Recombinant microorganisms for isoprene production

Abstract

Disclosed are recombinant microorganisms engineered to express heterologous polypeptides. The recombinant microorganisms can be used to produce isoprene from 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) by enzymatic activity of the heterologous polypeptides.

Description

RECOMBINANT MICROORGANISMS FOR ISOPRENE PRODUCTION1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63 / 666,846 filed July 2, 2024, the contents of which are incorporated herein in its entirety.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on June 12, 2025, is named BPC-025WO_SL.xml and is 37,046 bytes in size.3. BACKGROUND

[0003] Isoprene is an important feedstock with numerous applications in the chemical industry, such as the synthesis of polymers, synthetic rubber, and adhesives. About 800,000 metric tons of isoprene are produced annually worldwide. Currently, all commercial isoprene is derived from oil, a non-renewable resource.

[0004] In the interest of sustainability, it would be desirable to produce isoprene from renewable resources.

[0005] Attempts to produce isoprene using renewable resources have included engineering recombinant microorganisms to convert dimethylallyl pyrophosphate (DMAPP) to isoprene by activity of the enzyme isoprene synthase (ispS, EC 4.2.3.27). However, engineering recombinant microorganisms to express ispS may be complex and / or may interfere with natural pathways that require DMAPP for cell growth or maintenance.

[0006] Accordingly, there is a need in the art for improved and sustainable methods for isoprene production.4. SUMMARY

[0007] The present disclosure addresses this need and provides novel recombinant microorganisms engineered to produce isoprene.

[0008] In certain aspects, the present disclosure provides recombinant microorganisms engineered to express heterologous polypeptides that catalyze the conversion of (E)-4- hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) to isoprene. These heterologous polypeptides, referred to herein as ABH or BGH polypeptides, are based on ispH homologs from A. baylyi ADP1 (SEQ ID NO:7) and B. glumae BGR1 (SEQ ID NO:8). As shown herein, these ispH homologs are capable of effectively catalyzing the conversion of HMBPP to isoprene and thus are referred to herein as HMBPP-to-isoprene (“HTI”) polypeptides.

[0009] Examples of recombinant microorganisms of the present disclosure are described inSection 6.2 and numbered embodiments 1 to 61.

[0010] Recombinant microorganisms can be further engineered to maintain suitable ispH / HMBPP reductase activity during growth phases, e.g., as described in Section 6.3, enhance flux through the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway (also known as the MEP pathway) to HMBPP, e.g., as described in Section 6.4, and / or have reduced or no isoprene production from DMAPP, such as by having little or no ispS (EC 4.2.3.27) activity, as also described in Section 6.4, and numbered embodiments 30 to 61 .

[0011] Examples of HTI polypeptides that can be expressed by the recombinant microorganisms of the present disclosure are described in Section 6.5 and numbered embodiments 1 to 15.

[0012] Recombinant microorganisms of the present disclosure can be engineered from parental microorganisms using various methods. Examples of parental microorganisms and methods of engineering recombinant microorganisms of the present disclosure therefrom are described in Section 6.8 and numbered embodiments 16 to 21 .

[0013] Recombinant microorganisms of the present disclosure can be used to produce isoprene by culturing the recombinant microorganisms in appropriate culture media and under appropriate conditions. Examples of culture media and culture conditions for culturing recombinant microorganisms of the present disclosure are described in Section 6.9 and numbered embodiment 62. Examples of the production of isoprene and / or products thereof by recombinant microorganisms of the present disclosure are described in Section 6.10 and numbered embodiments 62 to 69.5. BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 : FIG. 1 schematically depicts the DXP pathway in E. coli, in context with pathways leading from pyruvate and glyceraldehyde-3-phosphate to DXP, and with pathways leading from DMAPP and IPP to isoprene, with certain enzymes assigned numbers and certain reactants and products assigned letters for ease of reference and convenience. Abbreviations and / or assignments used: GAP (A), glyceraldehyde-3- phosphate; pyruvate (B); DXP (C), 1-deoxyxylulose-5-phosphate; MEP (D), 2-C- methylerythritol 4-phosphate; CDP-ME (E), 4-diphosphocytidyl-2-C-methylerythritol; CDP- MEP (F), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MEcPP (G), 2-C-methyl- D-erythritol 2,4-cyclodiphosphate; HMBPP (H), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; DMAPP (I), dimethylallyl pyrophosphate; IPP (J), isopentenyl pyrophosphate; isoprene (K); Dxs (1), 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7); Dxr (2), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267); IspD(3), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60); IspE (4), 4- (cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148); IspF (5), 2-C-methyl- D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12); IspG (6), 4-hydroxy-3-methylbut-2- en-1-yl diphosphate synthase (EC 1.17.7.1); IspH or HMBPP reductase (7), 4-hydroxy-3- methylbut-2-enyl diphosphate reductase (EC 1.17.7.4); HTI, polypeptide catalyzing direct conversion of HMBPP (H) to isoprene (K); Idi (8), isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2). The pathway can be engineered to increase production of isoprene through the expression of a heterologous HTI polypeptide (10) or IspS (9) (EC 4.2.3.27), isoprene synthase. The use of multi-headed arrows (e.g., “^— ►—>•”) indicates one or more enzymatic activities are involved in converting the substrate (or one or more of multiple substrates) to one or more of the reactants in the depicted step. Not all enzymatic activities are shown, and single-headed arrows can be indicative of multi-step processes. Further, certain enzymes can have activity in more than one category (thus, for example, the annotation of an enzyme as an HTI does not preclude it having HMBPP reductase activity and vice versa). Because HTI polypeptides are based on ispH polypeptides, both HTI polypeptides and HMBPP reductases can be considered ispH polypeptides.

[0015] FIGS. 2A-2F: FIGS. 2A-2F schematically depict recombinant microorganisms engineered to express HTI polypeptides.

[0016] FIG. 2A schematically depicts a recombinant microorganism (shown as a thickwalled box with rounded corners) comprising a nucleotide sequence encoding an HTI polypeptide (HTI) operably linked to a single promoter (P). The recombinant microorganism further comprises a coding sequence encoding a native HMBPP reductase (nHMBPPR) operably linked to its native promoter (NP). As shown in FIG. 2A, the HTI coding sequence is on an extrachromosomal nucleic acid (NA), e.g., a plasmid, and the nHMBPPR coding sequence in the genome of the recombinant microorganism (shown as a ring). In an alternative embodiment, rather than NA being an extrachromosomal nucleic acid, it can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6. Exemplary promoters are disclosed in Section 6.7. In some embodiments, P is an inducible promoter.

[0017] FIG. 2B schematically depicts a recombinant microorganism comprising a nucleotide sequence encoding an HTI polypeptide (HTI) operably linked to a single promoter (P). The recombinant microorganism further comprises a coding sequence encoding a native HMBPP reductase (nHMBPPR) operably linked to a heterologous promoter (HP). As shown in FIG. 2B, the HTI coding sequence is on an extrachromosomal nucleic acid (NA), e.g., a plasmid, and the nHMBPPR coding sequence in the genome of the recombinant microorganism(shown as a ring). In an alternative embodiment, rather than NA being an extrachromosomal nucleic acid, it can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6. Exemplary promoters are disclosed in Section 6.7. In some embodiments, P is an inducible promoter.

[0018] FIG. 2C schematically depicts a recombinant microorganism comprising a nucleotide sequence encoding an HTI polypeptide (HTI) operably linked to two promoters (P1 and P2). As shown in FIG. 2C, the HTI coding sequence and two promoters are on an extrachromosomal nucleic acid (NA), e.g., a plasmid. In an alternative embodiment, rather than NA being an extrachromosomal nucleic acid, it can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6. Exemplary promoters are disclosed in Section 6.7. In some embodiments, P1 is an inducible promoter and P2 is a constitutive promoter. In the configuration shown in FIG. 2C, the recombinant microorganism may have a disruption or deletion of the native HMBPP reductase gene.

[0019] FIG. 2D schematically depicts a recombinant microorganism comprising a nucleic acid (NA) comprising a nucleotide sequence encoding a first HTI polypeptide (HT11 ) operably linked to a first promoter (P1) and a second HTI polypeptide (HTI2) operably linked to a second promoter (P2). As shown in FIG. 2D, NA is an extrachromosomal nucleic acid, e.g., a plasmid. In an alternative embodiment, rather than NA being an extrachromosomal nucleic acid, it can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. HTI1 and HTI2 can be the same or different. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6.Exemplary promoters are disclosed in Section 6.7. In some embodiments, P1 is an inducible promoter and P2 is a constitutive promoter. In the configuration shown in FIG. 2D, the recombinant microorganism may have a disruption or deletion of the native HMBPP reductase gene.

[0020] FIG. 2E schematically depicts a recombinant microorganism comprising a first nucleic acid (NA1) comprising a nucleotide sequence encoding a first HTI polypeptide (HT11 ) operably linked to a first promoter (P1) and a second nucleic acid (NA2) comprising a nucleotide sequence encoding a second HTI polypeptide (HTI2) operably linked to a second promoter (P2). As shown in FIG. 2E, NA1 and NA2 are extrachromosomal nucleic acids, e.g., plasmids. In an alternative embodiment, one or both of NA1 and NA2 can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. HTI1 and HTI2 can be the same or different. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6. Exemplary promoters aredisclosed in Section 6.7. In some embodiments, P1 is an inducible promoter and P2 is a constitutive promoter. In the configuration shown in FIG. 2E, the recombinant microorganism may have a disruption or deletion of the native HMBPP reductase gene.

