Engineered DXP pathway for improved isoprenoid production in e. coli

Abstract

The present disclosure relates to recombinant microorganisms, such as bacteria, e.g., E. coli, engineered to have improved flux and / or yield of the DXP pathway. Cells can be engineered to comprise nucleotide sequences comprising dxs polypeptides (EC 2.2.1.7); dxr polypeptides (EC 1.1.1.267); ispD polypeptides (EC 2.7.7.60); ispE polypeptides (EC 2.7.1.148); ispF polypeptides (EC 4.6.1.12); ispG polypeptides (EC 1.17.7.1 or EC 1.17.7.3); ispH polypeptides (EC 1.17.7.4); and idi polypeptides (EC 5.3.3.2). Cells can be further engineered to express redox polypeptides, e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin--NADP reductases (EC 1.19.1.1 or EC 1.18.1.2), e.g., to increase the activity of the ispG and / or ispH polypeptides. Some or all of the nucleotide sequences can be heterologous to the cells. The recombinant microorganisms can be used in methods of isoprenoid production.

Description

ENGINEERED DXP PATHWAY FOR IMPROVED ISOPRENOID PRODUCTION IN E COLI1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63 / 623,156 filed January 19, 2024 and United States provisional application no. 63 / 676,144, filed July 26, 2024, the contents of each of which are incorporated herein in their entireties.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 January 15, 2025, is named BPC-021WO_SL.xml and is 192,939 bytes in size.3. BACKGROUND

[0003] Isoprenoids are organic compounds having 5n carbon atoms, where n is an integer from 1 to 10 or more. Isoprenoids have potential uses as fuels, lubricants, and thermal management fluids. Because many bioindustrial microorganisms possess metabolic pathways that convert monosaccharides such as glucose or xylose to dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), and pathways that convert DMAPP and IPP to isoprenoids, isoprenoids are of particular interest in the development of renewable or “green” fuels.

[0004] Bacteria such as E. coli convert glucose or xylose to DMAPP and IPP via the 1- deoxyxylulose-5-phosphate (DXP) pathway (also known as the MEP pathway or the nonmevalonate pathway). However, in view of the variegated demands of other metabolic pathways, environmental demands such as carbon sources, and stages of the cell cycle, wild type E. coli generally produces relatively little DMAPP and IPP, and hence, has a relatively low yield of isoprenoids.

[0005] There is a need in the art for improved isoprenoid production in recombinant microorganisms, such as bacteria, e.g., E. coli.4. SUMMARY

[0006] In some aspects, the present disclosure relates to improved isoprene and / or isoprenoid production in recombinant microorganisms, such as bacteria, e.g., E. coli, by engineering cells to have improved flux and / or yield of the DXP pathway. Cells can be engineered to comprise nucleotide sequences comprising dxs polypeptides (EC 2.2.1.7); dxr polypeptides (EC 1.1.1.267); ispD polypeptides (EC 2.7.7.60); ispE polypeptides (EC 2.7.1.148); ispF polypeptides (EC 4.6.1.12); ispG polypeptides (EC 1.17.7.1 or EC 1.17.7.3); ispH polypeptides (EC 1.17.7.4); and idi polypeptides (EC 5.3.3.2). Some or all of the nucleotide sequences can be heterologous to the cells.

[0007] Surprisingly, polypeptides having amino acid sequences with relatively low sequence identity to native dxs, dxr, ispD, ispE, ispF, ispG, ispH, and / or idi polypeptides can replace native polypeptides and not only maintain, but increase, flux and / or yield of the DXP pathway. For some polypeptides, even 10-15% sequence identity can provide this result.

[0008] Examples of recombinant microorganisms with engineered DXP pathways are described in Section 6.2 and numbered embodiments 1 to 35, 177 to 455.

[0009] Some polypeptides of the DXP pathway comprise cofactors. For example, ispG polypeptides and ispH polypeptides native to E. coli and R. capsulatus comprise iron-sulfur clusters. The iron-sulfur clusters must be in their reduced form for these polypeptides to donate electrons to the substrates of their associated steps in the DXP pathway. Electron donation oxidizes the iron-sulfur clusters. Ongoing ispG and ispH activity requires recycling of the iron-sulfur clusters to their reduced form by interactions with redox polypeptides. It has been discovered that in some microorganisms, the native redox polypeptides may not provide desirable levels of recycling of the iron-sulfur clusters of a heterologous ispG and / or ispH, but that heterologous ispG and / or ispH activity can be increased by supplying one or more compatible heterologous redox polypeptides (e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin-NADP reductases (fpr)). Accordingly, the present disclosure provides recombinant microorganisms engineered to express heterologous redox polypeptides, e.g., in combination with heterologous ispG and / or ispH polypeptides.

[0010] Examples of recombinant microorganisms engineered to express heterologous redox polypeptides are described in Section 6.3 and numbered embodiments 407 to 435 and 447 to 455. In some embodiments, the heterologous redox polypeptides are derived from thesame genus or species as a heterologous ispG and / or ispH engineered into the microorganisms.

[0011] Flux and / or yield of the DXP pathway can further be improved by increasing flux into the DXP pathway. This can comprise disrupting genes containing coding sequences for polypeptides that catalyze reactions which convert precursors, such as glucose, or intermediates, such as gluconate or 2-keto-3-deoxy-gluconate (KDG), to products that are not useful in the DXP pathway. Examples of such polypeptides include gluconate kinases (gntK; EC 2.7.1.12), idonate kinases (idnK; EC 2.7.1.12), and other idonate pathway polypeptides.

[0012] Additionally or alternatively, this can comprise increasing expression or activity of polypeptides involved in conversion of glucose via intermediates to DXP. Examples of these latter polypeptides include gluconate dehydratases (EC 4.2.1.39 or EC 4.2.1.140), KDG kinases (EC 2.7.1.45), and KDG-phosphate aldolases (eda; EC 4.1.3.16).

[0013] Examples of recombinant microorganisms engineered to have increased flux into DXP pathways are described in Section 6.4 and numbered embodiments 36 to 67 and 450 to 454.

[0014] Isoprene and / or isoprenoid production can further be increased by engineering cells to gain or have increased activity of one or more isoprene and / or isoprenoid biosynthesis polypeptides. The isoprene and / or isoprenoid biosynthesis polypeptides can increase flux and / or yield of isoprene and / or isoprenoids such as limonene, beta-myrcene, nerol, geraniol, linalool, beta-ocimene,1 ,8-cineole, farnesene, and / or farnesol.

[0015] Examples of recombinant microorganisms engineered to have improved isoprene and / or isoprenoid production are described in Section 6.5 and numbered embodiments 68 to 176 and 455.

