein. Transcription of MmsR was upregulated by 3-HIB in the triple mutant, and 3-HIB also upregulated MmsR. Interestingly, the triple deletion mutant Transcription of mmsA in P. denitrificans is suppressed in either the absence or presence of 3-HP. was higher than that of the wild-type strain.

[0238] To confirm that MmsR is a transcriptional activator, deletion experiments and subsequent complementation experiments were performed. In the deletion mutant (ΔmmsR), the transcription of both mmsA and hbdH-4 was low (Fig. 2B). However, the level of mmsR was not affected by 3-HP. When reintroduced into the ΔmmsR mutant, the upregulation of both mmsA and hbdH-4 by 3-HP was completely blocked. These results suggest that MmsR is a transcriptional activator for the expression of mmsA and hbdH-4. Interestingly, the mmsA protein in the ΔmmsR mutant The basal level of transcription of hbdH-4 was ~2-fold higher than that of the wild-type or mmsR-complemented recombinant (mmR-C). This indicates that the MmsR protein without an inducer inhibits the transcription of mmsA and hbdH-4. This suggests that it can be suppressed to some extent.

[0239] hbdH-4 expression was regulated by 3-HP, but the promoter sequence was Because it lacks an operator site for binding of an activator protein, the promoter of mmsA is The promoter sequence was significantly different from that of the hbdH-4 promoter. The mmsA-hbdH-4 intergenic region was then transfected with a promoter containing green fluorescent protein (GFP) as a reporter. Furthermore, electrophoretic mobility shift assays (EMSA) were performed on the purified This was carried out using the isolated MmsR protein and a DNA fragment of the hbdH-4 promoter region. Our experiments also showed that the hbdH-4 promoter is constitutive and not induced by 3-HP. .P hbdH-4 GFP under the control of the MmsR protein and the hbdH-4 promoter region is constitutively expressed. Furthermore, no binding of the 3-HP domain was observed in cells grown in the presence of 3-HP (data not shown). In qRT-PCR experiments, abundant polycistronic mRNA transcripts of mmsA and hbdH-4 were detected. These results suggest that the transcription of hbdH-4 is essential for the 3-HP-induced P mmsA and constructive P hbdH-4 Toi hbdH-4 is controlled by two independent promoters, and the addition of 3-HP significantly up-regulated hbdH-4. Nodes (see Figures 1B and 2B) are P mmsA This suggests that the problem is due to skipping of transcription from the original text. The inventors also noticed that no terminator sequence was present in the intergenic region between mmsA and hbdH-4. It's attached. P hbdH-4is not inductive, so P mmsA was examined in detail.

[0240] Example 2. In silico analysis of the mmsR-mmsA intergenic operator-promoter region In silico analysis of the intergenic region between two divergently transcribed genes, mmsR and mmsA This study identified: (i) a putative upstream promoter of the mmsA transcription start site (TSS); (ii) two putative tandem operator sites O1 and O2, and (iii) two encompassing the two half-sites of O1; Three TNs, the last of which encompasses the O2 half-site 11 -A motif, cis-acting elements Each operator is located upstream of the putative TSS of the mmsA gene. Each contained a dyad-symmetric DNA sequence centered at positions -81 and -33. is common to many prokaryotic operators that are recognized by regulatory proteins. The distance between the centers of the palindromic regions is ∼50 bp, corresponding to a 5-turn helical DNA. Nucleotide sequence symmetry in the O1 site dyad, consisting of two 9-bp fragments separated by 15 bp, is observed. The inverted repeats of the O2 site separated by 11 bp are less symmetrical and have only one mismatch. , 6 out of 9 bases are mismatched. By alignment, a consensus sequence [ka] The presence of three bases, A, G, and T (Fig. 3B, bold), at positions 2, 4, and 5 was shown (Fig. 3B). Each of these fragments is completely conserved in all of them, with three bases, C, T, A ( The underlined regions were highly conserved in each of the three fragments. The identity of the putative O2 region was somewhat questionable. It should be noted that one of the half-sites shows very low conservation (3 / 9). The size of the spacer was 4 bp shorter than that in O1. Further evidence to confirm this should be obtained through in vivo and in vitro experiments ( See examples below).

[0241] LysR-type transcription regulators (LTTRs) are the largest family of transcription factors in prokaryotes. The LTTR-binding DNA sequences are called RBS (regulatory binding site) and ABS (activator binding site). The RBS has two symmetric operator regions, where the RBS has a larger pair compared to the ABS. Furthermore, the symmetric half-sites of the RBS and ABS often correspond to the T-(N11)-A motif. These sequence features, along with the structural features of MmsR, are known to be involved in the This indicates that the gene belongs to the LTTR family of transcriptional regulators. Our analysis of the transcription of mmsR (see Fig. 3A) revealed that the transcription of mmsR is mediated by its own product, MmsR. The O1 site is located in the putative -10 region upstream of mmsR. The O2 site completely overlapped with the putative -35 promoter region upstream of mmsR. The binding of the MmsR protein at the O2 site regulates the RNA polymerase activity at the mmsR promoter. This may interfere with binding and / or its movement along the DNA strand during transcription. Since the two half-sites are located in the 11-bp-long spacer region, we hypothesized that transcription of mmsA is mediated by MmsR. It was also noticed that the binding of proteins could be disrupted. The basal levels of transcription of mmsA and hbdH-4 in the sR mutant are comparable to those in the wild-type counterpart. This may explain why the levels are two-fold higher than those observed in the in silico predictions (see Figures 2A and 2B). To verify the validity of the measurement, we performed both in vivo and in vitro analyses of the intergenic region. Extensive studies have been carried out (see examples below).

[0242] Example 3. In vivo characterization of the mmsR-mmsA intergenic operator-promoter region Characterization of the promoter region controlling mmsA gene expression by 5' end mapping and in vivo To identify the -10 and -35 regions, the upstream region was examined by random mutagenesis in Various mutations were introduced into P, including sequential deletions in the P region, or randomization of the putative -10 and -35 regions. mmsA P The mutant promoter was fused to the gfp reporter gene. MmsR was able to bind to the promoter and affect the expression of the gfp reporter gene (Fig. 4). Therefore, P. denitrificans lacking the mmsR gene was used to test the mutant promoter. The host was used for the experiment. mmsA _Δ1) or -117 to -37 (P mmsA _Δ2)'s When the promoter region was deleted, gfp expression was significantly higher than that of the control containing the full-length promoter (P mmsA _ The concentration of α-glucan in the α-glucan-containing α-glucan was reduced by approximately 16% compared to the wt (Table 1). mmsA _Δ3) or -14(P mmsA _Δ4) When the longer upstream sequence before the promoter was deleted, the promoter strength was significantly reduced by 39% or 58%. From these results, PmmsA _Δ3 and P mmsA The sequence deleted in _Δ4 is ​​essential for transcription. Important elements, possibly the -10 and -35 regions, predicted by in silico analysis, were identified. Furthermore, it is suggested that it contains -10(P mmsA _Δ-10) and -35(P mmsA _Δ-35) region Randomization in P mmsA Compared with the promoter strength of _wt, the promoter strength 5'-end mapping and randomized mutagenesis revealed that P mmsA -10 and -3 The in silico predictions of five regions are confirmed. Table 1: GFP (AU / OD; In vivo mmsR-mmsA intergenic region analysis based on expression of AU (arbitrary units) [Table 1] a. Showing complementation of MmsR expression on the plasmid. b. Fluorescence was measured three times (n=3) 4 hours after induction and averaged. c. For induction, 25 mM 3-HP was added to the growth medium.

[0243] To investigate the location and content of the operator regions (O1 and O2) in vivo, 5' end mapping was performed. Mutations were also performed using genomic DNA and random mutagenesis. (See the following examples), mmsR is a weak P c1 Plus under the control of the promoter (hbdH-I promoter) As shown in Table 1, both operators (O1 and O2) ) showed high 3-HP induction (up to 34.6-fold). The first half-site of the constant O1 site (BS_Δ2) alone or in combination with the other half-sites of O1 or O2 (BS_Δ3 or When BS_Δ4 was deleted together with BS_Δ5, the inducibility was completely abolished.

[0244] We also investigated promoters with mutations in the O1 and / or O2 regions. The - sequence was randomized to break dyad symmetry, but the -35 region was left unchanged. As expected, randomization of O1 or both O1 and O2 completely abolished the inducibility of 3-HP. These results suggest that the O1 operator region is located downstream of -98. However, these results clearly define the role of the O2 region. When only the estimated O2 region was randomized (BS_ΔO2), the 3-H The promoter strength in the absence of P was significantly higher than that of the wild-type BS_wt. In the presence of 3-HP, the intensity was increased by 37% (compared to the intensity of wild-type BS_wt). Furthermore, both O1 and O2 were mutated simultaneously. When the promoter was induced in the presence of BS_ΔO1O2, the promoter strength was significantly reduced and the promoter was not induced. This indicates that the O2 site functions in close cooperation with the O1 site and that P mmsA Function and strength It is suggested that it plays an important role in regulating the

[0245] Overall, the in vivo studies on the operator area can be summarized as follows: (i) O1 alone can activate transcription (as in the case of the 3-HP-MmsR complex), whereas O1 and O2 can activate transcription. less efficient than when both are present, and (ii) MmsR without 3-HP for the O2 operator. The bond is P mmsA (iii) a ΔO2 mutation that can suppress transcription from the promoter High-strength promoters of the genus still require the presence of an O1 site. mmsA professional It is most likely that the motor is subject to positive regulation (in the presence of 3-HP) and negative regulation (in the absence of 3-HP). expensive.

[0246] Using a GFP reporter, we investigated the effect of MmsR binding within the operator region on mmsR expression. The results were also investigated. mmsR The promoter encodes its own protein product, the MmsR protein. When mmsR was deleted (P mmsR _wtΔmmsR), GFP expression was ~2-fold The mutation in O2 (P mmsR _ΔO2) did not affect GFP expression. and O1 mutation (P mmsR _ΔO1) slightly increased it by ~20%. These results , and MmsR occupies only the O1 region. mmsR Can repress transcription from promoters Furthermore, the presence of 3-HP does not affect the repression by the MmsR protein. This indicates that this repression is not 3-HP dependent. By ing, P mmsR Attempts to identify the -10 and -35 regions of P (Fig. 5A) were also unsuccessful. mmsR Promo The promoter strength was very weak, and differences in the mutant promoters were not achievable. (p>0.05).