[0021] FIG. 2F schematically depicts a recombinant microorganism comprising a first nucleic acid (NA1) comprising a nucleotide sequence encoding an HTI polypeptide (HTI) operably linked to a first promoter (P1) and a second nucleic acid (NA2) comprising a nucleotide sequence encoding a heterologous HMBPP reductase (hHMBPPR) operably linked to a second promoter (P2). As shown in FIG. 2F, NA1 and NA2 are extrachromosomal nucleic acids, e.g., plasmids. In an alternative embodiment, one or both of NA1 and NA2 can be integrated into the genome of the recombinant microorganism. Exemplary HTI polypeptides are disclosed in Section 6.5. Exemplary HMBPP reductase polypeptides are disclosed in Section 6.3. Exemplary extrachromosomal nucleic acids are disclosed in Section 6.6. Exemplary promoters are disclosed in Section 6.7. In some embodiments, P1 is an inducible promoter and P2 is a constitutive promoter. In the configuration shown in FIG. 2F, the recombinant microorganism may have a disruption or deletion of the native HMBPP reductase gene.

[0022] FIG: 3: FIG. 3 shows isoprene production of E. co / / strains expressing HTI polypeptides or HMBPP reductases, as described in Example 2.

[0023] FIG. 4: FIG. 4 shows growth of E. coli strains expressing HTI polypeptides or HMBPP reductases in LB medium, as described in Example 2.

[0024] FIG. 5: FIG. 5 shows optical density (600nm) of E. coli strains expressing HTI polypeptides or HMBPP reductases after 8-hour incubation in LB medium, as described in Example 2.6. DETAILED DESCRIPTION6.1. Definitions

[0025] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitutean admission that any of these documents forms part of the common general knowledge in the art.

[0026] ABH Polypeptide: As used herein, the term ABH polypeptide refers to a polypeptide that is related to or derived from the A. baylyi ADP1 ispH polypeptide (represented by the amino acid sequence of SEQ ID NO:7). An ABH polypeptide encompasses fragments and / or variants of SEQ ID NO:7 which are capable of catalyzing the production of isoprene from HMBPP. In some embodiments, the fragment and / or variant is capable of producing isoprene from HMBPP to an extent equal to or greater (e.g., at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold greater) than that produced by E. coli HMBPP reductase (SEQ ID NO:6) under the conditions of Example 2. In some embodiments, the fragment of SEQ ID NO:7 is at least 260 amino acids in length, at least 270 amino acids in length, at least 280 amino acids in length, at least 290 amino acids in length, at least 300 amino acids in length, or at least 310 amino acids in length. In some embodiments, the variants comprise an amino acid sequence having at least 90% (e.g., at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:7 or a fragment thereof. Thus the term “ABH polypeptides” encompasses polypeptides which are capable of catalyzing the production of isoprene from HMBPP and having at least 90% (e.g., at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) to the entirety of SEQ ID NO:7 or a fragment thereof of at least 260 amino acids in length, at least 270 amino acids in length, at least 280 amino acids in length, at least 290 amino acids in length, at least 300 amino acids in length, or at least 310 amino acids. In some embodiments, an ABH polypeptide has HMBPP reductase activity, e.g., sufficient HMBPP reductase activity to complement a deletion of the native ispH gene of the recombinant microorganism that recombinantly expresses the ABH polypeptide.

[0027] BGH Polypeptide As used herein, the term BGH polypeptide refers to a polypeptide that is related to or derived from the B. glumae BGR1 ispH polypeptide (represented by the amino acid sequence of SEQ ID NO:8). A BGH polypeptide encompasses fragments and / or variants of SEQ ID NO:8 which are capable of catalyzing the production of isoprene from HMBPP. In some embodiments, the fragment and / or variant is capable of producing isoprene from HMBPP to an extent equal to or greater (e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50- fold greater) than that produced by E. coli HMBPP reductase (SEQ ID NO:6) under the conditions of Example 2. In some embodiments, the fragment of SEQ ID NO:8 is at least 260 amino acids in length, at least 270 amino acids in length, at least 280 amino acids in length, at least 290 amino acids in length, at least 300 amino acids in length, at least 310 aminoacids in length, or at least 320 amino acids in length. In some embodiments, the variants comprise an amino acid sequence having at least 90% (e.g., at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:7 or a fragment thereof. Thus the term “BGH polypeptides” encompasses polypeptides which are capable of catalyzing the production of isoprene from HMBPP and having at least 90% (e.g., at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) to the entirety of SEQ ID NO:8 or a fragment thereof of at least 260 amino acids in length, at least 270 amino acids in length, at least 280 amino acids in length, at least 290 amino acids in length, at least 300 amino acids in length, at least 310 amino acids in length, or at least 320 amino acids. In some embodiments, a BGH polypeptide has HMBPP reductase activity, e.g., sufficient HMBPP reductase activity to complement a deletion of the native ispH gene of the recombinant microorganism that recombinantly expresses the BGH polypeptide.

[0028] Heterologous: As used herein, the term “heterologous,” when used to describe a first element in reference to a second element indicates that the first element and second element do not exist in nature disposed as described. For example, an HTI polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions, or (d) any combination of two or all of (a), (b) and (c). For example, a heterologous regulatory sequence (e.g., promoter, enhancer) can be used to regulate expression of a coding sequence in a way that is different than the coding sequence is normally expressed in nature. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation, wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently or semi-stably for more than one generation (e.g., episomal vector, plasmid or other self-replicating vector).

[0029] HTI Polypeptide: As used herein, the term “HTI polypeptide” refers to an ABH polypeptide or a BGH polypeptide.

[0030] HMBPP Reductase: As used herein, an “HMBPP reductase” is a polypeptide which catalyzes the conversion of HMBPP to DMAPP and IPP (EC 1.17.7.4).

[0031] Nucleic Acid: The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.

[0032] Operably Linked: The term “operably linked,” when used to describe the relationship between a first nucleic acid or nucleotide sequence and a second nucleic acid or nucleotide sequence, indicates that the first nucleic acid or nucleotide sequence is placed in a functional relationship with the second nucleic acid or nucleotide sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two proteincoding sequences, operably linked sequences may be in the same reading frame.

[0033] Operon: An “operon” as used herein refers to a nucleic acid sequence encoding multiple coding sequences which are transcribed in a single transcript. The coding sequences of the operon thus share regulatory sequences that are 5’-ward of the most upstream coding sequence (which may be termed “operon upstream regulatory sequences”) and 3’-ward of the most downstream coding sequence (which may be termed “operon downstream regulatory sequences”).

[0034] Parental Cell, Parental Microorganism: The terms “parental cell” or “parental microorganism” are used interchangeably to refer to unicellular organisms from which a recombinant microorganism can be derived by one or more engineering steps, even if the recombinant microorganism is not directly obtained through such engineering steps. For example, in the context of the present disclosure, if the recombinant microorganism is an E. co / / strain engineered as depicted in FIG. 2E, a parental recombinant microorganism can be a wild-type E. coli strain or an E. coli strain with only one of the first nucleic acid (NA1) or the second nucleic acid (NA2).

[0035] Polypeptide, Peptide, Protein: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.

[0036] Promoter: A “promoter” as used herein refers to a nucleic acid sequence which is capable of interacting with an RNA polymerase such that transcription of a sequence of interest begins. A typical prokaryotic promoter includes a -35 sequence (a region of about 6 nucleotides, the 5’ end of which is located from 30 to 40 nucleotides, such as 35 nucleotides, upstream ( / .e., 5’-ward) of the transcription start site) and a -10 sequence, also known as a Pribnow box (a region of about 6 nucleotides, the 5’ end of which is located from 5 to 15 nucleotides, such as 10 nucleotides, upstream of the transcription initiation site). A prokaryotic promoter typically has from 12 to 22 nucleotides, and in some embodiments 17 ±3 (e.g., 14, 15, 16, 17, 18, 19, or 20 nucleotides), intervening between the -35 sequence and the -10 sequence. A promoter may include at least a portion of a repressor binding site and / or an activator binding site.

[0037] Recombinant Microorganism: The terms “recombinant cell” and “recombinant microorganism” are used interchangeably to refer to a cell that has been genetically engineered. It should be understood that this term refers not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, a recombinant counterpart of a parental cell or parental microorganism includes progeny that are not identical to the initial recombinant cell or microorganism engineered from the parent cell or parental microorganism, but are still included within the scope of the terms “recombinant cell” or “recombinant microorganism” as used herein.

[0038] Regulatory Element, Regulatory Sequence: A “regulatory element” or “regulatory sequence” as used herein refers to non-coding sequences that influence the expression (e.g., transcription or translation) of a transcribed sequence. Regulatory sequences include different types of regulatory elements such as promoters, operator regions, terminator sequences, intergenic sequences encoding small regulatory RNAs (sRNAs), Shine-Dalgarno (SD) sequences, etc.

[0039] Sequence Identity: “Sequence identity” in relation to nucleotide or amino acid sequence of a nucleic acid or polypeptide molecule, refers to the overall relatedness between two such sequences. Calculation of the percent sequence identity (nucleotide or amino acid sequence identity) of two sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Percent sequence identity can be determined manually once an alignment of nucleotide or amino acid sequences is generated. An alignment of query nucleotide or amino acid sequence and a reference nucleotide or amino acid sequence can be generated using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more reference sequencesand aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a reference sequence, or both, to maximize sequence alignments. For fast pair wise alignment of nucleotide sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For fast pairwise alignment of amino acid sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. Unless indicated otherwise, the percent sequence identity between a reference nucleotide or amino acid sequence (e.g. a sequence with a defined SEQ ID NO as disclosed herein) and a query nucleotide or amino acid sequence is calculated across the entire length of the reference sequence.