[0016] In other aspects, the present disclosure relates to the production of isoprene and / or isoprenoids, e.g., using recombinant microorganisms described herein. Methods for producing isoprene and / or isoprenoids are described in Section 6.8 and numbered embodiments 456 to 473.5. BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1 schematically depicts the DXP pathway, in context with pathways leading from glucose or xylose to DXP, and with pathways leading from DMAPP and IPP to isoprene and / or isoprenoids, with certain enzymes assigned numbers and certain reactants and products assigned letters for ease of reference and convenience. Abbreviations and / or assignments used: glucose (A); gluconate (B); KDG (C), 2-keto-3-deoxygluconate; KDGP (D), 2-keto-3-deoxy-6-phosphogluconate; GAP (E), glyceraldehyde-3-phosphate; pyruvate (F); ribulose- 5-phosphate (G); DXP (H), 1 -deoxyxylulose- 5-phosphate; MEP (J), 2-C- methylerythritol 4-phosphate; CDP-ME (K), 4-diphosphocytidyl-2-C-methylerythritol; CDP- MEP (L), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MEcPP (M), 2-C-methyl- D-erythritol 2,4-cyclodiphosphate; HMBPP (N), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; DMAPP (P), dimethylallyl pyrophosphate; IPP (Q), isopentenyl pyrophosphate; xylose (R); xylulose (S); xylulose- 5-phosphate (T); 1 -deoxyxylulose (DX) (U); CTP, cytidine triphosphate; CMP, cytidine monophosphate; 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 (7), 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2); Idi (8), isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2); PEC (9), a protein electron carrier (e.g., ferredoxin or flavodoxin), in reduced (red) and oxidized (ox) forms; Fpr (10), flavodoxin / ferredoxin-NADP reductase EC 1.19.1.1 or EC 1.18.1.2), in reduced (red) and oxidized (ox) forms; RibB, 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12); YajO, 1-deoxyxylulose-5-phosphate synthase (EC 1.1.-.-); and XylB, xylulose kinase (EC 2.7.1.17). The use of multi-headed arrows (e.g.,indicates multiple 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.

[0018] FIG. 2 shows a protein alignment of 14 ispG protein sequences, as described in Example 3. Residues conserved relative to E. coli ispG are shown in bold. FIG. 2 showsSEQ ID NOS: 95-97, 27-28, 98, 29, 99, 73, 100, 30-31 and 101-102, respectively, in order of appearance.

[0019] FIG. 3A shows the 3D structure of the E. coli K-12 ispG enzyme, generated by PyMOL The ten conserved residues described in Example 3 are shown in black. The location of the iron-sulfur center of the enzyme, which is part of the active site, is also indicated. The first and last amino acids of the protein are shown by numbered boxes (residues 1 and 372).

[0020] FIG. 3B shows the region of FIG. 3A demarcated by the dotted box.

[0021] FIG. 3C shows the surface structure of the E. coli K-12 ispG enzyme, generated by PyMOL. The ten conserved residues described in Example 3 are shown in black. The location of the iron-sulfur center of the enzyme, which is part of the active site, is also indicated. The first and last amino acids of the protein are shown by numbered boxes (residues 1 and 372). Residue C124 is not visible in this representation; its position is indicated by a dotted arrow.

[0022] FIG. 3D shows the region of FIG. 3A demarcated by the dotted box. To visualize residue C124, the image was rotated on its Y-axis.6. DETAILED DESCRIPTION6.1. Definitions

[0023] 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 constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0024] Corresponding amino acid residues: An amino acid residue of a query amino acid sequence “corresponds” to an amino acid residue of a reference amino acid sequence when, upon alignment of the query and reference sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the query and the reference sequence for optimal alignment), the amino acid residue in the query sequence is aligned with the amino acid residue in the reference sequence. An alignment of a query amino acid sequence and a reference amino acid sequence can be generated using the computer program ClustalW (version 1.83, default parameters), which allows alignments of polypeptide sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more reference sequences and 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 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.

[0025] From: A coding sequence may be referred to herein as being “from” an organism if the coding sequence and a polypeptide encoded thereby have a high degree of sequence identity and functional similarity to the coding sequence and encoded polypeptide as isolated from the organism. A coding sequence “from” an organism does not have to directly obtained from the organism. A coding sequence “from” an organism also does not have to have 100% sequence identity at the nucleotide level, nor does the encoded polypeptide have to have 100% sequence identity at the polypeptide level, to coding sequence and encoded polypeptide as isolated from the organism. A coding sequence “from” an organism encompasses variants, truncated versions, etc. Typically, a coding sequence “from” an organism encodes a polypeptide having at least 90% sequence identity to that organism's native polypeptide. In some embodiments, the sequence identity to the organism’s native polypeptide is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0026] 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, a heterologous 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).

[0027] 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.

[0028] 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 protein-coding sequences, operably linked sequences may be in the same reading frame.

[0029] 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”).

[0030] Parental Microorganism: The terms “parental cell” or “parental microorganism” are used interchangeably to refer to unicellular organisms which can be engineered to increase flux and / or yield of the DXP pathway. The adjective “parental” indicates that a recombinant cell or recombinant microorganism can be engineered by the introduction into a parental cell or parental microorganism of a heterologous nucleic acid or plurality of heterologous nucleic acids, such as nucleic acid(s) each comprising a coding sequence or plurality of coding sequences each encoding a heterologous polypeptide, and / or by insertion, deletion, substitution, or other modification of coding sequences or regulatory sequences in the genome of the parental microorganism.

[0031] A parental microorganism can be a microorganism found in nature or a microorganism that is non-naturally occurring. In other words, a parental microorganism can comprise one or more genetic modifications (e.g., insertion, deletion, or modification of one or more coding sequences and / or regulatory sequences) relative to a strain thereof found in nature. In relationship to a recombinant microorganism of the disclosure generated through a series of engineering steps, the terms “parental cell” and “parental microorganism” can refer to an ancestral cell or organism incorporating any of the engineering steps, as well as a cell or microorganism without any of the engineering steps. Sometimes, for ease of reference and comparison, the terms “parental cell” and “parental microorganism” refer to a cell or microorganism which, if having genetic modifications, the genetic modification(s) do not relate to the microorganism engineering described in Section 6.2. Further, the term “parental cell” and “parental microorganism” is intended for use as a reference cell or microorganism and not that the cell or organism was used as a starting point for engineering a microorganism of the disclosure.

[0032] Polypeptide, Peptide, Protein: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide herein may be identified by a name or by a percentage of sequence identity to a reference amino acid sequence. When a polypeptide is identified by a name indicative of an activity performed or enabled by the polypeptide, the name refers to any polypeptide capable of performing or enabling the activity.

[0033] 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 (i.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.

[0034] 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.

[0035] Redox Polypeptide: A “redox polypeptide” is used herein to refer to any polypeptide capable of directly (via a single step) or indirectly (via more than one step) transferring electrons from NADPH to other polypeptides, e.g., to the iron-sulfur clusters of iron-sulfur cluster polypeptides. In some embodiments, a redox polypeptide is capable of transferring electrons to ispG and / or ispH. Examples of redox polypeptides include ferredoxins, flavodoxins, and flavodoxin / ferredoxin-NADP reductases (fpr; EC: 1.18.1.2).

[0036] Regulatory Sequence: A “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.

[0037] 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 sequences and 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.

[0038] 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 introducingnucleic 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.