[0247] The LTTR protein and its cis-acting elements have been studied in several microorganisms. In E. coli and Salmonella typhimurium LT2, the ilvC gene (acetohydroxy acid isomerase; IlvY protein (LysR-type transcriptional regulatory protein) that controls the expression of the IL-1 gene (encoding EC 1.1.1.86) In these studies, transcription of the ilvC gene was controlled by acetohydroxy acid isomerase. It is induced by acetohydroxybutyrate or acetolactate, which are substrates of the enzyme. The induction was mediated by the IlvY protein. mmsA The ilvY and ilvC genes, as well as the promoter, The promoter region of the gene has two operators, O1 and O2, each of which The repeats consisted of homologous sequences, [ka] and Salmonella typhimurium LT2 [ka] Furthermore, P. denitrificans P mmsA Like promoters, these The symmetry between the two dyads in the strain was higher in O1 (single mismatch) than in O2 (six mismatches). Another LysR-type activator, AtzR, present in Pseudomonas species, was strict in A It has been reported that AtzR shows differences in the BS operator region. However, ABS activates expression of the ATPase cleavage operon atzDEF. Three subunits designated ABS-1, ABS-2, and ABS-3 have different roles in the activation process. In vivo mutation analysis revealed that the ABS-1 and ABS-2 subsites contain the P motif. aztDEF In contrast, ABS-3 was shown to be involved in the full activation of the promoter. When AtzR is located at the ABS-2 and ABS-3 subsites, it functions as a "knit trap" and inhibits P aztDE F This results in the inactivation of P mmsA For the promoter, the O2 region is similar to many other LTTR-mediated The results showed that the two regions overlapped completely with the -35 region, as in the E. coli system. The transcriptional activators IlvY, ClcR, and AtuR from P. putida and P. aeruginosa also regulate the −35 promoter. The overlapping region has the same arrangement in which the overlapping region is located within the O2 subsite. The loop region binds to the LTTR dimer, thereby controlling the upregulation of downstream genes. However, the ability to repress downstream genes in the absence of inducer molecules has not been reported. not present.

[0248] Example 4. In vitro production of MmsR protein and its binding operator site For in vitro biochemical characterization, the protein was modified with six histidine residues at the C-terminus. Recombinant MmsR protein tagged with a nucleotide residue was produced and purified from recombinant E. coli. Not only the natural MmsR protein but also the His-tagged MmsR protein contains a His residue at the C-terminus of MmsR. Whether present at the nucleus or N-terminus, the assay was carried out as described in Example 1. The recombinant C-terminal His-tag appeared to function in the complementation experiments (see Figure 6C). Only MmsR was further investigated in vitro. Culture conditions (e.g., temperature, culture medium, IPTG concentration, The harvest time and 3-HP concentration, as well as various factors such as GroEL-ES, DnaKJ-GrpE, and trigger factors, were also investigated. After optimization of the co-expression with chaperones, recombinant MmsR was expressed in a soluble form at high levels in E. coli. The His-tagged MmsR protein was purified by affinity chromatography (Fig. 5A-E). The size of the protein was estimated to be 34.4 kDa, which is the size of the protein based on the 6×his-mmsR gene sequence. Native PAGE and / or gel filtration analysis revealed that MmsR The protein was monomeric at a low concentration of 65 nM, but dimeric at a high concentration of 550 nM (Figure 6B).

[0249] Binding of MmsR to the mmsR-mmsA intergenic region was examined in vitro by EMSA (Fig. 7A). The complete 130 bp DNA fragment of (F 12 fragments; see Figure 3B), 0–72.7n The purified MmsR protein of M was treated with the DNA probe in the presence of 3-HP as an inducer. The DNA fragments (F 1M2M )of The band shift in electrophoresis was determined by the presence of MmsR. When added, the intact 130 bp DNA fragment appeared, and the shifted DNA and the shifted The ratio of unbound DNA increased with increasing protein concentration during the incubation period. In contrast, such a migration delay was observed for the control DNA fragment at up to 72.7 nM of MmsR protein. This indicates that MmsR is a natural P mmsA Strength against It has been shown that MmsR has a high binding affinity and forms a binding complex in vitro. Proteins showed multiple bands migrating at shorter distances, which were the DNA fragments and the proteins. The results were attributed to the formation of various oligomeric complexes between the DNA proteins. The probe has two binding sites (O1 and O2) for MmsR, and MmsR binds to DNA as a dimer. Therefore, DNA and proteins form a variety of oligomeric complexes at high concentrations. Figure 7A shows the relationship between MmsR and P mmsA 3-H for binding affinity between promoters The effect of P is also shown. The band shift appears at lower MmsR concentrations when 3-HP is added. Also, oligomeric complexes that migrated shorter distances showed lower activity in the presence of 3-HP. This indicates that 3-HP appears faster at higher MmsR concentrations than at higher P mmsA Binding affinity between promoters It was suggested that this promotes Table 2: EMSA fragments used in this study [Table 2] a Bold letters indicate O1 operator half-sites; italic letters indicate O2 operator half-sites. The underlined letters indicated the site-directed mutagenesis region.

[0250] DNase I footprinting analysis to analyze MmsR binding regions in vitro The longer fragments, including the complete 130 bp intergenic region, were analyzed by capillary electrophoresis (Fig. 7B). A 169-bp DNA fragment (used in the EMSA experiment described above) was used as a probe (Table 2). The concentration of MmsR was varied from 0 to 1.2 μM. The results of the screening clearly showed the presence of two protected regions by MmsR, which , respectively, corresponding to operators O1 (centered at position -81) and O2 (centered at position -33) Protection became more pronounced with increasing MmsR concentrations. The exact region of protection has been difficult to pinpoint at the base pair level of the DNA sequence.

[0251] The EMSA experiment was repeated at a lower DNA concentration (0.4 nM) to determine the dissociation constant between the MmsR and PmmsA promoters. number K D One is the one that contains both the O1 operator site and the O2 operator site (F 12 ) The other two are F 12 has the same length as the O1 operator site (F 1M2 ) or O2 Operator Tar area (F 12M ) is mutated (randomized) in the palindromic region of Among these three DNA fragments, F 12 has the strongest affinity for MmsR followed by F 12M , then F 1M2 The MmsR binding (K D ) The dissociation constant for MmsR was calculated by plotting the MmsR-bound DNA fraction against the free MmsR concentration in the reaction buffer. The MmsR protein-DNA isotherm curve was determined from the MmsR protein (Fig. 9). When the quality was estimated in the absence of 3-HP, K D is F 12 10.7nM for F 12M About is 18.6nM, F 1M2 The binding affinity of 3-HP to the DNA fragment was 79.8 nM (Figure 9). In the presence of 3-HP, the K (at the monomeric MmsR concentration) D The value is F12 Two 5.4nM for F 12M 19.8nM for F 1M2 The value for F was estimated to be 316 nM (Figure 9). 12M or F 1M2 Higher than F 12 The affinity of MmsR to one operator (probably O1) to operator binding so that binding stimulates binding to another operator (perhaps O2). This suggests that some degree of cooperation exists. 12 About The presence of only one DNA-MmsR complex band indicates cooperativity in binding between the two operators. This supports the notion that there exists: if MmsR binding to O1 does not stimulate binding to O2 Two DNA-MmsR complex bands (one for O1 binding and another for O1 and O2 binding) were observed. It was supposed to appear.

[0252] Rhee et al. used either tandem operators (O1O2) or only O1 or O2 operators. The binding affinity of the E. coli IlvY protein to the DNA fragment containing is highest for fragments containing tandem O1O2 operators, and The Kapp of the monomeric IlvY was 4.4 nM for the tandem operator and 4.5 nM for O1. The values ​​were determined to be 35.2 nM for I and ~900 nM for O. However, Rhee et al. It has also been reported that the inducer does not affect the binding of IlvY protein to the operator. Here, in the presence of 3-HP, the two retarded bands were observed at the highest protein concentration. The changes in the properties of DNA-binding proteins in the presence of inducers were distinguished by This could have an effect on the formation of coalescence, possibly as explained in the above examples. As shown, when the concentrations of DNA and MmsR increase beyond a certain level, they undergo different Multiple complex oligomeric structures with mobility can be formed.

[0253] Example 5. Specificity of small molecule inducers for the HpdR transcriptional activator protein HpdR, like MmsR, recognizes 3-HP or similar small acids and is a ubiquitous nucleotide sequence in P. denitrificans. It is a transcriptional activator protein that can stimulate the expression of specific genes in Figure 10A shows the genes and intergenic regions in the HpdR-regulated operon in P. denitrificans. The arrangement of the regions is shown. The hpdR and hpdH genes are located in the Pseudomonas genome database. Conserved in all available sequenced Pseudomonas genomes, this The genomic region has the same structure. The hpdH gene encodes 3-hydroxybenzoate, an enzyme involved in 3-HP catabolism. The hpdR gene encodes the enzyme hydroxypropionate dehydrogenase (EC number 1.1.1.59). Compared with the structural gene hpdH, L, which is composed of 304 amino acid residues and is divergently transcribed, Sequence analysis revealed that the intergenic region between hpdR and hpdH is 124 nucleotides long. The HpdR promoter is nucleotide bp long and contains two promoters for the expression of hpdR and hpdH. The ribosomal binding sites (RBS and ABS, which are the regulatory and activating binding sites, respectively) and the ribosomal binding sites (RBS and ABS, which are the regulatory and activating binding sites, respectively) are also present. The DNA binding site of HpdR is probably threefold. It has putative tandem operator sites ABS-1, -2, and -3 and RBS-1 and -2, and two T-N11 Only the -A motif is located within this intergenic region, one at RBS-1 and the other at RBS-2. The presence of the T-N11-A motif is essential for the LTTR-mediated expression system. This is one of the typical features (see Fig. 10A ). The three operators are located at the hpdH transcription start site (TSS; upstream at positions -52, -36, and -21, respectively, relative to the NNPP tool (predicted using the NNPP tool). The expression of the hpdR gene was investigated using RBS-2. Pseudopalindromic repeats such as ABS-1 and ABS-2 mask the region between the transcription start site and the -35 region, which HpdR binding at these operator regions completely abolished RNA polymerase binding, and Therefore, it is possible to autorepress the transcription of its own gene, hpdR. On the other hand, the two 9-bp fragments at the RBS and ABS sites are separated by 5, 7, and 6 nucleotides, respectively. The nucleotide sequences of these three inverted dyads contain three mismatches at each subsite. There was a gap, so it wasn't completely symmetrical.