[0040] Transformation: The term “transformation” refers to the introduction of nucleic acid molecules into cells, e.g., into prokaryotic cells. In the context of the present disclosure, the term “transformation” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, e.g., into prokaryotic cells, such as into bacterial cells. Such methods encompass, for example, electroporation, calcium phosphate precipitation, or nanoparticle-based transformation, among other techniques known to the person of ordinary skill in the art having the benefit of the present disclosure.

[0041] Wild-type: The term “wild-type” as used herein to describe a microorganism species or strain refers to a defined species or strain, e.g., as deposited with a depositary such as the American Type Culture Collection (Manassas, Virginia). When describing a nucleic acid or polypeptide, “wild-type” indicates the nucleic acid or polypeptide has a sequence identical to that of the corresponding nucleic acid or polypeptide in a wild-type species or strain. An exemplary microorganism strain that is sometimes referenced herein as a “wild-type” strain is E. coli K12 substrain MG 1655. Another “wild-type” E. coli strain is E. coli K12 substrain BW25113.6.2. Recombinant Microorganisms Engineered to Express HTI Polypeptides

[0042] The present disclosure relates to recombinant microorganisms engineered to express HTI polypeptides, e g. for production of isoprene. In some embodiments, the recombinant microorganisms comprise heterologous nucleic acids comprising nucleotide sequences encoding the ABH polypeptides or the BGH polypeptides (“HTI coding sequences”).

[0043] The recombinant microorganisms can be any unicellular organisms, such as archaea, bacteria, or fungi, among others. In some embodiments, the bacteria are E. coli. In some embodiments, the E. coli are of strains MG1655, W3110, DH5alpha, W, or BL21.

[0044] The recombinant microorganisms can comprise one or more copies (e.g., one, two, three, four, or more copies, such as 1-6 copies, 1-10 copies, or 11-20 copies) of an HTI coding sequence. Where one or more HTI coding sequences are present in the recombinant microorganisms, the HTI coding sequences can be the same or different. In some embodiments, a recombinant microorganism comprising more than one copy of the same HTI coding sequence.

[0045] In some embodiments, the recombinant microorganism comprises one or more HTI coding sequences integrated into its genome. In some embodiments, genomic integrations of one or more HTI coding sequences are at positions that leave unchanged the expression or activity of native genes and polypeptides encoded by coding sequences thereof. In some embodiments, genomic integrations of one or more HTI coding sequences change the expression or activity of one or more native genes and / or polypeptides encoded by coding sequences thereof. In some embodiments, a change in expression or activity of a native gene comprises replacement of a native gene or a coding sequence thereof with an HTI coding sequence.

[0046] When the HTI coding sequences are integrated into the genome, in some embodiments, the recombinant microorganisms comprise one, two, three, four, five, or six copies of the HTI coding sequences.

[0047] Additionally or alternatively, the recombinant microorganism may comprise one or more HTI coding sequences on one or more extrachromosomal nucleic acids. Examples of extrachromosomal nucleic acids are described in Section 6.6.

[0048] When the HTI coding sequences are on extrachromosomal nucleic acids, in some embodiments, the recombinant microorganisms comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve copies of the HTI coding sequences. In some embodiments, when the HTI coding sequences are on extrachromosomal nucleic acids, the recombinant microorganisms comprise eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, 21 , or 22 copies of the HTI coding sequences.

[0049] Suitable regulatory sequences, including promoters, to regulate expression of HTI coding sequences are described in Section 6.7.

[0050] Recombinant microorganisms generally require one or more isoprenoids for optimal cell growth. Engineering a microorganism to express an HTI can reduce the production of isoprenoid by a native HMBPP reductase. Engineering HMBPP reductase activity to maintain the health or the recombinant microorganism and / or to optimize isoprene production while maintaining adequate isoprenoid production is described in Section 6.3.Different configurations of HTI and HMBPP reductase coding sequences in the recombinant microorganism of the disclosure are illustrated in FIGS. 2A-2F.

[0051] In addition or alternatively to HMBPP reductase engineering, the recombinant microorganisms of the disclosure can be engineered to enhance flux through the DXP pathway to HMBPP and / or have reduced or no isoprene production from DMAPP by the activity of ispS. Engineering a microorganism to increase flux through the DXP pathway and / or modulate ispS activity is described in Section 6.4.

[0052] Specific examples of HTI polypeptides which can be expressed by recombinant microorganisms engineered for that purpose are described in Section 6.5.

[0053] Recombinant microorganisms can be engineered from parental microorganisms, such as those described in Section 6.8, using methods such as those described in Section 6.8.1.

[0054] Recombinant microorganisms, such as those described in this section, can be used to produce isoprene. Recombinant microorganisms can be cultured in media as described in Section 6.9.1 and under conditions as described in Section 6.9.2. Isoprene and products thereof can be produced as described in Section 6.10.6.3. HMBPP Reductase Engineering

[0055] Recombinant microorganisms generally require one or more isoprenoids for optimal cell growth. Wild-type microorganisms generally produce isoprenoids starting from various proportions of the 5-carbon compounds DMAPP and IPP. As discussed above and shown in FIG. 1 , DMAPP (I) and IPP (J) are produced by the HMBPP reductase activity (EC 1.17.7.4) of a microorganism’s HMBPP reductase (7), which is typically an ispH gene. Direct conversion of HMBPP (H) to isoprene (K) can lead to reduced production of DMAPP and IPP, and reduced production of isoprenoids required for growth.

[0056] Recombinant microorganisms expressing HTI polypeptides of the present disclosure can be engineered to address the need for sufficient HMBPP reductase expression and / or activity during growth phases in a number of ways.

[0057] FIGS. 2A-2F schematically depict various ways recombinant microorganisms can be engineered for sufficient HMBPP reductase expression during growth phases.

[0058] In some embodiments, such as those schematically depicted in FIG. 2A, FIG. 2B, and FIG. 2F, the recombinant microorganism expresses both an HTI coding sequence and a HMBPP reductase coding sequence, which can be a native HMBPP reductase coding sequence (e.g., as depicted in FIG. 2A and FIG. 2B) or a heterologous HMBPP reductasecoding sequence (e.g., as depicted in FIG. 2F). A native HMBPP reductase coding sequence can be under the control of a native promoter (NP) as depicted in FIG. 2A or a heterologous promoter (HP) as depicted in FIG. 2B. In some embodiments, the HMBPP reductase coding sequence is under the regulation of a constitutive promoter (NP in FIG. 2A, HP in FIG. 2B, and P2 in FIG. 2F) providing sufficient expression of HMBPP reductase for adequate growth of the recombinant microorganism during the growth phase, while the HTI coding sequence is under the regulation of an inducible promoter (P in each of FIGS. 2A and 2B and P1 in FIG. 2F) that can be utilized to control HTI expression levels when isoprene production is desired.

[0059] In some embodiments, such as those schematically depicted in FIGS. 2C-2E, the native HMBPP reductase gene is disrupted or deleted, and the level of HMBPP reductase activity needed for growth can be provided by an HTI polypeptide which further has HMBPP reductase activity, with expression timing and levels of the HTI polypeptide tuned by selection of an appropriate pair of promoters (P1 and P2). In some embodiments, a constitutive promoter (P2) provides sufficient expression of the HTI polypeptide for adequate growth of the recombinant microorganism during the growth phase, while additional HTI expression is regulated by an inducible promoter (P1) that can be utilized to control HTI expression levels when isoprene production is desired. The HTI polypeptide expressed under the control of P1 can be the same or different from the HTI polypeptide expressed under the control of P2. P1 and P2 can drive expression of a single HTI coding sequence, e.g., as depicted in FIG. 2C, or different HTI coding sequences, e.g., as depicted in FIGS. 2D and 2E. Further, different HTI coding sequences can be on the same nucleic acid, e.g., as depicted in FIG. 2D, or different nucleic acids, e.g., as depicted in FIG. 2E.

[0060] HMBPP reductase activity can be assayed using the colorimetric method described by Grawert et al., 2010, Proc Nat Acad Sci 107:1077-1081 .

[0061] In some embodiments, heterologous HMBPP reductases include those disclosed in any one or more of US 10,480,015; US 10,774,346; US 11 ,352,648; WO 2007 / 140339; WO 2008 / 128159; WO 2010 / 148150; WO 2012 / 088450; WO 2012 / 088462; WO 2018 / 140778; WO 2012 / 135591 ; WO 2014 / 138419; and Wu et al., 2021 , Microbial Cell Factories 20:101 , each of which is hereby incorporated by reference in their entireties.6.4. DXP Pathway and / or ispS Engineering

[0062] As can be seen from FIG. 1 , HTI polypeptides act on HMBPP, which is the product provided by the first six steps of the DXP pathway. The production of isoprene from HMBPP catalyzed by HTI polypeptides of the present disclosure can be increased by increasing fluxthrough the DXP pathway relative to a native microorganism comprising only native DXP pathway enzymes expressed under native regulation.

[0063] Examples of microorganisms engineered to increase flux through the DXP pathway include those disclosed in US 10,480,015; US 10,774,346; US 11 ,352,648; WO 2007 / 140339; WO 2008 / 128159; WO 2010 / 148150; WO 2012 / 088450; WO 2012 / 088462; WO 2018 / 140778; WO 2012 / 135591 ; WO 2014 / 138419; and Wu et al., 2021 , Microbial Cell Factories 20:101 , each of which is hereby incorporated by reference in their entireties.