[0039] 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 MG1655. Another “wild-type” E. coli strain is E. coli K12 substrain BW25113.6.2. Recombinant Microorganisms With Engineered DXP Pathways

[0040] In some aspects, the present disclosure relates to recombinant microorganisms comprising an engineered DXP pathway, e.g., a DXP pathway comprising at least one heterologous coding sequence and / or regulatory sequence and / or at least one insertion, deletion, or substitution in at least one endogenous coding sequence and / or regulatory sequence relative to a wild-type parental microorganism. For example, recombinant microorganisms can comprise heterologous coding sequences for one, any two, any three, any four, any five, any six, any seven, or all eight of polypeptides (1)-(8) shown in FIG. 1. For another example, recombinant microorganisms can comprise at least one insertion, deletion, or substitution in at least one endogenous coding sequence for one, any two, any three, any four, any five, any six, any seven, or all eight of polypeptides (1)-(8). For a third example, recombinant microorganisms can comprise heterologous coding sequences for one, any two, any three, any four, any five, any six, or any seven of polypeptides (1)-(8) and at least one insertion, deletion, or substitution in at least one endogenous coding sequence for one, any two, any three, any four, any five, any six, or any seven of polypeptides (1)-(8).

[0041] For example, coding sequences encoding polypeptides (1)-(8), which may be termed the first through the eighth coding sequences or coding sequences, can be from at least two different organisms. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can from at least three different organisms. In someembodiments, the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from at least four different organisms.

[0042] In some embodiments, the third nucleotide sequence and the fifth nucleotide sequence, optionally together with the fourth nucleotide sequence, are derived from genes from the same organism, e.g., Rhodobacter capsulatus.

[0043] In some embodiments, the sixth nucleotide sequence and the seventh nucleotide sequence are derived from genes from the same organism, e.g., Shewanella oneidensis.

[0044] In some embodiments, the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are derived from genes from one organism, e.g., R. capsulatus.

[0045] The different organisms can include wild type parental microorganisms of the recombinant microorganisms. For example, when the recombinant microorganisms are E. coli, at least one of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli. In some embodiments, at least two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli. In some embodiments, at least three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli. In some embodiments, at least four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli.

[0046] In some embodiments, no more than two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli. In some embodiments, no more than three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli. In some embodiments, no more than four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences can be from E. coli.

[0047] Additionally or alternatively to the coding sequences, recombinant microorganisms can comprise heterologous regulatory sequences operably linked to heterologous and / or endogenous coding sequences for one, any two, any three, any four, any five, any six, any seven, or all eight of enzymes (1)-(8).

[0048] FIG. 1 schematically depicts the 1-deoxyxylulose-5-phosphate (DXP) pathway, in context with pathways leading from glucose (A) or xylose (R) to DXP (H), and with pathways leading from dimethylallyl pyrophosphate (DMAPP) (P) and isopentenyl pyrophosphate (IPP) (Q) to isoprene and / or isoprenoids.

[0049] The eight DXP pathway enzymes depicted in FIG. 1 include the following:

[0050] Dxs (1), 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7), catalyzes an acyloin condensation reaction between the position 2 and 3 carbons of GAP (E) and pyruvate (F) to DXP (H). Exemplary Dxs polypeptides are described in Section 6.2.1.

[0051] Dxr (2), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267) catalyzes the isomerization and reduction of DXP (H) to MEP (J). This activity of Dxr is NADPH- dependent. Exemplary Dxr polypeptides are described in Section 6.2.2.

[0052] IspD (3), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60) catalyzes the formation of CDP-ME (K) from MEP (J) and CTP. Exemplary IspD polypeptides are described in Section 6.2.3.

[0053] IspE (4), 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148) catalyzes the phosphorylation of a hydroxy group of CDP-ME (K) to yield CDP-MEP (L). Exemplary IspE polypeptides are described in Section 6.2.4.

[0054] IspF (5), 2-C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (EC 4.6.1.12), catalyzes the conversion of CDP-MEP (L) to MEcPP (M) with removal of CMP. Exemplary IspF polypeptides are described in Section 6.2.5.

[0055] IspG (6), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1), catalyzes the conversion of MEcPP (M) to HMBPP (N), with reduction of flavodoxin. Exemplary IspG polypeptides are described in Section 6.2.6.

[0056] IspH (7), 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2), converts HMBPP (N) to a mixture of DMAPP (P) and IPP (Q), with reduction of ferredoxin. Exemplary IspH polypeptides are described in Section 6.2.7.

[0057] Idi (8), isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2), catalyzes the interconversion of DMAPP (P) and IPP (Q). Exemplary Idi polypeptides are described in Section 6.2.8.

[0058] As can be seen in FIG. 1 , various enzymatic activities influence flux into the DXP pathway from glucose or xylose. Conversely, various other enzymatic activities downstream of the DXP pathway influence the production of isoprene and / or isoprenoids (e.g., limonene, beta-myrcene, nerol, geraniol, linalool, beta-ocimene,1,8-cineole, farnesene, and / orfarnesol) from DMAPP (P) and IPP (Q). Accordingly, production of isoprene and / or isoprenoids of interest can be increased by further engineering recombinant microorganisms by increasing flux into the DXP pathway (as described in Section 6.4) and / or increasing activity of one or more polypeptides involved in the conversion of DMAPP (P) and IPP (Q) to isoprene and / or isoprenoids of interest (as described in Section 6.5).

[0059] In some embodiments, in addition to being engineered to include heterologous DXP pathway components, recombinant microorganisms of the disclosure can have any one, any two, any three, any four, or all five of the following characteristics A-E:A. Regulated expression levels of the native isc operon and / or approximately native levels of production of iron-sulfur cluster polypeptides encoded by isc operon genes. In E. coli, the isc operon, which contains genes encoding Fe-S cluster assembly proteins, is regulated by IscR, a Fe-S cluster-containing transcription factor that represses the isc operon. Deletion / mutation of iscR has been used as a mechanism of constitutive / unregulated expression of the isc operon. Accordingly, in some embodiments, a recombinant organism of the disclosure comprises an intact iscR gene and / or expresses iscR at approximately native levels.B. Expression of ryhB. RyhB is a small RNA (94 nt long in E. coli K12 substr. MG1655) that down reg ulates expression of iron-containing proteins. Deletion / mutation of rhyB has been used as a mechanism to increase expression of ironsulfur cluster polypeptides. Accordingly, in some embodiments, a recombinant organism of the disclosure comprises an intact rhyB gene and / or expresses rhyB at approximately native levels.C. Expression of one or more (or all) components of the pyruvate dehydrogenase complex (PDC). In E. coli, the PDC, which catalyzed acetyl CoA production from pyruvate, comprises aceE (pyruvate dehydrogenase subunit E1), aceF (dihydrolipoyl transacetylase, E2), and IpdA (dihydrolipoyl dehydrogenase, E3). Accordingly, in some embodiments, a recombinant organism of the disclosure comprises intact PDC genes and / or expresses PDC components genes at approximately native levels.D. Lack of heterologous mevalonate (MVA) pathway coding sequences e.g., acetoacetyl-CoA thiolase, 3-hydroxy-3- methyl-glutaryl-CoA (HMG-CoA) synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase,diphosphomevalonate decarboxylase, and phosphomevalonate decarboxylase. The MVA pathway leads to the production of IPP from acetyl-CoA. Accordingly, in some embodiments, a recombinant organism of the disclosure comprises wild-type MVA pathway genes and / or expresses MVA pathway genes at approximately native levels. Some parental microorganisms may lack coding sequences encoding one or more of the mevalonate pathway polypeptides, in which case native expression of the polypeptide(s) is zero.E. Lack of a heterologous ispS (EC 4.2.3.27) coding sequence. IspS enzymes catalyze the conversion of DMAPP to isoprene. In some embodiments, a recombinant microorganism of the disclosure lack a nucleotide sequences encoding a heterologous ispS enzyme.