[0254] Example 6. Transcriptional activation of hpdH by HpdR HpdR is a transcriptional activator of the hpdH gene in Pseudomonas denitrificans To test this in vivo, deletion mutants were used. The mutant strains, i.e., PdΔhpdR and PdΔmmsR, and the recombinant strain P harboring pUCPK′ / Pc1-hpdR Several P. denitiliificans strains were generated, including dΔhpdR (for complementation tests), and the WT strain (ATCC13 867), and hpdH transcription was analyzed. To determine whether crosstalk exists between HpdR and MmsR, we investigated the putative C4 transcriptional activity. The PdΔmmsR strain, which has a deletion of the activator protein, was tested. The mRNA expression levels were determined. The effects of 3-HP on P. denitrificans were compared in the presence and absence of the inducer. The increase in hpdH expression from the canthus chromosome (WT) was 43.0±10.9-fold, but its transcriptional activation In the C4 operon, mmsA and hpdH-IV were not affected by 3-HP (Fig. 11A). The expression of m was also activated by 3-HP by 151.3±18.2-fold and 149.3±9.6-fold, respectively. The expression of msR was not activated at all (Fig. 11B). The genes for the proteins (i.e., hpdR and mmsR) were expressed at low levels, but these proteins Protein-regulated genes (hpdH, mmsA, and hpdH-IV) were highly expressed in the presence of 3-HP. In particular, the C4 operon mmsA and hpdH-IV showed high expression, and the high-strength promoter Once hpdR was deleted (ΔhpdR), transcription of hpdH was inactivated (1.2 0±0.58); when hpdR was complemented by a recombinant plasmid expressing HpdR (hpdR-C), The transcriptional activation was restored to the wild-type level (60.5±6.1). When present as a inducer, hpdH, one of the catabolic genes in the C3 operon of 3-HP, We concluded that the C3 operon is positively regulated by its transcriptional regulator HpdR. To investigate the crosstalk between the C4 operons, PdΔhpdR and hdpR complemented recombinant P. denito The expression of the C4 operon genes (mmsA and hbdH-IV) in the C. rificans strain was observed. The expression of hbdH-IV and hbdH-IV was not affected by the presence and / or deletion of hpdR, and the C3 transcriptional activator We showed that the C3 operon is specific for the C3 operon and actually regulates the C4 operon (Fig. 11B). Similarly, the C4 transcriptional activator (MmsR) does not affect the expression of the C3 operon genes. (Data not shown). Taken together, these results suggest that HpdR and MmsR share the same inductive Although they are activated by the 3-HP receptor and belong to the same LTTR family, Its binding to the operator site and subsequent transcriptional activation of the gene under its control It has been shown to be highly specific.

[0255] Example 7. Autoregulation of HpdR LTTR regulatory proteins regulate transcription by repressing transcription initiation in an inducer-independent manner. It is known to autoregulate its own promoter. The regulatory binding site (operator) of C3 has been implicated as a possible autoregulatory site. To understand the autoregulation of HpdR in peron, we investigated (i) PC1 or HpdR, constitutively expressed using the Pzwf promoter, hpdR G is expressed under the control of FP, and (ii) P eda and P fbp Several genes, including GFP, are constitutively expressed using a promoter. Several plasmids were constructed. GFP fluorescence was observed in P hpdR In this case, the gene is expressed using a promoter. In this case, HpdR is P zwf promoter or PC1(P zwf2 by one of the promoters (weaker than GFP is expressed at two different levels. Autoregulation of GFP was monitored over a 24-hour culture period. When HpdR was expressed using Pzwf or PC1, GFP reached saturation levels in 10 hours. However, despite the constitutive expression of HpdR, there was no further expression. Fluorescence is P eda and P fbp When regulated by a constitutive promoter such as The expression level of GFP fluorescence was determined based on the promoter strength, i.e., P hpdR , P eda , and P fbp This result suggests that the LTTR protein is affected by its own This was consistent with the fact that the promoter is autoregulated (Fig. 12).

[0256] Example 8. Inducer specificity of HpdR in the C3 operon Carbon number (C3 or 4), molecular mass, including 3-HP, L-val, BA, IBA, LAC, AA, IVA, and PA. and some acids and aldehydes selected according to their similarity to 3-HP in terms of chemical structure. The inducer specificity of HpdR was investigated using the hpdH and gfp gene transcription and G The relative induction was examined based on the fluorescence from FP. The transcription levels of HpdH and gfp, as well as GFP Based on fluorescence, 3-HP was determined to be the best inducer of HpdR. Compared to the control without inducer (Ctrl), transcription of hpdH was increased by 75±8.3-fold. was statistically significantly different from the results obtained with other chemicals (Figure 12A). The transcription of H also increased 52.8±27.4-fold upon exposure to L-valine, and L-valine significantly increased these It was the second most potent inducer in the experiment. However, other acids and propyl aldehyde is either less effective or disrupts the HpdR-mediated regulatory C3 gene expression system. Induction of 3-HP and L-valine using the C3 (hpdH and hpdR) regulon These findings regarding sex are described in the above example for the C4 regulon (mmsA and MmsR). The results were similar to those reported.

[0257] Structurally, 3-HP contains a carboxyl group that plays an important role in the binding ability of ligands to HpdR. It contains both a carboxyl group and a β-hydroxy group. hpdH encodes 3-HP dehydrogenase. is also involved in L-valine degradation, and its metabolites can exhibit inducing activity. L-valine, which is structurally different from 3-HP, induces the Cm operon (manuscript in preparation). It can be easily converted to 3-hydroxyisobutyric acid (3-HIB), which can be synthesized by L-val. The induction of 3-HIB is due to its conversion to 3-HIB. The hpdH gene was replaced with gfp in the plasmid pUCPK' / Pzwf-hpdR-PC3-gfp, and the transcription of g The reporter strain PdΔhpdRΔhpdH carrying gfp was heterogeneously expressed as described above. Treatment with several chemicals at 25 mM, including 3-HP, L-val, BA, IBA, LAC, AA, IVA, and PA. The expression of gfp mRNA was maximally induced by 3-HP, which was significantly higher than that of the control. This resulted in a 3.4-fold increase in expression, which was statistically significant relative to the control. Other chemicals tested did not induce mRNA expression (Fig. 12B). The induction was greatest by 3-HP (3-fold and 3.3-fold) at 6 and 11 hours after induction (Fig. 12C). This was consistent with the results of the gfp gene expression, which showed a 3-fold induction ratio. When complexed with HpdR in the C3 operon, it is the most dominant activator of target gene transcription. It is a specific inducer.

[0258] By varying the concentration of 3-HP in the range of 0.1 mM to 25 mM, the inducible promoter of hpdH was The dose effect of 3-HP on the system was also investigated. The fold increase in GFP fluorescence, expressed as the induction ratio, was linear. At 6 and 11 hours after induction, the levels were 0 to 0.5 mM. Concentrations higher than ∼25 mM showed a similar increase in induction (Fig. 12D). The C3 operon is regulated by HpdR in vivo and in vitro via a mediated induction process. The inducers were characterized through examination of inducer specificity at the transcriptional and translational levels. The enzyme specificity and strength of the hpdH promoter system were investigated. , the highest specificity when complexed with HpdR for induction of this gene expression system. and an overall 3-fold induction ratio of the hpdH promoter was observed in vivo for GFP expression. It was shown that this would be the case.

[0259] Example 9. In vivo analysis of HpdR binding sites In silico sequence analysis of the 5' region of PhpdH revealed five putative operators (RBS-1, RBS-2, A The presence of ABS-1, ABS-2, and ABS-3 was revealed. To elucidate the importance of the operators, we mutated the repeat sequences of each of the putative operators. One of the five operator repeats was a 9-bp random DNA sequence (conserved). Random DNA sequences that show neither the conserved T-N11-A motif nor sequence conservation with the actual subsites Created by the generator tool [ka] ) to replace the pUCPK' / Pzwf-hpdR-PC3-gfp plasmid. By constructing a recombinant plasmid and then measuring the GFP level (reporter), The inducibility of gene expression was assessed (data not shown). Mutation of ABS-1 significantly reduced 3-HP inducibility (data not shown). The effect of mutations at the ABS-3 site was less significant, while mutations at the RBS-2 site were , but only slightly affected the inducibility of the PC3 promoter. The RBS-1 and ABS-2 sites are important binding sites for HpdR to activate the transcription of hpdH (or gfp). , whereas the other operator sites are suggested to be less important for HpdR binding.

[0260] LTTR studies show that the LysR protein dimer binds cooperatively to two operator sites. Generally, when the RBS is first occupied, it is the ABS that helps recruit the second LTTR dimer. In this case, the RBS plays a key role in the efficient recruitment of LTTR dimers for the transcriptional activation of target genes. The RBS site is more important than the ABS site because of the functional role of the two reactions in one operator. Which of the repeats (i.e., RBS-1 and RBS-2) is blocked by the LTTR protein binding? It is unclear whether the repeats are essential for the helix-turn-helix structure of the LysR protein. However, the low PC3 Considering the promoter strength, the affinity of HpdR for the operator may be weak. This may be due to the lower contribution of RBS-2 in recruiting the HpdR protein. Although the RBS-2 mutation was examined, no inducible changes were observed. This suggests that the contribution of β-actin to LysR is not sufficiently high. Altering RBS-2 to have a more consensus sequence may improve LTTR binding. Further detailed studies on the role of RBS-2 in plaque formation can be performed. The modest decrease in expression after mutation of both repeats of hpdH suggests that this ABS is a putative promoter of hpdH. hpdH, which fits snugly into the motor site and whose mutations result in inducible fluctuations This may be due to the fact that the mutation on ABS-2 caused a change in the promoter. The mutation rendered PC3 constitutive and inducible for HpdR-mediated transcriptional activation in the presence of 3-HP. In summary, each of the operator mutations except for RBS-2 appeared to be related to the 3-HP In the case of HpdR complexed with α-HpdR, this caused a loss of inducibility, and this change This resulted in functional modification of the promoter from being constitutive to having constitutive characteristics. This change in gene expression due to the promoter mutation is due to (i) activation of hpdH (or gfp) transcription; (ii) reduced affinity of HpdR for its DNA binding site; (iii) DNA silencing leading to low transcription of target genes Inappropriate conformational changes of the HpdR protein-operator DNA complex on bending; (iii) expression This may be due to several hypothetical reasons, such as a significant change in the strength of the hpdH promoter, which results in a decrease in Therefore, operator mutations may alter inherent promoter characteristics. can bring about.