[0064] FIG. 1 also shows that DMAPP can be converted to isoprene in a reaction catalyzed by ispS. Recombinant microorganisms of the present disclosure engineered to express HTI polypeptides can produce isoprene in fewer steps than required for recombinant microorganisms relying on ispS activity to produce isoprene from DMAPP. Expression of native or heterologous ispS is thus not required for isoprene production by recombinant microorganisms of the present disclosure.

[0065] In some embodiments, the recombinant microorganisms of the present disclosure can produce isoprene without expression of an endogenous or a heterologous ispS. In some embodiments, recombinant microorganisms lack nucleotide sequences encoding endogenous ispS enzymes (EC 4.2.3.27). Alternatively or additionally, recombinant microorganisms can further lack nucleotide sequences encoding heterologous ispS enzymes (EC 4.2.3.27).6.5. HTI polypeptides

[0066] The recombinant microorganisms of the disclosure are engineered to express HTI polypeptides. The HTI polypeptides typically catalyze the conversion of HMBPP (H) to isoprene (K) (see FIG. 1) to a greater extent than the E. coli IspH polypeptide, which has predominantly EC 1 .17.7.4 HMBPP reductase activity (and thus is often referred to herein as an HMBPP reductase). HMBPP reductase activity catalyzes the conversion of HMBPP (H) to a mixture of DMAPP (I) and IPP (J).

[0067] While HTI polypeptides catalyze the conversion of HMBPP to isoprene, they may also possess HMBPP reductase activity.

[0068] The catalytic activity of any HTI polypeptide for the conversion of HMBPP to isoprene can be assayed according to the procedure of Example 2 below and normalized to the activity of E. co / / HMBPP reductase control (SEQ ID NO:6).

[0069] In some embodiments, HTI polypeptides comprise iron-sulfur cluster binding sites, such as [4Fe-4S] or [2Fe-2S] cluster binding sites.6.5.1. ABH polypeptides

[0070] SEQ ID NO:7 is the amino acid sequence of wild-type Acinetobacter baylyi ADP1 ispH (UniProt Accession No. Q9RBJ0). The wild-type polypeptide comprise three domains in a roughly Y-shaped rotational symmetry and a C-terminal tail of about six residues. Under current understanding, one cysteine residue per domain, specifically C12, C96, and C198, binds an iron atom of a [4Fe-4S] cluster at the junction of the three domains; residues H41 , H74, H124, S226, S227, N228, and S270 are involved in substrate binding; and residue E126 is at the proton donation site for HMBPP reductase activity. In some embodiments, ABH polypeptides comprise cysteine residues at positions corresponding to C12, C96, and C198 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise residues at positions corresponding to H41 , H74, H124, S226, S227, N228, and S270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise a residue corresponding to E126 of SEQ ID NO:7.

[0071] ABH polypeptides can be engineered at or near positions corresponding to the ironsulfur cluster binding site, the substrate binding site, or the proton donor site to modify the catalytic activity of HMBPP conversion to isoprene.

[0072] One, two, or all three of the domains can comprise one or more mutations which do not disrupt the catalytic activity of HMBPP conversion to isoprene. The C-terminal tail can be modified or deleted.

[0073] ABH polypeptides can comprise N-terminal truncations, such as truncations of one or more of amino acid residues in a range corresponding to amino acids 1-11 of SEQ ID NO:7, and / or C-terminal truncations, such as truncations of one or more of amino acid residues in a range corresponding to amino acids 271-316 of SEQ ID NO:7.

[0074] In some embodiments, ABH polypeptides comprise amino acid sequences having at least 90% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 92.5% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 95% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 96% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 97% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising aminoacids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 98% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having at least 99% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7. In some embodiments, ABH polypeptides comprise amino acid sequences having 100% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.6.5.2. BGH polypeptides

[0075] SEQ ID NO:8 is the amino acid sequence of wild-type Burkholderia glumae BGR1 ispH (UniProt Accession No. C5AC36). Similarly to the wild-type Acinetobacter polypeptide, the wild-type polypeptide comprises three domains in a roughly Y-shaped rotational symmetry. The wild-type polypeptide also comprises a C-terminal tail of nine residues, and an N-terminal tail of thirteen residues. Under current understanding, one cysteine residue per domain, specifically C27, C111 , and C209, binds an iron atom of a [4Fe-4S] cluster at the junction of the three domains; residues H56, H89, H 139, T179, S237, S238, N239, and S281 are involved in substrate binding; and residue E141 is at the proton donation site for HMBPP reductase activity. In some embodiments, BGH polypeptides comprise cysteine residues at positions corresponding to C27, C111 , and C209 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise residues at positions corresponding to H56, H89, H139, T179, S237, S238, N239, and S281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise a residue corresponding to E141 of SEQ ID NO:8.

[0076] BGH polypeptides can be engineered at or near positions corresponding to the ironsulfur cluster binding site, the substrate binding site, or the proton donor site to modify the catalytic activity of HMBPP conversion to isoprene.

[0077] One, two, or all three of the domains can comprise one or more mutations which do not disrupt the catalytic activity of HMBPP conversion to isoprene.

[0078] BGH polypeptides can comprise N-terminal truncations, such as truncations of one or more of amino acid residues in a range corresponding to amino acids 1-26 of SEQ ID NO:8, and / or C-terminal truncations, such as truncations of one or more of amino acid residues in a range corresponding to amino acids 282-326 of SEQ ID NO:8.

[0079] In some embodiments, BGH polypeptides comprise amino acid sequences having at least 90% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptidescomprise amino acid sequences having at least 92.5% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having at least 95% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having at least 96% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having at least 97% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having at least 98% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having at least 99% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8. In some embodiments, BGH polypeptides comprise amino acid sequences having 100% identity to SEQ ID NO:8.6.6. Extrachromosomal Nucleic Acids

[0080] The recombinant microorganisms of the disclosure are engineered to express HTI polypeptides.

[0081] In some embodiments, the recombinant microorganisms comprise one or more extrachromosomal nucleic acids comprising one or more HTI coding sequence and / or one or more HMBPP reductase coding sequences.

[0082] In some embodiments, the one or more extrachromosomal nucleic acids comprise a plasmid or a portion thereof.

[0083] In some embodiments, the one or more extrachromosomal nucleic acids comprise a bacterial artificial chromosome (BAC) or a portion thereof.

[0084] In some embodiments, the one or more extrachromosomal nucleic acids comprise a yeast artificial chromosome (YA) or a portion thereof.

[0085] Plasmids, BACs, and YACs can be selected to incorporate HTI coding sequence(s) and / or HMBPP reductase coding sequence(s) based at least in part on compatibility with the recombinant microorganism, the inclusion of selection markers, stability over multiple generations of the recombinant microorganism, and / or ease of insertion of the coding sequence therein, among other parameters that will be known to skilled persons.6.7. Regulatory Elements

[0086] The HTI coding sequences in the recombinant microorganisms of the disclosure are typically operably linked to one or more regulatory elements to control the expression of the HTI polypeptides. The recombinant microorganisms may also be engineered to express a HMBPP reductase (e.g., a native HMBPP reductase under the control of a heterologous promoter, e.g., as depicted in FIG. 2B, or a heterologous HMBPP reductase under the control of a native of heterologous promoter, e.g., as depicted in FIG. 2F).

[0087] Typically, the regulatory elements include a promoter.

[0088] Promoters can be identical to native promoters, identical to promoters native to viruses that infect the recombinant microorganisms, engineered variants of native or viral promoters, or fully synthetic promoters. In some embodiments, promoters are recognized by native transcriptional machinery of recombinant microorganisms.

[0089] A promoter can be chosen for operable linkage to any particular HTI and optionally HMBPP reductase coding sequence in view of the species and / or parental strain of the recombinant microorganism, the desired expression level of the HTI and optionally HMBPP coding sequence, and / or other parameters known to skilled persons.

[0090] The promoters can be constitutive or inducible.

[0091] Non-limiting examples of constitutive promoters suitable for use in E. coli include T7 promoters (for example, a T7 A1 promoter of SEQ ID NO:25), E. coli Spc promoters (e.g., a promoter of SEQ ID NO:26), and synthetic constitutive promoters (e.g., a synthetic consensus constitutive promoter of SEQ ID NO:27).

[0092] In some embodiments, the promoters are inducible promoters. Inducible promoters to allow production of the HTI polypeptides and optionally HMBPP reductase polypeptides by the recombinant microorganisms at desired times in the cell cycle and / or to desired level.

[0093] Inducers of the promoters can be compounds produced by recombinant microorganisms at one or more stages of recombinant microorganisms’ life cycle and / or can be compounds not produced by recombinant microorganisms and instead added to culture media comprising recombinant microorganisms.

[0094] In some embodiments the inducible promoters are part of an expression regulatory system, e.g., an operon. Non-limiting examples of inducible promoter systems include lactose regulated systems (e.g., lactose operon systems), sugar regulated systems, metal regulated systems, steroid regulated systems, alcohol regulated systems, IPTG inducible systems, arabinose regulated systems (e.g., arabinose operon systems, e.g., an ARA operon promoter, pBAD, pARA, PARAE, ARAE, ARAR-ParaE, portions thereof,combinations thereof and the like), synthetic amino acid regulated systems (e.g., see Rovner et al., 2015, Nature 518(7537):89-93), fructose repressors, a tac promoter / operator (pTac), tryptophan promoters, PhoA promoters, recA promoters, proU promoters, cst-1 promoters, tetA promoters, cadA promoters, nar promoters, PL promoters, cspA promoters, the like or combinations thereof. In certain embodiments, an inducible promoter is not a lac operon promoter.