[0060] In the foregoing aspects, reference to “native” expression levels of a gene or protein refers to expression levels of a gene or protein in the absence of genetic engineering or modification / mutation. When determining whether a gene or protein in an engineered microorganisms has “native” or “approximately native” expression levels as a reference (e.g., wild-type or parental) microorganism, the analysis is carried out under comparable circumstances (e.g., temperature, medium composition, culture conditions, etc.). In some embodiments, the term “approximately native” refers to expression levels that are within 25% of the expression levels of the gene or protein or protein in the reference microorganism and / or confer sufficient expression to perform the same activity or function as in the reference microorganism.

[0061] In some embodiments, a recombinant microorganism of the disclosure is characterized by A, and optionally one or more of B, C, D and E.

[0062] In some embodiments, a recombinant microorganism of the disclosure is characterized by B, and optionally one or more of A, C, D and E.

[0063] In some embodiments, a recombinant microorganism of the disclosure is characterized by C, and optionally one or more of A, B, D and E.

[0064] In some embodiments, a recombinant microorganism of the disclosure is characterized by D, and optionally one or more of A, B, C and E.

[0065] In some embodiments, a recombinant microorganism of the disclosure is characterized by E, and optionally one or more of A, B, C and D.

[0066] In some embodiments, a recombinant microorganism of the disclosure is characterized by A and B, and optionally one or more of C, D and E.

[0067] In some embodiments, a recombinant microorganism of the disclosure is characterized by D and E, and optionally one or more of A, B and C.6.2.1. Dxs Polypeptides

[0068] Recombinant microorganisms of the present disclosure comprise first nucleotide sequences encoding dxs polypeptides (1).

[0069] In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO: 19.

[0070] Surprisingly, the complementation studies of Example 1 , in which Adxs E. coli were rescued by expression of heterologous dxs, revealed that high (>90%) sequence identity to SEQ ID NO:19, the dxs polypeptide (1) of E. coli, was not required for complementation of Adxs E. coli. In fact, as shown in Example 2, heterologous dxs polypeptides (1) with low sequence identity to SEQ ID NO: 19 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0071] Dxs polypeptides can be obtained or derived from any source organism. In some embodiments, the source organism (1) produces at least twice the amount of pigments as E. coli on an average pigment: biomass weight ratio; (2) is photosynthetic; (3) is distantly related to E. coli', (4) is characterized by any combination of two or all three of (1), (2) and (3) (e.g., (1) + (2), (1) + (3), (2) + (3), or (1) + (2) + (3)). Though not to be bound by theory, a dxs polypeptide from such a source organism may be less susceptible than E. coli dxs to regulation by E. coli and / or has greater DXP pathway activity than that of E. coli.

[0072] In various embodiments, a distantly related source organism is not a member of at least one of (a) order Enterobacterales (b) class Gammaproteobacteria’, (c) phylum Pseudomonadota-, and (d) domain Bacteria.

[0073] In some embodiments, dxs polypeptides (1) comprise amino acid sequences having less than 65% sequence identity to the amino acid sequence of SEQ ID NO: 19. Dxs polypeptides (1) of these embodiments can comprise amino acid sequences having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:19. Dxs polypeptides (1) of these embodiments can comprise amino acid sequences having at least 40% sequence identity to the amino acid sequence of SEQ ID NO: 19. Dxs polypeptides (1) of these embodiments can comprise amino acid sequences having at least 45% sequence identity to the amino acid sequence of SEQ ID NO: 19.

[0074] Exemplary dxs polypeptides (1) able to complement dxs E. coli and lead to increased isoprene and / or isoprenoid production include those from Rhodobacter capsulatus (dxs1 , UniProt Accession No. D5AP89, SEQ ID NO:17; dxs2, UniProt Accession No. D5ASU5, SEQ ID NO:18).

[0075] In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:17. In some embodiments, dxs polypeptides (1) comprise the amino acid sequence of SEQ ID NO: 17.

[0076] In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:18. In some embodiments, dxs polypeptides (1) comprise the amino acid sequence of SEQ ID NO: 18.

[0077] In some embodiments, recombinant microorganisms are E. coli comprising first nucleotide sequences encoding dxs2 polypeptides (1) comprising the amino acid sequence of SEQ ID NO:18.

[0078] Another exemplary dxs polypeptide (1) is that from Pseudomonas fluorescens (UniProt Accession No. A0A379IJU9, SEQ ID NO:47).

[0079] In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:47. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:47. In some embodiments, dxs polypeptides (1) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:47. In some embodiments, dxs polypeptides (1) comprise the amino acid sequence of SEQ ID NO:47.6.2.2. Dxr Polypeptides

[0080] Recombinant microorganisms of the present disclosure comprise second nucleotide sequences encoding dxr polypeptides (2).

[0081] In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:20. In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:20. In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:20. In some embodiments, dxr polypeptides (2) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NQ:20.

[0082] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NQ:20, the dxr polypeptide (2) of E. coli, was not required for complementation of dxrE. coli.. The isoprenoid production studies of Example 2 showed that heterologous dxr polypeptides (2) may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0083] In some embodiments, dxr polypeptides (2) comprise amino acid sequences having less than 60% sequence identity to the amino acid sequence of SEQ ID NQ:20. Dxrpolypeptides (2) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:20. Dxr polypeptides (2) of these embodiments can comprise amino acid sequences having at least 35% sequence identity to the amino acid sequence of seq id no:20. Dxr polypeptides (2) of these embodiments can comprise amino acid sequences having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0084] Exemplary dxr polypeptides (2) able to complement dxrE. coli and lead to increased isoprene and / or isoprenoid production include those from Rhodobacter capsulatus (dxr, UniProt Accession No. D5ATT5, SEQ ID NO:21).

[0085] In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:21. In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:21. In some embodiments, dxr polypeptides (2) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:21. In some embodiments, dxr polypeptides (2) comprise the amino acid sequence of SEQ ID NO:21.

[0086] In some embodiments, recombinant microorganisms are E. coli comprising first nucleotide sequences encoding dxr polypeptides (2) comprising the amino acid sequence of SEQ ID NO:21.

[0087] Another exemplary dxr polypeptide (2) is that from Pseudomonas fluorescens (UniProt Accession No. A0A0P9ALW5, SEQ ID NO:48).

[0088] In some embodiments, dxr polypeptides (2) comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:48. In some embodiments, dxr polypeptides (2) comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:48. In some embodiments, dxr polypeptides (2) comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:48. In some embodiments, dxr polypeptides (2) comprise the amino acid sequence of SEQ ID NO:48.6.2.3. IspD Polypeptides

[0089] Recombinant microorganisms of the present disclosure comprise third nucleotide sequences encoding ispD polypeptides (3).

[0090] In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:22. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:22. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:22. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:22.

[0091] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NO:22, the ispD polypeptide (3) of E. coli, was not required for complementation of AispD E. coli. The isoprenoid production studies of Example 2 showed that heterologous ispD polypeptides (3) with low sequence identity to SEQ ID NO:22 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0092] In some embodiments, ispD polypeptides (3) can comprise amino acid sequences having less than 50% sequence identity to the amino acid sequence of SEQ ID NO:22. IspD polypeptides (3) of these embodiments can comprise amino acid sequences having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:22. IspD polypeptides (3) of these embodiments can comprise amino acid sequences having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:22. IspD polypeptides (3) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:22.