[0261] Example 10. Binding site randomization and modification of activator expression levels Gene expression can be modified at the transcriptional and / or translational level. modified the operator sites (O1 and O2) to form P mmsA Improving transcription from the promoter We attempted to identify the intergenic region of mmsR-mmsA, where the activator protein MmsR binds. The operator sites present in the promoter region regulate the affinity of the promoter region for RNA polymerase. The kgsA gene plays an important role in determining the function of the mmsR-mm The O2 gene was cloned downstream of the intergenic region of sA and consisted of half-sites of the operator. Two different parts of the operator sequence can be separated (P mmsA2a and P mmsA2b ) or in combination (P m msA2ab ) was randomized (Figure 15A). The transcriptional activator MmsR was inserted into the natural promoter (P mmsR ) and expressed from the bacterial chromosome. The intact intergenic sequences without mutations were used as controls. Used as. 2a Or O 2b Mutations in 3-HP increased the transcription level from 7.3 to 10.6 The first half of the O2 region (O 2a ) suddenly The mutation occurs in the second half (O 2b ) significantly improved transcription efficiency compared to the mutation in the O2 operator. When both half-sites of the 3-HP gene were randomized, the basal level of transcription (without 3-HP) was 2a Or O 2b The basal transcription levels were comparable to those obtained from either mutation (Fig. 15B). However, the maximum transcription level in the presence of 3-HP was reduced, and the level was higher than that of the wild-type promoter. The O2 region is where the σ-factor of RNA polymerase binds. The sequence changes around the -35 region are not related to the response to 3-HP. Regarding upregulation during mmsA Directly affects the affinity / function of MmsR for the promoter In this case, randomizing half of the O2 domain Reduction of the interaction greatly increased transcription levels in an inducible manner.

[0262] By using GFP as a reporter, the strength of wild-type and mutant promoters was The results clearly showed that the promoters with mutations in the first half of the O2 region Tar (P mmsA2a ) showed ~2-fold higher performance in expressing GFP during induction with 25 mM 3-HP. This result was consistent with the results of the transcription level shown in Figure 15B. This test modifies transcription levels by altering the operator binding site region. The present inventors have demonstrated that the basal expression level of the promoter (largely) Both the promoter induction level and expression (~2-fold) could be increased.

[0263] In bacteria, LysR-type transcriptional regulator (LTTR) proteins interact with RNA polymerase This effectively upregulates downstream genes. As a result, the amount of LTTR protein affects the transcription level of target genes. mmsA MmsR expression relative to promoter strength To investigate the effect of constitutive P zwf Developing a library of promoters (Figure 16A), P under the selected promoter zwf MmsR was expressed from the library (Figure 16B). In constructing this synthetic promoter library, P zwf -35 and -10 promoter We kept the consensus sequences constant and randomized the spacing around these sequences to vary their length. It changed.

[0264] Several synthetic promoters in the library were evaluated, and these are shown in Figure 16A. As a positive control, the constitutive promoter P zwf (P zw f-7 After normalizing the background signal, the library promoter is 11,500 (P zwf-1 ) ~ 51,000 (P zwf-12 ) relative fluorescence units (AU / OD 600 ) range of GFP fluorescence levels Thus, the level of promoter expression was significantly higher than that of wild-type P zwf-7 Based on (32,200) Three promoters from this library were selected: zwf-1 and P zwf-11is the wild-type P zwf These correspond to differences of 0.4, 1, and 1.7 times, respectively, compared to Derivative P zwf The transcriptional activator MmsR was placed under the control of each of the promoters. Produced at the P level mmsA It affected the transcription of the gene (kgsA) downstream of the promoter. Natural Promoter P mmsR A strain expressing MmsR from the chromosome under mmsA ) was used as a control At low MmsR levels (MmsR-1 strain), MmsR inhibits P zwf-1 When under the control of the promoter of kgsA mRNA expression was comparable to that of the control, whether in the absence or presence of 3-HP MmsR is two stronger P zwf-1 and P zwf-11 Place under the control of either of the promoters By increasing the amount of MmsR (MmsR-1 and MmsR-11 strains, respectively), the expression of kgsA was suppressed. Transcription was improved in the presence of 3-HP. However, the basal expression level of kgsA was significantly higher in the absence of 3-HP. This result is consistent with the previously reported suppression of apo-MmsR. For the inducible MmsR expression level, it was possible to understand that the higher the MmsR expression level, the higher the basal By minimizing the level, the difference between the uninduced and induced situations is increased. This indicates that the enhancement of the activator is a strong inducible promoter. This is an excellent strategy to prevent leakage of target genes before induction, which is often the case with catalysis. was shown.

[0265] Example 11. P mmsA Comparison of expression of the native mmsA gene and heterologous kgsA gene under promoters P. denitrificans can actively degrade 3-HP produced from glycerol. The enzymes 3-hydroxyisobutyric acid dehydrogenase (HbdH-4) and mmsA, respectively, are Dehydrogenase IV (HbdH-4) and methylmalonate semialdehyde dehydrogenase (MmsA) are 3- The mmsA promoter (P mmsA ) is an activator tamper This promoter was highly induced (>140-fold) by 3-HP with the aid of the protein MmsR. This 3-HP sensor can sensitively detect 3-HP at approximately 25 mM when added exogenously. We developed a new system.

[0266] P mmsA is highly induced by 3-HP, whereas P mmsA gfp or kgsA under the control of The expression levels of heterologous genes in P. denitrificans were mmsA MMS natively controlled by The expression level of A was much lower than that of P mmsA Before the modification of P mmsA Cloned under The expression of the homologous gene (mmsA) and the heterologous gene (kgsA) was quantitatively compared by RT-PCR. Transcription of the seed gene kgsA was increased by ∼100-fold when expressed from the chromosome, and by multiple copies (∼20 copies) of p When expressed episomally using the UCPK' plasmid, the native gene (mmsA) was ~40-fold lower. Crude cell KgsA activity clearly demonstrated the effects of 3-HP induction and gene dosage. When expressed from the chromosome (Int-1), the activity was 0.08 U / mg TNF-α without 3-HP, respectively. With 3-HP, the protein concentration was 0.15 U / mg protein. The activity was 1.02 U / mg protein without 3-HP and 3.15 U / mg protein with 3-HP, respectively. We found that KgsA activity determined either with or without 3-HP was significantly higher than that of the gene We noticed that the results accurately reflected the dosage effect and were roughly proportional to the gene copy number. KgsA was expressed together with glycerol dehydratase (DhaB) from a multicopy plasmid. When the crude cell activity was 2.5 U / mg protein, good 3-HP production resulted. This indicates that when kgsA is integrated into the chromosome, P mmsA The promoter amplifies the gene by ~20 times This suggests that the gene should be modified to express more highly. mmsA Promo Under the control of the β-actin, the homologous mmsA gene is transcribed ~100-fold more highly than the kgsA gene. The expression of kgsA was observed when the copy number of the kgsA gene was very low, such as 1 or 2 copies. Even in this case, the efficiency of transcription and / or translation can be improved by modifying the properties of the mmsA regulatory structure. This can be improved by increasing the

[0267] Example 12. Development of a tandem promoter system Attempts to optimize gene expression levels in bacterial hosts have involved altering transcription and translation levels. A rational and combinatorial approach is used. Strong promoters and / or Using a ribosome binding site (RBS) increases gene expression. A synthetic promoter library has been developed that provides The σ factor of RNA polymerase has a better response than the other in the vegetative and stationary phases. The promoters have also been identified. The aforementioned regulatory modules are being used to better understand the broader Regulation can help gene expression, which is at the gene level under dynamic growth conditions. To address this issue, the present inventors have previously described Tandem promoter incorporating the 3-HP inducible promoter expression module described in the Examples By creating a promoter, we developed a new gene expression regulatory module. Microorganisms adapt to various physiological conditions to maintain concentrations of specific molecules and during dynamic cell growth. The inventors have used tandem promoters to regulate proteins under Combining 3-HP or small acid-inducible promoters with various constitutive or inducible promoters These promoters were either natural or synthetic. can be either natural / synthetic and / or activate genes during exponential / stationary growth phase We developed a library of 3-HP inducible promoters fused to other promoters that can In some instances, the tandem promoter system comprises at least one inducible promoter. Each gene contained a promoter and at least one constitutive promoter.

[0268] Small acid-inducible promoters are designed to activate the activator protein (similar to LysR) once the promoter is activated. Upon binding, it is activated by 3-HP. 3-HP (or similar small acid) inducible promoter sequence After characterizing the activator proteins that regulate the stem, these promoters It expresses target proteins at low intensity and is therefore not suitable for use alone in recombinant protein expression. Therefore, random promoter mutations were generated. By combining these promoters, we developed a library of small acid-inducible promoters that exhibit various expression intensities. The mutant promoter was placed upstream of a reporter gene (green fluorescent protein (GFP)). The strength of each mutated promoter was analyzed by cloning. In vitro fluorescence measurements of strains carrying inducible derivative mutant plasmids were performed. Briefly, a 4-block sequence of 10 bp in length was analyzed at position -51 (the first Starting from the first 30 bp of the sequence (starting point of the repeat), the sequence was randomized from the predicted TSS to the -9 region. That is, the mutation from -51 to -21 did not result in a difference in fluorescence, but the mutation from -21 to -12 , significantly enhanced the fluorescence level (up to 6-fold). Therefore, random mutations around the -10 region The promoter strength was significantly enhanced by the addition of the nucleotide sequence (Figure 13). This result, along with the in silico prediction, , strongly confirming the location of promoter elements in our predicted 3-HP-inducible promoter. I support it.

[0269] A metabolic pathway can be augmented by an intermediate or end product of the pathway, and any additional The autoinducible promoters described herein do not require the addition of an inducer. -System useful for enzyme expression and regulation in biofuel and biofuel producing strains However, the types of autoinducible promoters are quite limited within cells. These promoters usually result in weak expression levels. For example, by modifying the promoter element or operator region, This can be done to achieve the desired functionality but maintain the inducible nature of the promoter. The present inventors have developed an expression vector containing a tandem promoter. By using a set, the strength of transcription from an inducible promoter can be increased while In addition, we sought to maintain the inducible nature of the promoter.

[0270] The present inventors have developed a tandem promoter that combines an inducible promoter and a constitutive promoter. A motor expression cassette was designed to control the kgsA gene. mmsA and P hbdH-4 promoter The tandem promoter expression cassette containing On the other hand, the MmsR activator was produced from its native promoter in the chromosome. 8 A is, A tandem promoter expression cassette and two mRNAs produced by the promoter. The transcript is shown schematically. By combining two promoters in tandem, ,Figure 1 8 As shown in A, two mRNA transcripts are produced, one of which is P mmsA From the promoter: The other is transcribed from the PhbdH-4 promoter, thereby increasing the number of kgsA mRNA transcripts. Figure 1 8 B shows that the tandem promoter system is a natural P mmsA Compared to promoter only The results show that the expression level of kgsA mRNA increased by 1.75-fold. 8 C is for tandem The use of promoter systems is mmsA Obtained by using only the promoter This shows that the enzyme activity of KgsA was increased by 3-fold compared to that of the control.