[0095] In some embodiments, promoters operably linked to HTI coding sequences are part of an expression system that is inducible by 2-keto-3-deoxy-D-gluconate (KDG). In some embodiments, the KDG-inducible expression system comprises a KDG repressor (KdgR) binding site. Examples of KDG-inducible promoters and expression systems include those found in the E. coli kdgK and eda genes (see, e.g., SEQ ID NO:21 and SEQ ID NO:22, respectively).

[0096] In some embodiments, promoters operably linked to HTI coding sequences are part of an expression system that is inducible by gluconate. In some embodiments, the gluconate-inducible expression system comprises a binding site for the gluconate repressor GntR. Examples of gluconate-inducible promoters and expression systems include those found in the E. coli gntK and gntT genes (see, e.g., SEQ ID NO:23 and SEQ ID NO:24, respectively).

[0097] In addition to promoters, the recombinant microorganisms can comprise other regulatory sequences that influence the expression of HTI and optionally HMBPP reductase coding sequences. As with promoters, particular instances of other regulatory sequences can be chosen for any particular HTI or HMBPP reductase coding sequence in view of the species and / or parental strain of the recombinant microorganism, the desired expression level of the HTI coding sequence, and / or other parameters.6.8. Parental Microorganisms

[0098] Any microorganisms can be parental microorganisms engineered to yield recombinant microorganisms described herein. The parental microorganisms include, but are not limited to, prokaryotes, such as bacteria, e.g., E. coli.

[0099] In some embodiments, the parental microorganism is E. coli. In particular aspects, the E. coli is E. coli strain K12 or a strain derived therefrom, such as E. coli K12 substrain MG1655.

[0100] Other E. coli strains from which recombinant microorganisms of the present disclosure can be engineered include, but are not limited to, E. coli K12 substrain W3110, E. coli K12 substrain DH5alpha, and non-K12 strains, such as E. coli BL21 and E. coli W.

[0101] Recombinant microorganisms of the present disclosure can be engineered from bacteria other than E. coli. Examples of such bacteria include, but are not limited to, Rhodobacter capsulatus, Bacillus subtilis, Pantoea ananatis, Tatumella citrea, Pseudomonas fluorescens, and Pseudomonas putida.

[0102] In some embodiments, parental microorganisms can be engineered to express HTI polypeptides, e.g., as described in Section 6.2, through the introduction of nucleic acids comprising HTI coding sequences.

[0103] In some embodiments, parental microorganisms are also engineered to express heterologous HMBPP reductases or to express native HMBPP reductases under heterologous control, e.g., as described in Section 6.3. In some embodiments, this is achieved through the introduction of nucleic acids comprising the heterologous HMBPP reductase coding sequences. In some embodiments, parental microorganisms are engineered to alter one or more regulatory sequences (e.g., promoters) operably linked to native HMBPP reductase coding sequences, e.g., through the introduction of nucleic acids comprising the regulatory sequences designed to integrate the native HMBPP reductase loci such that the regulatory sequences become operably linked to the native HMBPP reductase coding sequences.

[0104] In some embodiments, parental microorganisms are further engineered to increase flux through the DXP pathway and or alter ispS activity, e.g., as described in Section 6.4.6.8.1. Engineering Methods

[0105] Parental microorganisms can be engineered using techniques known in the art.

[0106] In some embodiments, nucleic acids are introduced into microorganisms by any appropriate transformation technique. Nucleic acids can be extrachromosomal, on a vector (such as a plasmid or a phage), such as a low copy number vector, an intermediate copy number vector, or a high copy number vector. Nucleic acids can be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, nucleic acids can be integrated in one or more copies into the genome of the cell. Integration into the cell’s genome can occur at random by non- homologous recombination, or at selected locations by homologous recombination (e.g., to replace an endogenous coding sequence and / or regulatory sequence with a modified one, a replacement therefor, or a partial or complete deletion thereof), as is well known in the art.

[0107] When multiple coding sequences are to be introduced into a cell, such as for integration into the genome, one or more coding sequences can be included in each of one or more expression cassettes. For example, two expression cassettes can be used. The expression cassettes can be operons, e.g., one promoter and associated regulatorysequences can be operably linked to and enable expression of multiple coding sequences. Independently of one another, each expression cassette can be integrated into the cell’s genome. For example, if two expression cassettes are used, either or both can be integrated into the cell’s genome.

[0108] Various genome editing techniques, including but not limited to homologous recombination, CRISPR, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), can be used to delete or disrupt genes in a parental microorganism or to operably link a coding sequence to a regulatory sequence to which it is not operably linked in a parental microorganism (which may change promoter strength, change whether a promoter is constitutive or inducible, or change which inducer molecule induces transcription of a coding sequence from an inducible promoter), to reduce or increase enzymatic activity of polypeptides encoded by those genes.

[0109] RNAi techniques can be used to reduce activity of enzymes in prokaryotes by regulating gene expression (Waters et al., 2009, Cell 136(4) :615-628) or interfering with translation of RNAs encoding the enzymes. Nucleic acids can be introduced into or engineered in recombinant microorganisms to produce regulatory RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), antisense RNAs (asRNAs), and / or single guide RNAs (sgRNAs) for CRISPR interference.

[0110] Engineering methods can reduce activity of an endogenous polypeptide relative to a wild-type microorganism. For example, all or a portion of coding sequences can be mutated, optionally wherein the mutation can be a deletion; all or a portion of regulatory sequences can be mutated, optionally wherein the mutation can be a deletion; heterologous sequences can be introduced into endogenous loci; interfering RNA (RNAi) systems that reduce activity can be engineered into recombinant microorganisms; or any two, any three, or all four thereof, among other techniques.6.9. Methods of Use6.9.1. Culture Media

[0111] Generally, methods disclosed herein comprise growing cells of a recombinant microorganism in a growth medium suitable for growth to a desired cell density, and culturing the cells in a production medium suitable for production of isoprene. Culturing can be in a batch mode or a continuous mode.

[0112] Examples of media that can be used in batch mode culturing include M9 medium and Hi-Def medium. In some embodiments, M9 medium comprises the following: sodium phosphate dibasic heptahydrate, 1 .28 w / v%; potassium phosphate monobasic, 0.3 w / v%; sodium chloride, 0.05 w / v%; ammonium chloride, 0.1 w / v%; glucose, 0.4 w / v%; MgSO4,0.024 w / v%; and CaCI2, 0.001 w / v%. In some embodiments, Hi-Def medium comprises ingredients known to the person of ordinary skill in the art, and it is commercially available (Teknova Inc. Hollister, CA). Other suitable growth media include, but are not limited to, MOPS, LB, and TB.

[0113] In some embodiments, a culture medium comprises at least 0.1 w / v% sucrose, at least 0.2 w / v% sucrose, at least 0.3 w / v% sucrose, at least 0.4 w / v% sucrose, at least 0.5 w / v% sucrose, at least 0.6 w / v% sucrose, at least 0.7 w / v% sucrose, at least 0.8 w / v% sucrose, at least 0.9 w / v% sucrose, or at least 1 w / v% sucrose. A culture medium typically comprises less than 5 w / v% sucrose, more typically less than 2 w / v% sucrose (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% sucrose; from 0.1 w / v% to 2 w / v% sucrose; from 0.1 w / v% to 1 w / v% sucrose; or from 1 w / v% to 2 w / v% sucrose, among other possible ranges).

[0114] In some embodiments, culture media comprise at least 0.1 w / v% glucose, at least 0.2 w / v% glucose, at least 0.3 w / v% glucose, at least 0.4 w / v% glucose, at least 0.5 w / v% glucose, at least 0.6 w / v% glucose, at least 0.7 w / v% glucose, at least 0.8 w / v% glucose, at least 0.9 w / v% glucose, or at least 1 w / v% glucose. A culture medium typically comprises less than 5 w / v% glucose, more typically less than 2 w / v% glucose (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% glucose; from 0.1 w / v% to 2 w / v% glucose; from 0.1 w / v% to 1 w / v% glucose; or from 1 w / v% to 2 w / v% glucose, among other possible ranges).

[0115] In some embodiments, culture media comprise at least 0.1 w / v% gluconate, at least 0.2 w / v% gluconate, at least 0.3 w / v% gluconate, at least 0.4 w / v% gluconate, at least 0.5 w / v% gluconate, at least 0.6 w / v% gluconate, at least 0.7 w / v% gluconate, at least 0.8 w / v% gluconate, at least 0.9 w / v% gluconate, or at least 1 w / v% gluconate. A culture medium typically comprises less than 5 w / v% gluconate, more typically less than 2 w / v% gluconate (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% gluconate; from 0.1 w / v% to 2 w / v% gluconate; from 0.1 w / v% to 1 w / v% gluconate; or from 1 w / v% to 2 w / v% gluconate, among other possible ranges).