[0093] Exemplary ispD polypeptides (3) able to complement AispD in E. coli and lead to increased isoprene and / or isoprenoid production include those from Rhodobacter capsulatus (UniProt Accession No. Q08113, SEQ ID NO:23; amino acids 1-222 of UniProt Accession No. Q08113, SEQ ID NO:65).

[0094] In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23. Insome embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, ispD polypeptides (3) comprise the amino acid sequence of SEQ ID NO:23.

[0095] In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:65. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:65. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:65. In some embodiments, ispD polypeptides (3) comprise the amino acid sequence of SEQ ID NO:65.

[0096] In some embodiments, recombinant microorganisms are E. coli comprising third nucleotide sequences encoding ispD polypeptides (3) as domains of ispDF fusion polypeptides, the ispD polypeptide (3) domains comprising the amino acid sequence of SEQ ID NO:23.

[0097] Another exemplary ispD polypeptide (3) is that from Pseudomonas (GenBank Accession No. QQU70697.1 , SEQ ID NO:49).

[0098] In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, ispD polypeptides (3) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, ispD polypeptides (3) comprise the amino acid sequence of SEQ ID NO:49.6.2.4. IspE Polypeptides

[0099] Recombinant microorganisms of the present disclosure comprise fourth nucleotide sequences encoding ispE polypeptides (4).

[0100] In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24. In someembodiments, ispE polypeptides (4) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:24. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:24. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0101] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NO:24, the ispE polypeptide (4) of E. coli, was not required for complementation of AispE E. coli. The isoprenoid production studies of Example 2 showed that heterologous ispE polypeptides (4) with low sequence identity to SEQ ID NO:24 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0102] In some embodiments, ispE polypeptides (4) comprise amino acid sequences having less than 60% sequence identity to the amino acid sequence of SEQ ID NO:24. IspE polypeptides (4) of these embodiments can comprise amino acid sequences having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:24. IspE polypeptides (4) of these embodiments can comprise amino acid sequences having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:24. IspE polypeptides (4) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0103] Exemplary ispE polypeptides (4) able to complement AispE E. coli and lead to increased isoprene and / or isoprenoid production include those from Rhodobacter capsulatus (UniProt Accession No. D5AMY7, SEQ ID NO:25).

[0104] In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NO:25. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, ispE polypeptides (4) comprise the amino acid sequence of SEQ ID NO:25.

[0105] In some embodiments, recombinant microorganisms are E. coli comprising fourth nucleotide sequences encoding ispE polypeptides (4) comprising the amino acid sequence of SEQ ID NO:25.

[0106] Another exemplary ispE polypeptide (4) is that from Pseudomonas fluorescens (GenBank Accession No. QQU66123.1 , SEQ ID NO:50).

[0107] In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NQ:50. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:50. In some embodiments, ispE polypeptides (4) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:50. In some embodiments, ispE polypeptides (4) comprise the amino acid sequence of SEQ ID NQ:50.6.2.5. IspF Polypeptides

[0108] Recombinant microorganisms of the present disclosure comprise fifth nucleotide sequences encoding ispF polypeptides (5).

[0109] In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:26. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:26.

[0110] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NO:26, the ispF polypeptide (5) of E. coli, was not required for complementation of AispF E. coli. The isoprenoid production studies of Example 2 showed that heterologous ispF polypeptides (5) with low sequence identity to SEQ ID NO:26 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0111] In some embodiments, ispF polypeptides (5) comprise amino acid sequences having less than 75% sequence identity to the amino acid sequence of SEQ ID NO:26. IspF polypeptides (5) of these embodiments can comprise amino acid sequences having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:26. IspF polypeptides (5) of these embodiments can comprise amino acid sequences having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:26. IspF polypeptides (5) of these embodiments can comprise amino acid sequences having at least 45% sequence identity to the amino acid sequence of SEQ ID NO:26.

[0112] Exemplary ispF polypeptides (5) able to complement AispF E. coli and lead to increased isoprene and / or isoprenoid production include those from Rhodobacter capsulatus (UniProt Accession No. Q08113, SEQ ID NO:23; amino acids 223-379 of UniProt Accession No. Q08113, SEQ ID NO:66).

[0113] In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 90% identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 95% identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 97% identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having 100% identity to the amino acid sequence of SEQ ID NO:23.

[0114] In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 90% identity to the amino acid sequence of SEQ ID NO:66. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 95% identity to the amino acid sequence of SEQ ID NO:66. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 97% identity to the amino acid sequence of SEQ ID NO:66. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having 100% identity to the amino acid sequence of SEQ ID NO:66.

[0115] In some embodiments, third nucleotide sequences and fifth nucleotide sequences are operably linked so as to encode fusion proteins comprising ispD polypeptides (3) and ispF polypeptides (5).

[0116] Another exemplary ispF polypeptide (5) is that from Pseudomonas fluorescens (GenBank Accession No. QQU70693.1 , SEQ ID NO:51).

[0117] In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 90% identity to the amino acid sequence of SEQ ID NO:51. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 95% identity to the amino acid sequence of SEQ ID NO:51. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having at least 97% identity to the amino acid sequence of SEQ ID NO:51. In some embodiments, ispF polypeptides (5) comprise amino acid sequences having 100% identity to the amino acid sequence of SEQ ID NO:51 .6.2.6. IspG Polypeptides

[0118] Recombinant microorganisms of the present disclosure comprise sixth nucleotide sequences encoding ispG polypeptides (6).

[0119] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0120] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NO:27, the ispG polypeptides (6) of E. coli, was not required for complementation of AispG E. coli. The isoprenoid production studies of Example 2 showed that heterologous ispG polypeptides (6) with low sequence identity to SEQ ID NO:27 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest

[0121] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having less than 100% sequence identity to the amino acid sequence of SEQ ID NO:27. IspG polypeptides (6) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:27. IspG polypeptides (6) of these embodiments can comprise amino acid sequences having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:27. IspG polypeptides (6) of theseembodiments can comprise amino acid sequences having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0122] Exemplary ispG polypeptides (6) able to complement AispG E. coli and lead to increased isoprene and / or isoprenoid production include those from Pantoea ananatis (UniProt Accession No. A0A0H3KYT9, SEQ ID NO:28); Proteus mirabilis (UniProt Accession No. B4EZT3, SEQ ID NO:29); Serratia marcescens (UniProt Accession No. A0A0P0QHY9 with K365N substitution, SEQ ID NQ:30); and Shewanella oneidensis (UniProt Accession No. Q8EC32, SEQ ID NO:31).

[0123] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NO:28. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:28. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:28. In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NO:28.

[0124] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:29. In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NO:29.

[0125] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:30. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:30. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:30. In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NQ:30.

[0126] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:31. In someembodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:31 . In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:31 . In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NO:31.

[0127] Another exemplary ispG polypeptide (6) is that from Synechococcus elongatus (SEQ ID NO:52).

[0128] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:52. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:52. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:52. In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NO:52.

[0129] Another exemplary ispG polypeptide (6) able to lead to increased isoprene and / or isoprenoid production is that from R. capsulatus (UniProt Accession No. D5AT87, SEQ ID NO:73).