[0271] When examining the translation efficiency of each promoter in the tandem construct, the present inventors The P hbdH-4One unit of transcription from the promoter produces two units of protein / activity (ratio is 1:2) whereas P mmsA One unit of transcription from the promoter produces one unit of protein It was found that the activity of P hbdH-4 The 5' untranslated region (5'UTR) of the promoter is P mmsA Compared with the 5'UTR region of the promoter, We demonstrated that a tandem promoter expression cassette can result in a higher translation level than that of the control. Each transcript produced by hbdH-4 This allows for significant translation. The promoters (P mmsA or P hbdH- 4) Higher KgsA compared to only Therefore, compared to either single promoter alone, , P hbdH-4 The inducibility of the promoter is (even if slightly reduced) mmsA Promoter's contribution The basal expression level without inducer was maintained by the administration of ATP. Expression was enhanced by a constitutive promoter.

[0272] Figure 55A shows a tandem promoter containing at least one 3-HP inducible promoter. The four different expression constructs constructed are shown. These are the phpdH and Pc1 promoters. The promoters were either Pzwf or Pzwf promoters. This indicates that the dem promoters had varying strengths based on DhaB activity.

[0273] Example 13. Modification of UTRs The 5'-untranslated region (5'-UTR) and 5'-proximal coding sequence are responsible for the ribosomal transport of these elements. plays an important role in regulating the rate of translation initiation and mRNA stabilization based on the accessibility of The inventors have demonstrated that transcription factors produced by genes under the control of this system To increase the translation of mAdH-4 5'UTR and 5' of the tandem promoter expression cassette A library of mutations in the proximal coding sequences was generated. The entire length of these regions Several factors contribute to the mutation of these regions, including the fact that the translation efficiency can be easily lost. The RBS modification randomized the RBS, making it difficult to induce ribosome binding to the RBS. However, the 6-9 nucleotides in the RBS are an easy way to adjust the affinity of the Only the nucleotides are modified by this method, and no other important components for translation (5'-UTR or 5'-UTR) are modified. '-coding sequence) should have been considered.

[0274] An in silico tool called UTR Designer has been developed for accurate prediction of translation initiation levels. This tool is used to analyze the 5' untranslated region of mRNA to understand the translational regulation of target genes. The folded structure of the (5'UTR) (35 bp), the 5'-proximal coding sequence of the structural gene (25 bp), and the ribosome The affinity of the Shine-Dalgarno region to the ATPase inhibitors was examined, and the level of translation was therefore altered. Using this tool, the inventors have been able to design modifications to the proteins they have created. The constructs carrying Opt-3 and Hyb-20 along with chromosomal expression of MmsR (the present inventors UTR-0) and P hbdH-4 Transcription and translation of tandem promoter expression cassettes As shown in Figure 19C, the UTR designer tool When this system was fully expressed under normal conditions (200 rpm, 37°C, 25 mM 3-HP), predicted a 12-fold increase, which is greater than the 1.5- to 2-fold improvement we observed (data not shown). To clarify the effect of UTR modifications on translation initiation, we analyzed the relevant gene (in this case The expression level of KgsA was measured. Protein expression levels were measured on SDS-PAGE gels. However, we have developed an inducer to allow easy differentiation of the various UTRs. The addition of serotonin was intentionally avoided to maintain expression at low levels. This experiment was performed to determine the translation initiation effect. Therefore, basal expression without inducer was evaluated by the present inventors. This should provide better and more complete evidence for the effect of the serotonin gene on the growth of the serotonin gene; The rotation speed was limited by using a rotor (150 rpm) to minimize protein expression limits.

[0275] As shown in Figures 19A-C, UTR modifications increase the levels of target proteins. Figure 19A shows that the extensive translation levels of P hbdH-4 Tandem promoter expression cassette This was achieved with a library of mutations made to the 5'UTR and 5' proximal coding sequence of the ribosomal RNA. This was observed when the bacteria were grown under normal induction conditions (200 rpm, 37°C, 25 mM 3-HP). The present inventors have shown that the efficacy is comparable to that of natural P mmsA significantly (~30 It was found that the activity of the ATP can be enhanced (3-fold).

[0276] The UTR-6 construct showed the greatest fold change, as determined by SDS-PAGE analysis (BioR ad Image Lab 2.2 software), UTR-0 and P mmsA Compared to The levels of UTR-6 were increased by 12.5-fold and 30.8-fold compared to those of the recombinant P. denitrificans. The strain was selected for integration at the mmsA chromosomal location (the mmsR activator gene system Finally, expression of the target protein from the chromosome was confirmed as shown in Figures 18B and 18C. As shown, the transcription (mRNA) and translation (enzyme activity) levels exceeded the expected levels. The results showed that UTR designers could successfully achieve 2 and 1.7 times the target gene expression, respectively. Enhance expression over a wide range and achieve desired levels of target genes with fewer mutations It has been shown to be a promising tool for

[0277] Example 14. Codon optimization and protein hybridization Even with an effective transcription system, if the translation system does not work well, the protein may not be produced efficiently, thus resulting in sufficient protein levels. To achieve this, optimization of transcriptional and translational control is often used.

[0278] Codon preference is related to tRNA composition and controls the rate of translation propagation. In this case, it affects protein folding. In such cases, it is usually recommended to use preferred codons, but this can lead to misfolding. This can even result in the production of inactive, oxidized proteins or inclusion bodies. Fusion systems (hybrid proteins) increase protein expression and enhance protein synthesis. Considerations should be taken to prevent solution, improve solubility, and aid in purification.

[0279] As described in the examples above, P mmsA Expression system The use of the genotype resulted in different expression levels between the homologous mmsA gene and the heterologous kgsA gene. P in the control of SA mmsA The poor performance of the expression system may be due to the nature of the kgsA and mmsA genes (secondary structure). These differences may be due to differences in the structure, translation initiation ability, codon bias, and solubility. To achieve this, the present inventors modified the kgsA gene.

[0280] Codon optimization of whole genes is a common method to increase translation. However, this is an expensive and time-consuming process. The study focused on optimizing only the first 10 codons of the heterologous gene to enhance translation initiation. The inventors used the GenScript bioinformatics tool to A codon usage table was created for the native mmsA gene expressed in The codon frequency levels of each amino acid in MmsA are shown. (i) use codons that show high levels of frequency in mmsA; (ii) use codons that show intermediate levels of frequency; and (iii) use codons that exhibit the same frequency level as the first 10 codons of MmsA. and exclude rare codons (0% frequency) in KgsA. The first 10 codons of gsA (heterologous protein) were modified (Table 3).

[0281] The present inventors have demonstrated that codon optimization with Option 3 (Opt-3) is effective in the absence or presence of 3-HP. It was found that the KgsA activity could be increased by 30% in both cases. They maintained the frequencies of all the first 10 codons at the same frequency as that of the mmsA gene (Opt-3 ) improved the translation of kgsA. In contrast, some codons, e.g. For example, increasing the frequency of the 1st, 4th, 7th, and 9th genes (Table 3) in Opt-1 and Opt-2 resulted in increased expression. Instead of increasing, it even decreased slightly. Table 3: Wild-type and optimized kgs based on mmsA codon usage in P. denitrificans Frequency of the first 10 codons of A [Table 3] a Use codons that show high frequency levels b Use codons that show intermediate frequency levels c Codons with the same frequency level as the first 10 codons of mmsA were used, and rare codons were not used. exclude

[0282] The results were confirmed by protein production on SDS-PAGE (FIG. 21B).

[0283] The fusion protein enhances protein production, reduces proteolysis, and It is often used in heterologous gene expression to improve binding and solubility. They found that the N-terminus of MmsA produced by the highly expressed mmsA gene of P. denitrificans is Kg The present inventors fused a part of the 5' coding region of mmsA to the enzyme P mmsA Promoter's By adding a downstream gene, translation initiation of the heterologous gene (kgsA) transcript becomes more efficient. The present inventors reasoned that fusing a portion of the MmsA protein to KgsA would allow the We were interested in the effect of the length of MmsA on the activity of the enzyme. , 5, 10, 15, and 20 ami, corresponding to strains Hyb-5, Hyb-10, Hyb-15, and Hyb-20, respectively. The results showed that as the length of MmsA fused to KgsA increased, the KgsA activity However, once the 15 amino acid MmsA was used (Hyb-15), this effect plateaued. The Hyb-15 fusion protein significantly increased the activity of the ATP-dependent ATPase compared to the control KgsA (no MmsA fusion) (Fig. 17A). In the presence of 3-HP, production increased by ~3-fold. Further extension to the amino acid sequence MmsA (Hyb-20) did not improve expression; The basal level of protein production in the culture medium was enhanced. This result was confirmed by the KgsA band on SDS-PAGE. This was confirmed by the intensity (Figure 17B).

[0284] In addition to its involvement in translation initiation, the 5' end of target genes also regulates the level of transcripts through regulation of mRNA concentration. It plays an important role in the regulation of the β-amyloid ... The stability of the kgsA transcript with the N-terminal fusion (mmsA(20)kgsA) was measured (Fig. 17C). The half-lives of kgsA and mmsA(20) kgsA were 2.7 and 5.4 minutes, respectively. , the mmsA hybrid fusion kgsA mRNA is more stable than the wild-type kgsA mRNA. The half-life of the mmsA transcript was found to be approximately 6 min, so mmsA(20) kgs The improvement in A stability is due to the protection of the 5'-end coding portion (20 amino acids) of mmsA fused to the kgsA gene. Interestingly, among the genes we tested, The most stable transcript appeared to belong to mmsR, with a half-life of 9.5 min (FIG. 17D).