[0116] In some embodiments, culture media comprise at least 0.1 w / v% cellulose-derived sugars, at least 0.2 w / v% cellulose-derived sugars, at least 0.3 w / v% cellulose-derived sugars, at least 0.4 w / v% cellulose-derived sugars, at least 0.5 w / v% cellulose-derived sugars, at least 0.6 w / v% cellulose-derived sugars, at least 0.7 w / v% cellulose-derived sugars, at least 0.8 w / v% cellulose-derived sugars, at least 0.9 w / v% cellulose-derived sugars, or at least 1 w / v% cellulose-derived sugars. A culture medium typically comprises less than 5 w / v% cellulose-derived sugars, more typically less than 2 w / v% cellulose-derivedsugars (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% cellulose-derived sugars; from 0.1 w / v% to 2 w / v% cellulose-derived sugars; from 0.1 w / v% to 1 w / v% cellulose-derived sugars; or from 1 w / v% to 2 w / v% cellulose-derived sugars, among other possible ranges). The concentrations of cellulose-derived sugars listed here are the sum of the concentrations of all cellulose-derived sugars (which may be one or more cellulose-derived sugars, e.g., glucose and / or xylose) in the media.

[0117] In some embodiments, a production medium comprises sucrose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises gluconate. In some embodiments, a production medium comprises one or more cellulose-derived sugars. In some embodiments, a production medium comprises any two, and three, or all four of sucrose, glucose, gluconate, or cellulose-derived sugars. The inclusion of gluconate in production media can induce expression of sequences of interest in recombinant microorganisms comprising nucleic acids comprising coding sequences operably linked to gluconate-inducible promoters.

[0118] In some embodiments, a production medium comprises an inducer. In some embodiments, a production medium comprises KDG. The inclusion of KDG in production media can induce expression of sequences of interest in recombinant microorganisms comprising nucleic acids comprising coding sequences operably linked to KDG-inducible promoters.

[0119] In some embodiments, wherein culture media comprise two or more carbon sources, culture media comprise at least 0.5 w / v% total carbon sources, at least 0.6 w / v% total carbon sources, at least 0.7 w / v% total carbon sources, at least 0.8 w / v% total carbon sources, at least 0.9 w / v% total carbon sources, or at least 1 w / v% total carbon sources. A culture medium typically comprises less than 5 w / v% total carbon sources, more typically less than 2 w / v% total carbon sources (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% total carbon sources; from 0.1 w / v% to 2 w / v% total carbon sources; from 0.1 w / v% to 1 w / v% total carbon sources; or from 1 w / v% to 2 w / v% total carbon sources, among other possible ranges).

[0120] In some embodiments of some methods described herein, it may be desirable to allow growth of a recombinant microorganism without expression of one or more genes until a desired biomass of the recombinant microorganism has been reached. For example, such growth can be encouraged or effected by use of a growth medium comprising glycerol, such as at least 0.1 w / v% glycerol, at least 0.2 w / v% glycerol, at least 0.3 w / v% glycerol, at least 0.4 w / v% glycerol, at least 0.5 w / v% glycerol, at least 0.6 w / v% glycerol, at least 0.7 w / v% glycerol, at least 0.8 w / v% glycerol, at least 0.9 w / v% glycerol, or at least 1 w / v% glycerol. Agrowth medium typically comprises less than 5 w / v% glycerol, more typically less than 2 w / v% glycerol (e.g., in some embodiments, growth media comprise from 0.1 w / v% to 5 w / v% glycerol; from 0.1 w / v% to 2 w / v% glycerol; from 0.1 w / v% to 1 w / v% glycerol; or from 1 w / v% to 2 w / v% glycerol, among other possible ranges).

[0121] Although glycerol can provide a carbon source for growth of a recombinant microorganism in a growth medium, glycerol can be included in a production medium. Typically, glycerol is included in a production medium at the same or lower concentration than in a growth medium.

[0122] In some embodiments, a growth medium lacks added glucose and / or sucrose, i.e., one or both of these sugars is not intentionally included in a growth medium. In particular embodiments, a growth medium comprises no more than 0.1 w / v% each of glucose and / or sucrose.

[0123] The selection of particular concentrations of sucrose, glucose and / or glycerol to include in a production medium and / or a growth medium can be made by the person of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

[0124] For fed-batch and / or continuous mode culturing, the ranges of sucrose, glucose, gluconate, glycerol, or combinations thereof given above can be initially provided to the medium. The consumption of the carbon source(s) during culturing can be repeatedly or continuously monitored and additional carbon source(s) can be provided as needed to sustain a desired respiratory coefficient, growth rate, rate of expression of sequences of interest, and / or a rate of production of desired compound(s). The feed rate may be adjusted to avoid accumulation of carbon source(s), which may maximize output of desired compound(s) and minimize waste of carbon source(s). The person of ordinary skill in the art having the benefit of the present disclosure can select the medium composition and the amount of carbon source added thereto during the process to enable the expression of sequences of interest to a desired level and / or production of desired product(s) to a desired concentration, such as at least 20 g / L, at least 50 g / L, or at least 100 g / L.6.9.2. Culture Conditions

[0125] Recombinant cells comprising expression systems of the disclosure may be cultured under suitable conditions in a medium, such as a medium described in Section 6.9.1 . In some embodiments, recombinant cells undergo fermentation. Fermentation conditions include batch, fed-batch and continuous fermentation. Classical batch fermentation is a closed system, wherein the composition of the medium is not subject to artificial alterations during fermentation. In fed-batch fermentation, the substrate is added in increments as fermentation progresses. In both classical batch fermentation and batch-fed fermentation,the product(s) remain in the bioreactor until the end of the process. Batch and fed-batch fermentation are common and well-known in the art. In continuous fermentation, a defined medium is added continuously to the bioreactor and an equal volume of product containing medium is removed simultaneously. Continuous fermentation aims to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology. The fermentation process is typically an aerobic fermentation process.

[0126] The fermentation process is typically run at a temperature that is optimal for growth of a recombinant microorganism. Fermentation for a mesophilic microorganism is typically carried out at a temperature within the range of from 20°C to 45°C, from 25°C to 40°C, from 35°C to 40°C, or from 30°C to 37°C. In some embodiments wherein a recombinant microorganism is derived from one of the exemplary microorganisms described herein, culturing comprises maintaining the recombinant microorganism at a mesophilic temperature. In some embodiments, the mesophilic temperature is selected from any of the foregoing ranges.

[0127] Fermentation is typically carried out at a pH in the range of 4 to 8, in the range of 5 to 7, or the range of 5.5 to 6.5. For example, fermentation can be carried out for a period of time within the range of from 8 to 240 hours, from 12 hours to 168 hours, from 16 hours to 144 hours, from 20 hours to 120 hours, from 24 hours to 72 hours, or from 36 to 48 hours.6.10. Methods for Producing Isoprene and Isoprene Products

[0128] In some aspects, the present disclosure relates to methods for producing isoprene that makes use of recombinant microorganisms. In some embodiments, the methods comprising culturing recombinant microorganisms as described in Section 6.2 under conditions in which isoprene is produced. The recombinant microorganisms can be E. coli, including but not limited to E. coli described in Section 6.8.

[0129] The conditions can include culturing recombinant microorganisms in appropriate media, e.g., media comprising glucose.

[0130] Cells can be grown to desired cell concentrations in appropriate media. After growth to desired cell concentrations, cells in which one or more nucleotide sequences encoding HTI polypeptides are operably linked to inducible promoters can be cultured in media comprising the inducer. Induction of expression of HTI polypeptides leads to the enzymatic conversion of HMBPP to isoprene.

[0131] Isoprene is insoluble in water, and has a boiling point of 34°C, which is in or below the ranges of typical fermentation temperatures described in Section 6.9.2 for mesophilic organisms. Isoprene produced in the methods of the disclosure will typically exit cells of the recombinant microorganism, pass through the medium, and enter a headspace above the medium. Recovery of isoprene can be from a headspace above the medium.

[0132] Isoprene can be recovered using standard techniques, such as gas stripping, membrane enhanced separation, fractionation, adsorption / desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent. In some embodiments, isoprene is recovered by absorption stripping. In some embodiments, isoprene is recovered by extractive distillation with an alcohol, such as ethanol, methanol, propanol, or a combination thereof. In some embodiments, isoprene is recovered by isolation in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). In some embodiments, isoprene is recovered by gas stripping, in which isoprene vapor is removed from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation by cooling and / or pressurization. In some embodiments, isoprene is recovered by membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor, resulting in the condensation of liquid isoprene. In some embodiments, isoprene is compressed and condensed.

[0133] Isoprene recovery can be performed in one step or in multiple steps. In some embodiments, the removal of isoprene vapor from fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent. In one embodiment, the isoprene is recovered by absorption stripping. Suitable methods to recover isoprene from fermentation broths have been described in US 9,751 ,820.

[0134] In some embodiments, methods further comprise purifying isoprene. Isoprene can be purified using techniques known to the skilled person. Examples of purification methods include distillation and chromatography. Isoprene purity can be assayed by any appropriate method, such as column chromatography, HPLC analysis, or GC-MS analysis.

[0135] In some embodiments, methods further comprise polymerizing the recovered isoprene to yield a polymer comprising an isoprene unit. The polymerizing can behomopolymerization of isoprene or copolymerization of isoprene with one or more nonisoprene monomers. Homopolymerization and copolymerization techniques known to the skilled person can be used to form cis-polyisoprene, other isoprene homopolymers, and isoprene copolymers.

[0136] Isoprene homopolymers comprise at least two isoprene units. Known isoprene homopolymers can comprise on the order of 10,000 isoprene units. Isoprene copolymers comprise at least one isoprene unit and one non-isoprene unit. Isoprene copolymers can comprise block copolymers comprising blocks of isoprene units and blocks of non-isoprene units; alternating copolymers comprising an alternating pattern of an isoprene unit and a non-isoprene unit, graft copolymers; or star copolymers, among other copolymers known to the skilled person.