[0130] In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NO:73. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:73. In some embodiments, ispG polypeptides (6) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:73. In some embodiments, ispG polypeptides (6) comprise the amino acid sequence of SEQ ID NO:73.

[0131] IspG polypeptides (6) that are heterologous to E. coli can comprise one, any combination of two or more, or all of: (i) an arginine at the position corresponding to R14 of SEQ ID NO:27; (ii) an aspartic acid at the position corresponding D24 of SEQ ID NO:27; (iii) a glycine at the position corresponding to G25 of SEQ ID NO:27; (iv) a cysteine at the position corresponding to C124 of SEQ ID NO:27; (v) an asparagine at the position corresponding to N129 of SEQ ID NO:27; (vi) a glutamine at the position corresponding to Q175 of SEQ ID NO:27; (vii) a serine at the position corresponding to S191 of SEQ IDNO:27; (viii) an alanine at the position corresponding to A213 of SEQ ID NO:27; (ix) an arginine at the position corresponding to R364 of SEQ ID NO:27; and (x) an isoleucine at the position corresponding to I365 of SEQ ID NO:27.

[0132] In some embodiments, IspG polypeptides (6) that are heterologous to E. coli comprise an aspartic acid at the position corresponding D24 of SEQ ID NO:27 and a glycine at the position corresponding to G25 of SEQ ID NO:27, and optionally one, any combination of one or more, or all of: an arginine at the position corresponding to R14 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; a serine at the position corresponding to S191 of SEQ ID NO:27; an alanine at the position corresponding to A213 of SEQ ID NO:27; an arginine at the position corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to I365 of SEQ ID NO:27.

[0133] In other embodiments, IspG polypeptides (6) that are heterologous to E. coli comprise an arginine at the position corresponding to R364 of SEQ ID NO:27 and an isoleucine at the position corresponding to I365 of SEQ ID NO:27, an optionally one, any combination of two or more, or all of an arginine at the position corresponding to R14 of SEQ ID NO:27; an aspartic acid at the position corresponding D24 of SEQ ID NO:27; a glycine at the position corresponding to G25 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; a serine at the position corresponding to S191 of SEQ ID NO:27; an alanine at the position corresponding to A213 of SEQ ID NO:27; an arginine at the position corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to I365 of SEQ ID NO:27.

[0134] In further embodiments, IspG polypeptides (6) that are heterologous to E. coli comprise an arginine at the position corresponding to R14 of SEQ ID NO:27; an aspartic acid at the position corresponding D24 of SEQ ID NO:27; a glycine at the position corresponding to G25 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; an arginine at theposition corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to 1365 of SEQ ID NO:27, and optionally one or both of a serine at the position corresponding to S191 of SEQ ID NO:27 and an alanine at the position corresponding to A213 of SEQ ID NO:27.6.2.7. IspH Polypeptides

[0135] Recombinant microorganisms of the present disclosure comprise seventh nucleotide sequences encoding ispH polypeptides (7).

[0136] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0137] The complementation studies of Example 1 indicated that high (>90%) sequence identity to SEQ ID NO:32, the ispH polypeptides (7) of E. coli, was not required for complementation of AispH E. coli. The isoprenoid production studies of Example 2 showed that heterologous ispH polypeptides (7) with low sequence identity to SEQ ID NO:32 may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0138] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having less than 100% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 10% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 15% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can compriseamino acid sequences having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 50% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:32. IspH polypeptides (7) of these embodiments can comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0139] Exemplary ispH polypeptides (7) able to complement AispH E. coli and lead to increased isoprene and / or isoprenoid production include those from Acinetobacter baylyi (UniProt Accession No. Q9RBJ0, SEQ ID NO:33); Burkholderia glumae BGR1 ispH1 , UniProt Accession No. C5AC36, SEQ ID NO:34); Caulobacter crescentus (UniProt Accession No. A0A0H3CCC9, SEQ ID NO:35); Pantoea ananatis (UniProt Accession No. D4GJM9 with D148E substitution, SEQ ID NO:36); Pseudomonas fluorescens (UniProt Accession No. C3KDX7, SEQ ID NO:37); Proteus mirabilis (UniProt Accession No. B4F2T8, SEQ ID NO:38); P. sitchensis (UniProt Accession No. C0PR44 with AM1-A44 truncation, SEQ ID NO:39); P. trichocarpa (UniProt Accession No. B3GEM6, with AC1 , A2M, and A67T mutations, SEQ ID NQ:40); Serratia marcescens (UniProt Accession No. A0A0P0QA59 with D149E and N188S substitutions, SEQ ID NO:41); Shewanella oneidensis (UniProt Accession No. Q8EBI7, SEQ ID NO:42); and Synechococcus sp. (UniProt Accession No. B1XPG7, SEQ ID NO:43).

[0140] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NO:33. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:33. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:33. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:33.

[0141] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:34. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:34. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:34. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:34.

[0142] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:35. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:35.

[0143] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:36. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:36. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:36. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:36.

[0144] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:37. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:37. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:37. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:37.

[0145] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:38. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:38. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:38. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:38.

[0146] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:39. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:39. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:39. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:39.

[0147] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:40. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:40. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:40. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NQ:40.

[0148] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:41. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41 . In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:41 . In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:41.

[0149] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:42. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequenceidentity to the amino acid sequence of SEQ ID NO:42. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:42.

[0150] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:43.

[0151] Other exemplary ispH polypeptides (7) include those from Nostocaceae (GenBank Accession No. RUR80075, SEQ ID NO:53); Synechococcus elongatus (GenBank Accession No. Q5N249.1 , SEQ ID NO:54); and Pseudomonas (GenBank Accession No. QQU66087.1 , SEQ ID NO:55).

[0152] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:53. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:53. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:53. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:53.

[0153] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:54. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:54. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:54. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:54.

[0154] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:55. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:55. In some embodiments,ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:55. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:55.

[0155] Another exemplary ispH polypeptide (7) able to lead to increased isoprene and / or isoprenoid production is that from R. capsulatus (UniProt Accession No. D5AT87, SEQ ID NO:74).

[0156] In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 90% sequence identity to SEQ ID NO:74. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:74. In some embodiments, ispH polypeptides (7) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:74. In some embodiments, ispH polypeptides (7) comprise the amino acid sequence of SEQ ID NO:74.

[0157] In some embodiments, ispH polypeptides (7) are not capable of directly converting HMBPP (N) to isoprene to an appreciable extent, e.g., the ispH polypeptides (7) have an activity level of HMBPP to isoprene conversion that is no more than 30% (e.g., no more than 20%, no more than 10% or no more than 5%) of their activity level of HMBPP conversion to DMAPP (P) and IPP (Q).6.2.8. Idi Polypeptides

[0158] Recombinant microorganisms of the present disclosure comprise eighth nucleotide sequences encoding idi polypeptides (8).

[0159] In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:44. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:44. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:44. In some embodiments, idi polypeptides (8) comprise amino acid sequences having 100% sequence identity to the amino acid sequence of SEQ ID NO:44.

[0160] The isoprenoid production studies of Example 2 showed that heterologous idi polypeptides (8) with low sequence identity to SEQ ID NO:44, the idi polypeptide (8) of E. coli, may lead to increased flux through the DXP pathway and a concomitant increase in production of isoprene and / or isoprenoids of interest.