[0285] Example 15. Coenzyme B12 Production Coenzyme B 12 is a glycerol dehydratase, methionine synthase, or methylmalonate. It is an essential cofactor for many enzymes, including glycerol dehydrogenase (CoA) mutase. Hydratase (or alternatively, diol dehydratase) converts glycerol to 3-hydroxybenzoates. It is involved in the conversion of glycerolipids to hydroxypropanealdehyde (3-HPA). 3-HPA is an essential enzyme for the production of 3-HP (or its salts) or 1,3-PDO from ethanol. It has been further developed into other industrially important chemicals such as 1,3-PDO or 3-HP (or their salts). These biochemicals can be produced continuously from glycerol on a commercial scale. To do this, coenzyme B 12 A continuous supply of coenzyme B is essential. 12 is very expensive, so The present inventors have found that natural coenzyme B 12 The production of 3-HP from glycerol was investigated using the production material. Coenzyme B 12 There are several microorganisms that have been reported to naturally produce , Enterobacter species including Klebsiella species, Streptococcus species including Streptococcus pneumoniae, P. denis Pseudomonas species, including S. trificans, Rhizobium species, and alfalfa rhizobia (Sm Sinorhizobium species, including R. eliloti, and R. capusu Rhodobacter species, including R. latus and R. sphaeroides, are also included in this invention. The authors transformed some of these microorganisms into host cells for the production of 3-HP from glycerol. When tested primarily, the produced coenzyme B 12 is sufficient to synthesize high titers of 3-HP. (each produced <30 g / L).

[0286] P. denitrificans can tolerate ∼20.8 g / L of 3-HP (or its salts) without the external addition of this cofactor. However, this strain was not able to produce saturating amounts of coenzyme B12. When supplemented, it was able to produce ~100 g / L of 3-HP. Coenzyme B12, naturally produced by certain microorganisms, is sufficient to support 3-HP production at high titers. After careful analysis, it was found that the regulatory (secondary mRNA) involved in coenzyme B12 synthesis By removing the 2-amino acid residue (substrate structure), a slight improvement in its production was observed. The improvement in coenzyme B12 was This recombinant strain was confirmed by various assays mentioned in the previous section. When used as a host for the production of 3-HP from ethanol, the yield was more than double that from glycerol. The results showed an improvement in 3-HP production of 38.6 g / L.

[0287] In the next experiment, we added a small amount of coenzyme B 12 By replenishing The results are shown in Figure 50. The inventors added a small amount of cofactor to the culture. Basic B 12When 3-HP production was increased to 100 g / L, the 3-HP production improved significantly from 38.6 g / L to 100 g / L. This result indicates that the coenzyme B produced by the modified strain (IR1-RS1) 12 The amount of It is shown that although the improvement was not sufficient to support 3-HP production at high titers.

[0288] Furthermore, to improve coenzyme B12 production, the present inventors have investigated the effects of coenzyme B12 synthesis pathway on the It is necessary to enhance the activity of the enzyme that is responsible for the supplementation. This can be achieved by enhancing the enzyme B12 synthesis pathway. Enzyme activity is determined by its physical properties. and by enhancing kinetic properties or improving its expression and increasing its amount. This pathway involves many enzymes. Therefore, the present inventors first attempted to increase the amount of the enzyme and enhance the synthesis of coenzyme B12. To enhance the amount of enzyme in cells, the natural promoter was modified by the above synthetic method. The promoter was replaced with a new one.

[0289] In this study, the inventors investigated the synthesis of coenzyme B by microorganisms. 12 Improve production and make it suitable for commercialization We sought to support the production of biochemicals from glycerol on a reasonable scale. To achieve this, the present inventors have investigated the effects of various coenzymes B 12 - Riboswitch (B 12 -riboswitch)-mediated transcription and coenzyme B 12 Natural microbial producers of, e.g., P. We investigated the cobalamin gene cluster in B. denitrificans. , using software, coenzyme B 12We identified five secondary structures that can be regulated by (See Figure 23.) We found that four of these structures are riboswitches. In vitro characterization revealed that the cobG and cbtB genes were Only three of the riboswitches in the stream were shown to be transcriptionally regulated (Figure 23). The riboswitch is activated by the addition of cobalamin B12 (at a low concentration of about 5 nM). Coenzyme B, resulting in a significant decrease in translation 12 (See Figure 28).

[0290] Furthermore, we have identified six characterized cob gene clusters located in cob gene clusters I and II. The missing genes (gst, xre, dahp, gntR, bgpM, cobA) 12 Whether it is involved in synthesis (See Figure 23.) The present inventors found that the bgpM gene product binds to coenzyme B 12 Essential for biosynthesis cobA, which encodes uroporphyrinogen methyltransferase, was found to be involved in the The gene is coenzyme B 12 It is involved in a key branch point in the biosynthetic pathway and therefore The inventors have shown that the deletion of cobA results in the loss of coenzyme B 12 However, this invention The authors found that the cobA deletion mutant produced significant amounts of coenzyme B 12 It was found that it is possible to synthesize When the present inventors analyzed the genome of P. denitrificans, they found that We noticed the existence of several CobA isozymes that could potentially substitute for CobA activity.

[0291] In the first part of this study, we 12 -Used to identify riboswitches The method used and coenzyme B 12 Describe its regulatory role in transcription / translation involved in synthesis. The present inventors have demonstrated that coenzyme B12 production can be controlled by adjusting the riboswitch. We also investigated methods to enhance the expression of the native promoter as described in the above examples. Synthetic expression modules (tandem promoters, UTRs, and regulatory tags for highly expressed genes) Coenzyme B 12 This further improves production. In these modified strains, coenzyme B 12 Enhanced production of coenzyme B 12 External addition of This promoted increased production of 3-HP (or its salts) from glycerol without ATP.

[0292] The strain with the modified promoter showed good coenzyme B12 production, and analysis showed that this The strain produced four times more coenzyme B12 than the wild-type strain and two times more coenzyme B12 than the riboswitch-modified strain. This strain was transformed with a plasmid containing the 3-HP synthesis pathway enzymes to produce enzyme B12. When transfected with 3-HP, this showed a significant improvement in 3-HP production without the addition of coenzyme B12. The strain produced over 90 g / L of 3-HP from glycerol, but its growth was slower than that of the wild-type strain. This was slightly slower than that observed in the control group, which may be due to modifications in the coenzyme B12 synthesis pathway. In this study, the inventors found that coenzyme B12 can be produced in larger amounts and with higher potency. We have successfully developed a mutant strain capable of supporting 3-HP production at high titers.

[0293] Example 16. In silico analysis and comparison of B12 genes among various Pseudomonas species. To understand the gene arrangement and its regulation, we have investigated the function of various Pseudomonas sp. Coenzyme B present in the strain 12 We compared gene clusters. So far, 31 genes have been identified. Aerobic coenzyme B 12 Coenzyme B involved in the biosynthetic pathway 12 Identified in the biosynthetic gene cluster Among them, coenzyme B from P. denitrificans 12 Biosynthetic pathway examined at the enzyme level Coenzyme B from various Pseudomonas species 12 Based on comparative analysis of gene composition The genes of P. denitrificans ATCC 13867 and P. aeruginosa PAO1 were highly similar (Fig. 24). The results showed that these strains were similar, with an average sequence identity of 73%. four additional uncharacterized genes in the coenzyme B12 gene cluster: gst, xre , dahp, and bgpM. Unlike other Pseudomonas strains, The gene encoding chelatase (chlID) was expressed in these two strains along with the xre gene. , located far apart. Furthermore, P. entomophila, P. putida, and P. fluorescens The genes and gene organization in this strain are similar to those in P. denitrificans. These strains contain coenzyme B 12 ton-dependent B, which is known to encode a transport system 12 Transporters ( btuB). In addition, the operon cobGHIJ in these microorganisms is 12 Dependence Rebosui It is not controlled by the pitch structure (Figure 24).

[0294] Nitrogen-fixing bacteria such as Ensifer / Rhizobium species also have high B 12 Production capacity Interestingly, P. denitrificans SC510 (and industrial B 12 producer) and P. denitrificans ATCC 13867 and Ensifer meliloti (Ens A comparison of the genetic makeup between P. denitrificans SC510 and Ensif P. meliloti is more closely related to P. denitrificans ATCC 13867 strain. These differences were found to be distinct from those of the whole genome of P. denitrificans SC510. The unavailability of the nome sequence prevented further investigation.

[0295] In addition to understanding the genetic organization in P. denitrificans, we also investigated the Rfam Using the database / tool, four potential B 12 -riboswitches were identified. One representative example of a riboswitch structure is shown in Figure 25A, which is involved in the regulation of cobalamin biosynthesis. These B 12 -riboswitches exist in E. coli, Similar to that of Salmonella enterica btuB B 12 -Ribos Cobalamin-binding consensus domains (regulatory and receptor domains) similar to those of itch One riboswitch (RS1) regulates the synthesis of hydrogenobyrinic acid from the precorrin-2 intermediate. Two divergently located cobGHIJ and cobLFK operons encode enzymes involved in the formation of It is located between the ribosomal genes (excluding the cobM gene) (Fig. 25A; see Fig. 23). Based on the importance of the genes at this locus, we have identified the coenzyme B 12 Node and Shiro Strict regulation of intergenic region 1 (IR1) by RS1 in flux diversion between heme biosynthetic nodes The two B operons located between the cobWN operon and the cbtBA-cobEM operon are assumed to be highly regulated. 12 Specific riboswitch structures (RS2 and RS3) regulate the expression of both operons independently. Finally, RS4 encodes the enzyme responsible for the formation of Ado-Cbl from hydrogenobyrinic acid. It is located upstream of the cob operon (btuB-cobOBRDCQUP-bgpM-cobV) which contains 11 genes that encode It is placed.

[0296] The present inventors have investigated the mechanism by which these riboswitch structures control the expression of the cob operon genes. Briefly, the interaction between the conserved regions J6 / 3 and J11 / 10 of the riboswitch The adenosyl moiety makes each riboswitch unique and binds coenzyme B 12 To bind to While this allows the riboswitch to function, the interaction between loops L5 and L13 also prevents this. The adjustment status of the switch is changed (see Figures 25A to 25D).

[0297] Example 17. Coenzyme B12-sensing riboswitch structure and its promoter system characteristics analysis) Riboswitch structures regulate gene expression at either the level of transcription elongation or translation initiation. The location of riboswitches within intergenic regions influences the manner of regulation by these riboswitch structures. We determined the B 12 In addition to the riboswitch structure Most overlap with promoter elements, not untranslated regions (UTRs). This suggests that these riboswitches may be regulated at the transcriptional level. Therefore, we investigated the effect of coenzyme B12 on the growth of yeast cells harvested at late logarithmic phase. Selection in the cob operon using real-time PCR on P. denitrificans cultures The mRNA levels of the genes (first gene in each operon) were analyzed. The mRNA levels of the four genes in the cob operon (cobG, cobW, cbtB, and btuB) were significantly higher than those of the coenzyme Transcription of the operon cbtBA-cobEM under the control of RS3 was significantly suppressed in the presence of B12 (Fig. 26). The present inventors found that most of the genes in this operon were repressed by 3.3-fold. 12 Biosynthesis We noticed that the putative structural genes of the pathway (cobE and cbtBA, respectively) were not encoded by the cobGHIJ, cobWN, and cobH ... Transcription of the structural genes encoding btuB, btuB-cobOBRDCQUP-bgpM-cobV) is 12 (uninhibited form) In the presence of It is noteworthy that RS1 has cobGHIJ / cob It is unclear whether it regulates the expression of either or both LFK operons. Based on mRNA levels, RS1 regulates the expression of the cobGHIJ operon but not the cobLFK operon. The present inventors have identified the cob gene cluster, in descending order of importance, as -Various B 12 We observed transcriptional repression by -RS: cbtB-RS3>cobG-RS1>cobW-RS2>btuB-R S4. No repression was observed in the cobLFK, chlID-xre, and dahp operons. The result is that they lack the riboswitch structure upstream of these operons. correlates with reality.