[0137] Isoprene can also be efficiently converted to 1 ,6-dimethyl-1 ,5-cyclooctadiene (DMCOD), that can be used for the production of high-performance bio-based jet fuel (Rosenkoetter K. et al., Green Chem., 2019, 21 : 5616-5623).

[0138] In some embodiments, methods further comprise using the recovered isoprene to react with non-isoprene molecules, to yield molecules of higher molecular weight. An example of such reaction is the cross-coupling of the terpenoid myrcene with isoprene to form high-performance jet and diesel blendstocks, as described by Woodroffe and Harvey (Energy Fuels 2022, 36: 2630-2638).

[0139] Because isoprene is flammable, appropriate precautions should be taken during fermentation, recovery, purification, and handling.7. SPECIFIC EMBODIMENTS

[0140] The present disclosure is exemplified by the specific embodiments below.1 . A recombinant microorganism engineered to express an HTI polypeptide, optionally wherein the HTI polypeptide comprises an amino acid sequence:(a) having at least 90% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7, or(b) having at least 90% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.2. The recombinant microorganism of embodiment 1 , wherein the amino acid sequence has at least 92.5% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.3. The recombinant microorganism of embodiment 1 or embodiment 2, wherein the amino acid sequence has at least 95% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.4. The recombinant microorganism of any one of embodiments 1 to 3, wherein the amino acid sequence has at least 96% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.5. The recombinant microorganism of any one of embodiments 1 to 4, wherein the amino acid sequence has at least 97% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.6. The recombinant microorganism of any one of embodiments 1 to 5, wherein the amino acid sequence has at least 98% identity to SEQ ID NO:7 or to a fragment thereof, e g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.7. The recombinant microorganism of any one of embodiments 1 to 6, wherein the amino acid sequence has at least 99% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.8. The recombinant microorganism of any one of embodiments 1 to 7, wherein the amino acid sequence has 100% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.9. The recombinant microorganism of embodiment 1 , wherein the amino acid sequence has at least 92.5% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.10. The recombinant microorganism of embodiment 1 or 9, wherein the amino acid sequence has at least 95% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.11 . The recombinant microorganism of any one of embodiments 1 or 9 to 10, wherein the amino acid sequence has at least 96% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.12. The recombinant microorganism of any one of embodiments 1 or 9 to 11 , wherein the amino acid sequence has at least 97% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.13. The recombinant microorganism of any one of embodiments 1 or 9 to 12, wherein the amino acid sequence has at least 98% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.14. The recombinant microorganism of any one of embodiments 1 or 9 to 13, wherein the amino acid sequence has at least 99% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.15. The recombinant microorganism of any one of embodiments 1 or 9 to 14, wherein the amino acid sequence has 100% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.16. The recombinant microorganism of any one of embodiments 1 to 15, which is an E. coli.17. The recombinant microorganism of embodiment 16, which is an E. coli MG1655.18. The recombinant microorganism of embodiment 16, which is an E. coli K12 substrain W3110.19. The recombinant microorganism of embodiment 16, which is an E. coli K12 substrain DH5alpha.20. The recombinant microorganism of embodiment 16, which is an E. coli \N.21 . The recombinant microorganism of embodiment 16, which is an E. coli BL21 .22. The recombinant microorganism of any one of embodiments 1 to 21 , which further comprises a first nucleic acid comprising a first nucleotide sequence encoding the HTI polypeptide.23. The recombinant microorganism of embodiment 22, wherein the first nucleic acid is genomically integrated.24. The recombinant microorganism of embodiment 22, wherein the first nucleic acid is extrachromosomal.25. The recombinant microorganism of any one of embodiments 22 to 24, wherein the first nucleic acid comprises a first promoter that is operably linked to the first nucleotide sequence.26. The recombinant microorganism of embodiment 25, wherein the first promoter is an inducible promoter.27. The recombinant microorganism of embodiment 26, wherein the first promoter is not the lac operon promoter.28. The recombinant microorganism of embodiment 26 or embodiment 27, wherein the first promoter is inducible by 2-keto-3-deoxy-D-gluconate (KDG).29. The recombinant microorganism of embodiment 26 or embodiment 27, wherein the first promoter is inducible by gluconate.30. The recombinant microorganism of any one of embodiments 26 to 29, wherein the first nucleic acid further comprises a second promoter operably linked to the first nucleotide sequence.31 . The recombinant microorganism of embodiment 30, wherein the second promoter is a constitutive promoter.32. The recombinant microorganism of embodiment 30 or embodiment 31 , wherein the second promoter drives expression of the first nucleotide sequence during a growth phase.33. The recombinant microorganism of any one of embodiments 26 to 32, further comprising a second nucleotide sequence encoding the HTI polypeptide.34. The recombinant microorganism of embodiment 33, further comprising a second promoter operably linked to the second nucleotide sequence.35. The recombinant microorganism of embodiment 34, wherein the second promoter is a constitutive promoter.36. The recombinant microorganism of embodiment 34 or embodiment 35, wherein the second promoter drives expression of the second nucleotide sequence during a growth phase.37. The recombinant microorganism of any one of embodiments 33 to 36, wherein the second nucleotide sequence is on the first nucleic acid.38. The recombinant microorganism of any one of embodiments 33 to 36, wherein the second nucleotide sequence is on a second nucleic acid.39. The recombinant microorganism of embodiment 38, wherein the second nucleic acid is genomically integrated.40. The recombinant microorganism of embodiment 38, wherein the second nucleic acid is extrachromosomal.41 . The recombinant microorganism of any one of embodiments 1 to 40, further comprising a third nucleotide sequence encoding a 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (EC: 1.17.7.4) having less than 90% identity to SEQ ID NO:7 and to SEQ ID NO:8.42. The recombinant microorganism of embodiment 41 , further comprising a third promoter operably linked to the third nucleotide sequence.43. The recombinant microorganism of embodiment 41 or embodiment 42, wherein the third promoter is a constitutive promoter.44. The recombinant microorganism of embodiment 43, wherein the third promoter which drives expression of the third nucleotide sequence during a growth phase.45. The recombinant microorganism of any one of embodiments 41 to 44, wherein the HMBPP reductase encoded by the third nucleotide sequence is native to the recombinant microorganism.46. The recombinant microorganism of embodiment 45, wherein the third promoter is natively operably linked to a nucleotide sequence encoding the HMBPP reductase in the recombinant microorganism.47. The recombinant microorganism of any one of embodiments 41 to 44, which is engineered to express the third nucleotide sequence.48. The recombinant microorganism of any one of embodiments 41 to 47, wherein the third nucleotide sequence is on the first nucleic acid.49. The recombinant microorganism of any one of embodiments 41 to 47, wherein the third nucleotide sequence is on the second nucleic acid.50. The recombinant microorganism of any one of embodiments 41 to 47, wherein the third nucleotide sequence is on a third nucleic acid.51 . The recombinant microorganism of embodiment 50, wherein the third nucleic acid is genomically integrated.52. The recombinant microorganism of embodiment 50, wherein the third nucleic acid is extrachromosomal.53. The recombinant microorganism of any one of embodiments 1 to 52, in which flux through the DXP pathway is increased relative to a native microorganism comprising only native DXP pathway enzymes expressed under native regulation.54. The recombinant microorganism of any one of embodiments 1 to 53, which is engineered to not express an endogenous ispS enzyme (EC 4.2.3.27).55. The recombinant microorganism of any one of embodiments 1 to 54, which is not engineered to express a heterologous ispS enzyme (EC 4.2.3.27).56. The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2A.57. The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2B.58. The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2C.59. The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2D.60. The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2E.61 . The recombinant microorganism of any one of embodiments 1 to 55, which has the genetic configuration depicted in FIG. 2F.62. A method for producing isoprene, comprising:(a) culturing the recombinant microorganism of any one of embodiments 26 to 61 in a medium; and(b) inducing expression of the first nucleotide sequence during a production phase.63. The method of embodiment 62, further comprising recovering isoprene from a headspace above the medium.64. The method of embodiment 63, further comprising purifying the recovered isoprene.65. The method of embodiment 63 or embodiment 64, further comprising polymerizing the recovered isoprene, to yield a polymer comprising an isoprene unit.66. The method of embodiment 63 or embodiment 64, further comprising converting the recovered isoprene to 1 ,6-dimethyl-1 ,5-cyclooctadiene (DMCOD).67. The method of embodiment 63 or embodiment 64, further comprising reacting the recovered isoprene with a non-isoprene organic compound.68. The method of embodiment 67, wherein the non-isoprene organic compound is a terpenoid.69. The method of embodiment 68, wherein the terpenoid is myrcene.8. EXAMPLES8.1. Example 1. Cloning of IspH homologs.

[0141] PCR amplification of Escherichia coli K12 MG1655 ispH was performed with primer PRSA110 EcispH_HindlllFor (SEQ ID NO: 1) and PRSA111 EcispH_SallRev (SEQ ID NO: 2) using genomic DNA as a template. Codon-optimized Rhodobacter capsulatus SB1003 ispH was PCR-amplified from a synthetic construct using primer PRSA1 14 RcispH_HindlllFor (SEQ ID NO: 3) and PRSA115 RcispH_SallRev (SEQ ID NO: 4).Amplified products were digested with Hind 111 and Sall, ligated to pBAD-crtEIB plasmid (SEQ ID NO: 5), and transformed into E. coli DH5alpha using standard protocol. The resulting plasmid constructs were purified and confirmed by Sanger sequencing.