[0161] In some embodiments, idi polypeptides (8) comprise amino acid sequences having less than 40% sequence identity to the amino acid sequence of SEQ ID NO:44. Idi polypeptides (8) of these embodiments can comprise amino acid sequences having at least 15% sequence identity to the amino acid sequence of SEQ ID NO:44. Idi polypeptides (8) of these embodiments can comprise amino acid sequences having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:44. Idi polypeptides (8) of these embodiments can comprise amino acid sequences having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:44. Idi polypeptides (8) of these embodiments can comprise amino acid sequences having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:44.

[0162] Exemplary idi polypeptides (8) expected to lead to increased isoprene and / or isoprenoid production include those from R. capsulatus (idi1, UniProt Accession No. D5AKF1 , SEQ ID NO:45; idi2, UniProt Accession No. P26173, SEQ ID NO:46).

[0163] In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:45. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:45. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:45. In some embodiments, idi polypeptides (8) comprise the amino acid sequence of SEQ ID NO:45.

[0164] In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:46. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:46. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:46. In some embodiments, idi polypeptides (8) comprise the amino acid sequence of SEQ ID NO:46.

[0165] In some embodiments, recombinant microorganisms are E. coli cell and eighth nucleotide sequences encode idi1 polypeptides (8) comprising the amino acid sequence of SEQ ID NO:45.

[0166] Another exemplary idi polypeptide (8) is that from Pseudomonas (GenBank Accession No. WQ21040.1 , SEQ ID NO:56).

[0167] In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:56. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:56. In some embodiments, idi polypeptides (8) comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:56. In some embodiments, idi polypeptides (8) comprise the amino acid sequence of SEQ ID NO:56.6.2.9. Recombinant Microorganisms Comprising Combinations of Polypeptides

[0168] Recombinant microorganisms, such as recombinant E. coli, can comprise various combinations of nucleotide sequences encoding polypeptides (1 )-(8).

[0169] In some embodiments, the first nucleotide sequence encodes a dxs2 polypeptide (1) comprising the amino acid sequence of SEQ ID NO:18; and the second nucleotide sequence encodes a dxr polypeptide (2) comprising the amino acid sequence of SEQ ID NO:21. The first and second nucleotide sequences can be included in an expression cassette comprising, in order from 5’ to 3’, 5’ untranscribed and / or untranslated regulatory sequences (e.g., a promoter), the first nucleotide sequence, and the second nucleotide sequence. The promoter can be a synthetic T5 gluconate-inducible promoter. The promoter, the first nucleotide sequence, and the second nucleotide sequence can be integrated in the cell’s chromosome downstream of the gluconate repressor gene gntR.

[0170] In some embodiments, the eighth nucleotide sequence encodes an idi1 polypeptide (8) comprising the amino acid sequence of SEQ ID NO:45; the third and fifth nucleotide sequences encode an ispDF fusion polypeptide (3 + 5) comprising the amino acid sequence of SEQ ID NO:23; and the fourth nucleotide sequence encodes an ispE polypeptide (4) comprising the amino acid sequence of SEQ ID NO:25. The third, fourth, fifth, and eighth nucleotide sequence can be in an expression cassette comprising in order a promoter, theeighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence. The promoter can be a T7 A3 constitutive promoter. The promoter, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence can be integrated in the cell’s chromosome at the insHIO locus.

[0171] In some embodiments, the sixth nucleotide sequence encodes an ispG polypeptide (6) comprising the amino acid sequence of SEQ ID NO:31; and the seventh nucleotide sequence encodes an ispH polypeptide (7) comprising the amino acid sequence of SEQ ID NO:42. The sixth and seventh nucleotide sequences can be in an expression cassette comprising in order a promoter, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence. The promoter can be a T7 A3 constitutive promoter. The promoter, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence can be integrated in the cell’s chromosome at the insHIO locus.

[0172] The combinations of nucleotide sequences and expression cassettes described in this section can further comprise regulatory components, including, but not limited to, antitermination sites, gt10 sequences, ribosome binding sites / Shine-Dalgarno sequences, or spacer sequences, among others.

[0173] The particular combinations of nucleotide sequences and expression cassettes described in this section are exemplary. Other combinations, expression cassettes, promoters, and sites of integration are contemplated.6.3. Increasing IspG and / or IspH Activity by Expression of Redox Polypeptides

[0174] Both ispG and ispH (6)-(7) contain [4Fe-4S] iron-sulfur clusters which must be in reduced form for the polypeptides to transfer electrons to the substrates MEcPP (M) and HMBPP (N), respectively. After transferring electrons, the polypeptides are subsequently rereduced by interactions with redox polypeptides, such as flavodoxins (fid) or ferredoxins (fdx), in reduced form. The electron transfer oxidizes the redox polypeptides, which have to be re-reduced in order to be capable of transferring electrons to other polypeptides. In the case of flavodoxins and ferredoxins, flavodoxin / ferredoxin-NADP reductases (fpr;EC:1.18.1.2) re-reduce the redox polypeptides from oxidized form to reduced form.

[0175] Microorganisms, such as E. coli, natively express one or more fid and / or fdx polypeptides along with fpr polypeptides. In principle, recombinant microorganisms expressing only native redox polypeptides may provide electrons to heterologous ispG and / or ispH polypeptides. However, not all heterologous ispG and / or ispH polypeptides provide desirable levels of activity in all such recombinant microorganisms. Though not to be bound by theory, low activity of at least some heterologous ispG and / or ispH polypeptides in at least some recombinant microorganisms may result from interactions between the heterologous polypeptides and native redox polypeptides that are weaker than those of the native redox polypeptides with native ispG and / or ispH.

[0176] The activity of heterologous ispG and / or heterologous polypeptides can be increased by engineering recombinant microorganisms to express one or more compatible heterologous redox polypeptides (e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin-NADP reductases (fprs)) to improve iron-sulfur recycling in the heterologous ispG and / or ispH polypeptides in the microorganisms.

[0177] In some embodiments, the redox polypeptides comprise flavodoxins. In some embodiments, the redox polypeptides comprise ferrodoxins. In some embodiments, the redox polypeptides comprise fpr.

[0178] In some embodiments, the redox polypeptides do not comprise pyruvate:flavodoxin oxidoreductases (PFORs). PFORs are polypeptides capable of transferring electrons from pyruvate to flavodoxin (EC:1.2.7.-). In some embodiments, the redox polypeptides do not comprise a PFOR capable of transferring electrons from NADP to flavodoxin or ferredoxin at a level that is greater than 30% (e.g., greater than 20%, greater than 10% or greater than 5%) of their activity of transferring in transferring electrons from pyruvate to ferrodoxin. Enzymatic assays for ferredoxins, flavodoxins and flavodoxin / ferredoxin--NADP reductases based on cytochrome c reduction by NADPH can be performed as described in, e.g., McIver et al., 1998, Eur. J. Biochem. 257:577-585). Enzymatic assays for PFORs can be performed as described in, e.g., Nakayama et al., 2013, Genes Genet. Syst. 88:175-188.