[0298] The present inventors have demonstrated that these riboswitches regulate transcription at the initiation of translation in addition to transcriptional regulation. Furthermore, the present inventors have speculated that the promoter of the cob operon may also be regulated by and the enzyme coding region (upstream) into a plasmid. We attempted to predict the regulatory strength of these riboswitches. They inserted a reporter protein (fluorescent Green fluorescent protein (GFP) was added as a marker. The first 35 bp of each sequence encoding the native protein was used as a primer. One version was fused to the gene encoding GFP in a plasmid, and the other version did not have this fusion. One version was a fusion form of cobG (the first 35 bp of cobG fused to gfp). It showed 4.2-fold higher fluorescence compared to the unfused gfp construct (Figure 27, Table 6). However, we found that when the first 35 residues of cobG were fused to gfp, other promoters Fluorescence provided by the intergenic segment from another B12 operon We observed no significant difference in the levels of α- and β-glucanase, indicating that they are not regulated at the translation initiation level. These results suggest that P cobG -RS1 is inherited at both the transcription and translation initiation levels These findings further suggest that the expression of the gene is tightly controlled. This is an indication of the importance of regulated expression of the genes in the cobGHIJ operon.

[0299] Estimation of natural promoter strength can be used for any pathway in which several genes are involved, e.g. , Vitamin B 12 When overexpressing a pathway, or a pathway involving several membrane proteins, It is extremely important that overexpression of pathways with many genes is detrimental to cell proliferation. Imbalanced expression of pathway genes / operons can lead to the accumulation of toxic pathway intermediates. Analysis and quantification of the strength of the native promoter may result in overexpression of the pathway enzyme during the engineering. Without expressing the operon, we redesigned it with a synthetic promoter. Table 5 shows the results of our experiments with various native cob promoters in an operon. The estimated promoter strength (expressed in relative florescence units) observed for The promoter P controls the expression of the cobalt transporter gene. cbtB has the highest strength, and P cobL The promoter has the lowest strength of this group. Enzyme-encoding genes usually appear to be driven by weak promoter systems. It was said. Table 6. Characterization of the cob regulon intergenic region in P. denitrificans [Table 4]

[0300] (Modification of the coenzyme B12 production pathway) Rational modification of the B12 promoter, along with its UTR structure (fusion and non-fusion versions), Quantification of promoter strength is required. Therefore, The strength of the native promoter of the ob operon was carefully quantified (Figure 27, Tables 6 and 10). To increase the expression level of these (cob) genes (3-5 fold compared to their natural expression) The expression of the cob operon was modified by replacing it with a suitable constitutive promoter (Figure 57). ) Constitutive promoters are similar in strength to some native Pseudomonas promoters. The results were selected by screening and identifying the following (Table 10). For example, a 3-fold improved protease inhibitor was To develop a Pedd-IR13-PsucA recombinant strain with motor strength, PcobG and PcobL were The promoters were replaced with the constitutive promoters Pedd and PsucA, respectively (see Table 1 for further details). See 8). Table 10. Strategy for B12 promoter replacement [Table 5]

[0301] Example 18. Development of a riboswitch-based B12 sensing system Vitamin B 12 Quantitative methods typically include microbiological assays and high performance liquid chromatography with UV detection. The HPLC method was used to detect high concentrations (above μM levels) of B 12 Although microbial assays can detect very low concentrations (as low as nM levels), B 12 Microbiological assays can detect specific mutations in Salmonella typhimurium. Variant strain metE - cbiB - B as a growth factor in 12 In short, this The growth of these microorganisms is 12However, the B 12 of Quantitation is achieved depending on several physiological factors, such as the culture medium and incubation time. Therefore, cell proliferation is based on coenzyme B 12 Quantitation of is tedious and difficult to reproduce. is difficult.

[0302] In this study, we investigated five putative B 12 In the riboswitch structure, the promoter P cobG ' and P cbtB Two of them are coenzyme B 12 In the presence and absence of We found that differential expression of downstream genes could be observed in these ribosomal proteins. The dynamic range of the chromatogram was investigated by measuring the concentration of coenzyme B 12 (up to 100 nM) to develop a novel B 12 Its potential as a sensor was evaluated. 12 Concentration vs. GFP fluorescence Cobalamin B with a shape correlation of approximately 5 nM 12 This was observed at the switch-off point (Figure 28). 12 Riboswitch P cobG ' and P cbtB is B 12 The differential expression was ~2.1- and 1.7-fold in the presence and absence of α-glucan. Breeding-based B 12 In contrast to fluorescence-based detection, B 12 The detection method is reproducible and easy to use , and hassle-free.

[0303] Example 19. Coenzyme B 12 Quantitative bioassay of :) P. denitrificans was cultured in 0.1 mM 5,6-dimethylbenzimidazole (DMB), 1 g / L beta and trace elements 0.232 g / L H3BO3, 0.174 g / L ZnSO4 7H2O, and 0.116 g / L Fe(NH4 )2SO4·6H2O, 0.025 g / L CoCl2·6H2O, 0.022 g / L (NH4)6Mo7O 24 4H2O, and 0.008g / L Supplemented with CuSO4·5H2O, 0.008 g / L MnSO4·4H2O, 1 g / L NaCl, 1 g / L NH4Cl, 2 mM Mg SO4, an initial OD of 0.1 in M9 minimal medium containing 10 g / L sodium gluconate 600 Propagate with Cells were harvested in the logarithmic phase and homogenized using a FastPrep-24™ 5G homogenizer system (MP Biomedicine). The cell lysate was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was collected. The resulting lysate solution was sterilized by passing it through a 0.2 μm membrane filter. Used in B. Coenzyme B 12 , mutant strain Salmonella typhimurium metE - cbiB - To use Briefly, in Salmonella typhimurium, metE is expressed in B 12 independent methio cbiB encodes the ribosomal synthase, and cbiB encodes the B 12 Adenosylcobalamin, an enzyme essential for the synthesis pathway Mutations in these genes result in murine typhus Bacteria is B 12 Or it becomes an auxotroph for methionine. The indicator strain, Salmonella typhimurium metE - cbi B - , supplemented with 50 mg / L methionine; 0.5 g / L NaCl; 6 g / L Na2HPO4; 3 g / L KH2PO4; 1 Preculture in minimal medium containing 0.5 g / L NH4Cl; 4 g / L glucose; 2 mM MgSO4, 0.1 mM CaCl2. The overnight broth was centrifuged and washed with water. The washed cells were soaked in minimal medium with 0.01 ml of PBS. Initial OD 600 B 12 The cell lysate from which the activity needed to be measured was added to the culture broth. The growth of the standard strain reflects the concentration of coenzyme B12 in the sample. 12 The concentration of A quasi-curve was created. B 12 The dynamic range of concentration is 0-100 pM B 12 It was. B 12 Concentration of 1 OD 600 Expressed as nM per 1000 mg / mL.

[0304] Example 20. Identification of bgpM as a gene essential for B12 synthesis. To gain a complete insight into the essentiality of genes within the cob biosynthetic operon, B 12 Biosynthesis Several uncharacterized genes in the cob gene cluster for, e.g., gnt The essentiality of the genes gst, xre, dahp, and bgpM was investigated. 12 Required for biosynthesis To elucidate whether the genes are involved in the regulation of ATP production, we performed single knockout mutants of each of these genes. These mutant strains were 12 Biosynthetic ability was measured using the B 12 Special Alternative riboswitch sensing systems and conventional Salmonella typhimurium metE - cbiB - B on the bass 12 Assay Both systems (see Materials) were investigated. 12 Biosynthesis involves attaching a metal center to the Since the mutant strains require CoCl2, their biosynthesis is dependent on the concentration of CoCl2. Increased concentrations of CoCl (up to 50 μg L -1 ), GFP fluorescence was significantly higher than that of the ΔbgpM mutant. These results suggest that each of these mutant strains Coenzyme B in wild-type strains, except for ΔbgpM 12 Coenzyme B, similar to 12 that it is possible to synthesize The gene product of bgpM is coenzyme B in P. denitrificans. 12 In the biosynthesis of However, B 12 Its precise role in biosynthesis The essentiality analysis was performed using Salmonella typhimurium as an indicator strain. The coenzyme B synthesized by these mutant strains 12 The intracellular concentration of was quantified (Figure 29B). Similar to the results in P. denitrificans, only the ΔbgpM mutant expressed coenzyme B 12 Combined (or traces of coenzyme B 12 (It could only be synthesized in the wild) ~7 times less B than type strains 12 Based on these results, the gene located in IR2 (see Figure 23) (Reference) is B 12 Therefore, overexpression of these genes is not essential for the biosynthesis of Enzyme B 12 Surprisingly, B 12 Biosynthetic pathway A key branching point, the cobA deletion mutant (uroporphyrinogen methyltransferase The enzyme that encodes coenzyme B 12 We were able to synthesize (Figure 29B). Based on this analysis, we hypothesized that P. denitrificans plays a role in place of CobA. The presence of several potential CobA isozymes has been identified in the genome of P. denitrificans.