[0142] Codon-optimized ispH homologs from Acinetobacter baylyi ADP1 , Burkholderia glumae BGR1 , Caulobacter crescentus NA1000, Pantoea ananatis PA13, Pseudomonas fluorescens SBW25, Proteus mirabilis HI4320, Picea sitchensis, Populus trichocarpa, Ricinus communis, Serratia marcescens subsp. marcescens ATCC 13880, Shewanellaoneidensis MR-1 , Synechococcus PCC 7002, and Wigglesworthia glossinidia were synthesized and cloned into pBAD-crtEIB plasmid at the Hind I II and Sall sites by GenScript (Piscataway, NJ).8.2. Example 2. Isoprene production of heterologous IspH homologs in E. coli host.

[0143] E. coli AispH mutants expressing heterologous IspH polypeptides which complemented the deletion of the native gene were grown in Lysogeny Broth (LB) containing carbenicillin (100 mg / L) in a shaking incubator at 37°C. To assess isoprene production, overnight cultures were diluted in 0.5 ml Terrific Broth (TB) containing carbenicillin (100 mg / L) such that the optical density at 600 nm (ODeoo) was at 0.05. TB cultures were transferred into 20-ml sealed headspace vials (Agilent, Santa Clara, CA) and incubated in a shaker at 37°C. After 24 h, a sample from the headspace of the vial was taken, and isoprene concentration was measured using gas chromatography flame ionization detector (GC-FID) method and normalized to ODeoo of each culture. To assess growth, overnight cultures were diluted 100-fold in 0.2 ml LB containing carbenicillin (100 mg / L) and incubated at 37°C in a 96-well plate shaker. Growth profile of each strain was monitored by measuring ODeoo at 30- minute intervals over 12 h.

[0144] FIG. 3 shows the comparison of isoprene produced by E. coli strains expressing native and heterologous IspH polypeptides. The highest isoprene producers were strains expressing HTI polypeptides from B. glumae and A. baylyi, producing 41.6 and 8.3-fold more isoprene, respectively. The remaining strains expressing HMBPP reductase polypeptides showed a 1 .5 to 3.6-fold improvement compared to their E. coli counterpart. The results shown in FIG. 3 are the averages of three runs per strain; Table 1 shows the results for each run. For each strain, the results for each run shown in Table 1 were normalized to the native enzyme.* Calculated by dividing isoprene titer (normalized to OD6oo) of each strain to that of the native E. coli IspH control.

[0145] FIG. 4 shows OD6oo (in logarithmic scale) time curves over 12 h and FIG. 5 shows OD6OO values (t = 8 h) for the test strains and the E. coli control grown in LB. Strains expressing heterologous IspH homologs from P. ananatis, P. mirabilis, S. marcescens, S. oneidensis, and Synechococcus exhibit comparable growth to the native E. coli IspH control.9. SEQUENCES

[0146] Exemplary sequences referred to herein are provided in Table 2 below (where “SEQ” refers to the SEQ ID NO).10. CITATION OF REFERENCES

[0147] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

Claims

WHAT IS CLAIMED IS:1 . A recombinant microorganism engineered to express an HTI polypeptide, optionally wherein the HTI polypeptide comprises an amino acid sequence:(a) having at least 90% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7, or(b) having at least 90% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.

2. The recombinant microorganism of claim 1 , wherein the amino acid sequence has at least 95% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.

3. The recombinant microorganism of claim 1 or 2, wherein the amino acid sequence has at least 99% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.

4. The recombinant microorganism of any one of claims 1 to 3, wherein the amino acid sequence has 100% identity to SEQ ID NO:7 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 12-270 of SEQ ID NO:7.

5. The recombinant microorganism of claim 1 , wherein the amino acid sequence has at least 95% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.

6. The recombinant microorganism of any one of claims 1 or 5, wherein the amino acid sequence has at least 99% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.

7. The recombinant microorganism of any one of claims 1 , 5 or 6, wherein the amino acid sequence has 100% identity to SEQ ID NO:8 or to a fragment thereof, e.g., a fragment thereof comprising amino acids 27-281 of SEQ ID NO:8.

8. The recombinant microorganism of any one of claims 1 to 7, which is an E. coli.

9. The recombinant microorganism of claim 8, which is an E. coli MG 1655, an E. coli K12 substrain W3110, an E. coli K12 substrain DH5alpha, an E. coli \N, or an E. coli BL21.

10. The recombinant microorganism of any one of claims 1 to 9, which further comprises a first nucleic acid comprising a first nucleotide sequence encoding the HTI polypeptide.11 . The recombinant microorganism of claim 10, wherein the first nucleic acid is genomically integrated.

12. The recombinant microorganism of claim 10, wherein the first nucleic acid is extrachromosomal.

13. The recombinant microorganism of any one of claims 10 to 12, wherein the first nucleic acid comprises a first promoter that is operably linked to the first nucleotide sequence.

14. The recombinant microorganism of claim 13, wherein the first promoter is an inducible promoter.

15. The recombinant microorganism of claim 14, wherein the first promoter is not the lac operon promoter.

16. The recombinant microorganism of claim 14 or claim 15, wherein the first promoter is inducible by 2-keto-3-deoxy-D-gluconate (KDG).

17. The recombinant microorganism of claim 14 or claim 15, wherein the first promoter is inducible by gluconate.

18. The recombinant microorganism of any one of claims 14 to 17, wherein the first nucleic acid further comprises a second promoter operably linked to the first nucleotide sequence.

19. The recombinant microorganism of claim 18, wherein the second promoter is a constitutive promoter.

20. The recombinant microorganism of claim 18 or claim 19, wherein the second promoter drives expression of the first nucleotide sequence during a growth phase.21 . The recombinant microorganism of any one of claims 14 to 20, further comprising a second nucleotide sequence encoding the HTI polypeptide.

22. The recombinant microorganism of claim 21 , further comprising a second promoter operably linked to the second nucleotide sequence.

23. The recombinant microorganism of claim 22, wherein the second promoter is a constitutive promoter.

24. The recombinant microorganism of claim 22 or claim 23, wherein the second promoter drives expression of the second nucleotide sequence during a growth phase.

25. The recombinant microorganism of any one of claims 21 to 24, wherein the second nucleotide sequence is on the first nucleic acid.

26. The recombinant microorganism of any one of claims 21 to 24, wherein the second nucleotide sequence is on a second nucleic acid.

27. The recombinant microorganism of claim 26, wherein the second nucleic acid is genomically integrated.

28. The recombinant microorganism of claim 26, wherein the second nucleic acid is extrachromosomal.

29. The recombinant microorganism of any one of claims 1 to 28, further comprising a third nucleotide sequence encoding a 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (EC: 1 .17.7.4) having less than 90% identity to SEQ ID NO:7 and to SEQ ID NO:8.

30. The recombinant microorganism of claim 29, further comprising a third promoter operably linked to the third nucleotide sequence, wherein the third promoter is a constitutive promoter which drives expression of the third nucleotide sequence during a growth phase.31 . The recombinant microorganism of claim 29 or claim 30, wherein the HMBPP reductase encoded by the third nucleotide sequence is native to the recombinant microorganism and wherein the third promoter is natively operably linked to a nucleotide sequence encoding the HMBPP reductase in the recombinant microorganism.

32. The recombinant microorganism of any one of claims 29 to 31 , which is engineered to express the third nucleotide sequence.

33. The recombinant microorganism of any one of claims 29 to 32, wherein the third nucleotide sequence is on the first nucleic acid.

34. The recombinant microorganism of any one of claims 29 to 32, wherein the third nucleotide sequence is on the second nucleic acid.

35. The recombinant microorganism of any one of claims 29 to 32, wherein the third nucleotide sequence is on a third nucleic acid.

36. The recombinant microorganism of claim 35, wherein the third nucleic acid is genomically integrated.

37. The recombinant microorganism of claim 35, wherein the third nucleic acid is extrachromosomal.

38. The recombinant microorganism of any one of claims 1 to 37, in which flux through the DXP pathway is increased relative to a native microorganism comprising only native DXP pathway enzymes expressed under native regulation.

39. The recombinant microorganism of any one of claims 1 to 38, which is engineered to not express an endogenous ispS enzyme (EC 4.2.3.27).

40. The recombinant microorganism of any one of claims 1 to 39, which is not engineered to express a heterologous ispS enzyme (EC 4.2.3.27).41 . The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2A.

42. The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2B.

43. The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2C.

44. The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2D.

45. The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2E.

46. The recombinant microorganism of any one of claims 1 to 40, which has the genetic configuration depicted in FIG. 2F.

47. A method for producing isoprene, comprising:(a) culturing the recombinant microorganism of any one of claims 14 to 46 in a medium; and(b) inducing expression of the first nucleotide sequence during a production phase.

48. The method of claim 47, further comprising recovering isoprene from a headspace above the medium and purifying the recovered isoprene.

49. The method of claim 48, further comprising polymerizing the recovered isoprene, to yield a polymer comprising an isoprene unit.

50. The method of claim 48, further comprising converting the recovered isoprene to 1 ,6-dimethyl-1 ,5-cyclooctadiene (DMCOD).51 . The method of claim 48 or claim 49, further comprising reacting the recovered isoprene with a terpenoid.

52. The method of claim 51 , wherein the terpenoid is myrcene.

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