[0305] Example 21. Coenzyme B12 production is improved by deleting the riboswitch structure. B 12 To improve production, the identified negative regulatory domains (P5 The riboswitch-deficient mutants were individually deleted. Coenzyme B mRNA levels 12 Coenzyme B was measured in the presence and absence of >5 nM (Table 7). 12 The existence In the presence of β-actin, cob operon transcription is repressed by a riboswitch in P. denitrificans strains However, in the riboswitch mutant strain, coenzyme B 12 With the presence of Amount of coenzyme B 12 Therefore, these mutant strains can result in the production of The synthesized B 12 By measuring the amount of coenzyme B, the riboswitch mutation 12 Raw materials It would be easier to assess the enhancement of the gene. , ΔRS1 (riboswitch 1 deletion) mutant and ΔRS2 / RS3 (riboswitch 2 and 3 deletion) mutant There was no significant difference in the production of coenzyme B12 in the body. However, when compared to the wild type, , coenzyme B in the ΔRS4 (riboswitch 4 deletion) mutant 12 There was a significant difference in coenzyme B 12 The production of ΔRS1 and ΔRS2 / RS3 was significantly suppressed in the ΔRS4 strain. Defective coenzyme B 12The ΔRS4 deletion did not affect coenzyme B production. 12 Significantly increase production One possibility is that the mutations were randomly selected. The regions in riboswitches 1, 2, and 3 that were removed were not sufficient to abolish function. Therefore, we hypothesized that the coenzyme B 12 production On the other hand, the ΔRS4 mutation caused the riboswitch to overlap with the coding region. Since the deletion mutants were inserted into the nuclease, it is likely that they affect protein expression. To overcome this limitation without causing any problems, the present inventors have investigated whether the secondary structure in mRNA is altered by mutation. The ribosomal switch is neutralized by codon optimization, while the protein sequence is maintained by codon optimization. The chi region was modified. Table 7. B in the presence and absence of 25 mg / L CoCl2 12 Transcription levels of synthetic genes [Table 6]

[0306] The inventors designed the sequence using software, chemically synthesized the nucleic acid, and synthesized coenzyme B 12 If cob, which encodes an enzyme in the synthesis pathway, has been exchanged in the genome to avoid secondary structure formation in mRNA. The inventors have found that by varying individual riboswitches or combinations of riboswitches, Interestingly, we developed seven recombinant strains lacking riboswitch 1. When the coenzyme B 12 A slight decrease in production was observed This may possibly be due to the accumulation of some toxic intermediates. However, deletion of riboswitches 2 and 3 (ΔRS2 / RS3) or riboswitch 4 (ΔRS4) did not result in complementation. Enzyme B 12 This was due to the limitation of carbon flux in the upper pathway. However, riboswitch 1 can be riboswitches 2 and 3 (ΔRS1 ΔRS2 / RS3) or When either riboswitch 4 (ΔRS1 ΔRS4) was deleted, coenzyme B 12 Only a small amount of production Furthermore, mutant strains lacking all riboswitches showed similarly high complementarity. Enzyme B 12 The production was shown (Table 8).

[0307] This study demonstrated the activity of coenzyme B by P. denitrificans. 12 The cob mRNA is then derivatized into a secondary structure. It was shown that the effect can be improved by removing coenzyme B. 12 Mutant strains with improved production When 3-HP (or its salts) was produced from glycerol using the host, it was found to be <38 The mutant strain produced 0.6 g / L of 3-HP (Table 8). This mutant strain produced significantly more 3-HP than the wild-type strain, which produced <30 g / L. The mutant strain produced slightly higher 3-HP titers from glycerol compared to the wild-type strain. The improvement of 3-HP production in 12 This may be due to the improved pathway. Coenzyme B 12 When exogenously supplemented in the medium, this strain produced ~100 g / L of 3-HP from glycerol. These experiments demonstrated that the mutant strains were able to produce coenzyme B 12 Production Although this strain has been shown to increase viability, it was further refined to produce higher titers of 3-HP. It was clearly shown that the nucleotide sequence can be further modified.

[0308] Coenzyme B 12 To further improve production, we performed a gene mutation of the cob operon in the riboswitch mutant strain. The natural promoter was substituted with the synthetic expression module (highly expressing gene) described in the above examples. The cob gene was replaced with the tandem promoter, UTR, regulatory protein, and N-terminal region of the gene. Subcluster analysis revealed five promoters that control the expression of genes in the B12 biosynthetic pathway. The presence of the natural promoters was demonstrated, either individually or in combination as shown in Table 8. Several recombinant strains were developed by combining and exchanging the promoters. A selection of promoters that can be used is shown in Table 10. Each promoter exchange is performed using coenzyme B 12 production showed a slight improvement in the phenotype, but replacing all promoters with synthetic gene expression modules When coenzyme B 12 This strain (PhpdH-Pedd-IR15-PsucA-PhpdH: Php dH-Ptkt-IR45-Psp2-PhpdH) as a host, was converted from glycerol to This strain was able to produce 3-HP at ~100 g / L, which is sufficient for industrial production. To produce 3-HP, wild-type P. denitrificans was cultured with coenzyme B 12 (Maximum 50mg / L) This is similar to the amount of 3-HP that can be produced when externally fed into the reactor (Figure 50 Therefore, we have found that the amount of coenzyme B 12 can be synthesized, As a result, coenzyme B 12 commercial-scale production of 3 from waste glycerol without the need for external supplementation of We have successfully developed a recombinant P. denitrificans strain that supports 3-HP production. Approximately 38% of the cost is coenzyme B 12 This is due to the cost of externally supplementing the culture medium with The cost of producing 3-HP from glycerol has been significantly reduced. Table 8. Summary of development and evaluation of various coenzyme B12-overproducing P. denitrificans strains. [Table 7]

[0309] Example 22. Development of an aldehyde dehydrogenase expression cassette. Aldehyde dehydrogenase (ALDH) converts 3-HPA to 3-HP (in the 3-HP synthesis pathway). To minimize the accumulation of toxic intracellular 3-HPA, For example, expression of ALDH (by the kgsA gene) is higher than that of its glycerol dehydratase (DhaB). Furthermore, in order to integrate ALDH into the chromosome, its expression must be maintained at a high level. to even higher levels (to track observed gene expression from the plasmid). To achieve this, the inducible tandem promoter described in the examples above is used. The expression module / cassette containing the motor system was used as the base promoter. The ALDH expression cassette was then used to drive a gene expressing ALDH (e.g., kgsA) and further developed. The strength of the β-glucanase was altered / improved by various genetic modifications described in the examples above. In summary, an expression module / cassette has the following characteristics: (i) one promoter is inducible; Tandem promoters, where one promoter is constitutive and the other promoter is inducible ( The promoter may be either native to the host microorganism or synthetic. (ii) an activator protein binding (operator) site in the promoter region; (iii) alteration of the expression level of the activator protein; (iv) various fusions of a suitable length (wherein the N-terminal portion of a highly expressed native protein, e.g., MmsA, is , fused to ALDH), and codon optimization of the first 10 codons of ALDH (host microorganism, (v) a 5'-untranslated region (UTR) (See Figure 30A for a schematic of genetic modifications to the expression system; see Figure 30B for a schematic of genetic modifications to the ALDH expression gene.) For the expression system used to integrate the gene into the chromosome, see Figure 30B. was.

[0310] Example 23. Development of a DhaB-GdrAB expression cassette The dhaB and gdrAB genes were integrated into the chromosome of P. denitrificans, and the transcription and translation efficiencies were investigated. The expression level of 3-HPA was improved by enhancing both the 3-HPA production rate and the 3-HPA accumulation rate. In order to achieve this, dhaB and gdrAB should be expressed at a lower level than ALDH. To achieve this, the promoter used is 3-HP, which is of moderate strength compared to the promoter used to produce ALDH. An inducible promoter was used to drive expression of the dhaB and gdrAB genes. , were designed as follows: (i) UTR modification; (ii) one promoter was constitutive and the other (iii) the use of tandem promoters where the promoter is inducible; (iv) the modification of constitutive promoters. (iv) an inducible promoter (-10 to -35 box) of the activator protein HpdR and (v) modification of the binding (operator) site; and (v) variation in the expression level of the activator protein ( For a schematic of genetic modifications to the expression system, see Figure 31A; (See Figure 31B for the expression system used to incorporate the vector.)

[0311] Thus, the aldehyde dehydrogenase expression cassette described in Example 22, and the DhaB-gdrAB expression cassette described herein, allowing recombinant strains to express glycerol 3-HP was produced from the le.

[0312] Example 24. ALDH and DhaB from Plasmids in Pseudomonas denitrificans Expression of P. denitrificans Δ3hpdhΔ3hibdhIVΔ3hibdhIpUCPK...

Claims

1. A method for extracting 3-hydroxypropionic acid (3-HP) from an aqueous solution containing 3-HP using a liquid continuous extractor comprising a solvent vessel containing a first solvent, an extraction vessel, a condenser, and a side tube located above the extraction vessel, comprising: evaporating the first solvent; condensing the evaporated first solvent; and redirecting the flow of the condensed first solvent in the extraction vessel to extract the 3-HP from the aqueous solution; Including, the 3-HP is extracted from the aqueous solution without countercurrent liquid flow; The first solvent is C 1-6 Alkyl acetate, C 4-6 Alcohol, and C 1-6 at least one solvent selected from the group consisting of alkyl ethers; the aqueous solution containing the C 1-3 alcohol and the 3-HP is placed in the extraction vessel; the first solvent evaporates in the solvent container, flows through the side tube to the condenser, and condenses in the condenser; the condensed first solvent flows from the condenser downwardly into the extraction vessel; the condensed first solvent forms an organic phase in the extraction vessel; redirecting the flow of the first solvent from the bottom to the top of the extraction vessel, thereby extracting the 3-HP from an aqueous phase formed from the aqueous solution containing the C 1-3 alcohol and the 3-HP into an organic phase; the organic phase from which the 3-HP has been extracted flows from the extraction vessel through the side tube to the solvent vessel; The aqueous solution is 1-3 extracting the 3-HP from the aqueous phase into the organic phase by including an alcohol; and The C 1-3 The method, wherein the amount of alcohol is 10 v / v % to 50 v / v % of the amount of the aqueous solution.

2. 2. The method of claim 1, wherein the aqueous solution has a pH of 3 to 7.

3. the first solvent comprises at least one solvent selected from the group consisting of 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; or 10. The method of claim 1, wherein the first solvent comprises at least one solvent selected from the group consisting of n-butyl alcohol, isobutyl alcohol, tert-butyl alcohol, n-pentyl alcohol, n-hexyl alcohol, diethyl ether, dipropyl ether, ethyl propyl ether, methyl tert-butyl ether, and methyl hexyl ether.

4. Said C 1-3 10. The method of claim 1, wherein the alcohol comprises at least one solvent selected from the group consisting of methanol, ethanol, n-propanol, and isopropanol.

5. Said C 1-3 2. The method of claim 1, wherein the amount of alcohol is 10% to 20% v / v of the amount of the aqueous solution.

6. 1) the first solvent and the C 1-3 2) the ratio of the combined volume of alcohol to the volume of said aqueous solution is 1:1 to 2:1.

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