[0179] A heterologous redox polypeptide can be considered compatible with a heterologous ispG polypeptide and / or a heterologous ispH polypeptide if increases ispG and / or ispH activity as compared to the corresponding redox polypeptide native to a recombinant microorganism. For example, in the case of a flavodoxin or ferredoxin, a heterologousflavodoxin or ferrodoxin can be considered compatible with the heterologous ispG polypeptide and / or ispH polypeptide if it reduces the oxidized form of the ispG polypeptide and / or the ispH polypeptide to a greater degree than a native flavodoxin or ferrodoxin (e.g., where the recombinant microorganism is E. coli, wild-type E. coli flavodoxin or ferrodoxin). Alternatively or additionally, a heterologous redox polypeptide can be considered compatible with a heterologous ispG polypeptide and / or a heterologous ispH polypeptide if flux through the DXP pathway in a recombinant microorganism expressing the heterologous ispG and / or ispH polypeptide and the heterologous redox polypeptide is at least 50% greater (e.g., at least 2-fold greater, at least 3-fold greater, or at least 4-fold greater) than the flux in a recombinant microorganism expressing the heterologous ispG and / or ispH polypeptide but only native redox polypeptides. Flux through the DXP pathway can be determined according to Example 5.

[0180] In some embodiments, (a) heterologous redox polypeptides and (b) heterologous ispG and / or ispH polypeptides are derived from the same genus. In some embodiments, (a) heterologous redox polypeptides and (b) heterologous ispG and / or ispH polypeptides are derived from the same species of microorganism, whether the same strain or a different strain of the microorganism.

[0181] In some aspects, the present disclosure relates to recombinant microorganisms comprising engineered DXP pathways as described in Section 6.2, wherein sixth nucleotide sequences and / or seventh nucleotide sequences encode heterologous ispG and / or ispH polypeptides, respectively, and further comprising a nucleotide sequence (e.g., a ninth nucleotide sequence encoding a compatible heterologous redox polypeptide. In some embodiment, the recombinant microorganisms further comprise more than one additional nucleotide sequence (e.g., a ninth nucleotide sequence, a tenth nucleotide sequence, and optionally an eleventh nucleotide sequence) encoding compatible heterologous redox polypoeptides. In some embodiments, the one or more additional nucleotide sequences encode a flavodoxin, a ferrodoxin, a flavodoxin / ferredoxin-NADP reductase, or a combination of two or more of the foregoing (e.g., (a) a flavodoxin and a ferrodoxin or (b) a flavodoxin / ferredoxin-NADP reductases and either a flavodoxin, a ferrodoxin, or both).

[0182] In some embodiments, a recombinant microorganism comprises ninth (and optionally tenth and further optionally eleventh) nucleotide sequences encoding redox polypeptidesfrom an organism in the same genus as the organism from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.

[0183] In some embodiments, a recombinant microorganism comprises ninth (and optionally tenth and further optionally eleventh) nucleotide sequences encoding redox polypeptides from an organism in the same species as the organism from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.

[0184] In some embodiments, a recombinant microorganism comprises ninth (and optionally tenth and further optionally eleventh) nucleotide sequences encoding redox polypeptides from the same strain from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.

[0185] In some aspects, a recombinant microorganism comprises a ninth nucleotide sequences encoding a flavodoxin. In some embodiments, the recombinant microorganism further comprises a tenth nucleotide sequence encoding a ferrodoxin and optionally an eleventh nucleotide sequence encoding a flavodoxin / ferredoxin-NADP reductase. In some embodiments, the recombinant microorganism further comprises a tenth nucleotide sequence encoding a flavodoxin / ferredoxin--NADP reductase and optionally an eleventh nucleotide sequence encoding ferrodoxin.

[0186] In other aspects, a recombinant microorganism comprises a ninth nucleotide sequences encoding a ferrodoxin. In some embodiments, the recombinant microorganism further comprises a tenth nucleotide sequence encoding a flavodoxin and optionally an eleventh nucleotide sequence encoding a flavodoxin / ferredoxin-NADP reductase. In some embodiments, the recombinant microorganism further comprises a tenth nucleotide sequence encoding a flavodoxin / ferredoxin-NADP reductase and optionally an eleventh nucleotide sequence encoding flavodoxin.

[0187] In some embodiments, the heterologous flavodoxins and / or ferrodoxins cannot complement native fldA genes of the recombinant microorganisms. Though not to be bound by theory, redox polypeptides (e.g., flavodoxins and ferredoxins) encoded by the ninth nucleotide sequences of these embodiments may have minimal redox interactions with native polypeptides.

[0188] In other embodiments, the heterologous flavodoxins and / or ferrodoxins can complement native fldA genes of the recombinant microorganisms, e.g., where the heterologous ispG and / or ispH polypeptides are sufficiently dissimilar from native ispG and / or ispH polypeptides that native redox polypeptides have minimal interactions with the heterologous ispG and / or ispH polypeptides.

[0189] In either or both scenarios, negative impacts on cell growth or maintenance arising from increased or decreased supply of redox partners to native polypeptides may be reduced.

[0190] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, the ninth nucleotide and the optional tenth nucleotide sequence and / or eleventh nucleotide sequence are derived from genes from organisms in the same genus.

[0191] In some embodiments, the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, the ninth nucleotide and the optional tenth nucleotide sequence and / or eleventh nucleotide sequence are derived from genes from the same organism.

[0192] Though not to be bound by theory, in these embodiments, by being derived from the same genus and / or species, polypeptides (1)-(9) (and optionally (10) and / or (11) may be evolutionarily tuned for higher interaction than if polypeptides (1)-(9) (and optionally (10) and / or (11) were derived from different organisms.

[0193] In particular embodiments, sixth nucleotide sequences encode an ispG polypeptide derived from Rhodobacter capsulatus (e.g., polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73), seventh nucleotide sequences encode an ispH polypeptide also derived from R. capsulatus (e.g., polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74), and ninth nucleotide sequences encode a redox polypeptide also derived from R. capsulatus.

[0194] An exemplary ferredoxin derived from R. capsulatus is fdxA, UniProt Accession No. D5AP15 (SEQ ID NO:75). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:75. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:75. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:75. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NO:75.

[0195] Another exemplary ferredoxin derived from R. capsulatus is fdxB, NCBI Accession No. WP_013068971.1 (SEQ ID NO:76). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:76. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:76. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:76. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NO:76.

[0196] An exemplary ferredoxin derived from R. capsulatus is fdxC, NCBI Accession No. WP_013068981.1 (SEQ ID NO:77). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:77. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:77. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:77. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NO:77.

[0197] An exemplary ferredoxin derived from R. capsulatus is fdxD, NCBI Accession No. WP_013066317.1 (SEQ ID NO:78). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:78. In some embodiments, the ninth nucleotide sequences encodepolypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:78. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:78. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NO:78.

[0198] An exemplary ferredoxin derived from R. capsulatus is fdxE, NCBI Accession No. WP_013068113.1 (SEQ ID NO:79). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:79. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:79. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:79. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NO:79.

[0199] An exemplary ferredoxin derived from R. capsulatus is fdxN, NCBI Accession No. WP_013068980.1 (SEQ ID NQ:80). In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NQ:80. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:80. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:80. In some embodiments, the ninth nucleotide sequences encode polypeptides comprising the amino acid sequence of SEQ ID NQ:80.

[0200] In particular embodiments, sixth nucleotide sequences encode an ispG polypeptide derived from Rhodobacter capsulatus (e.g., polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73), seventh nucleotide sequences encode an ispH polypeptide also derived from R. capsulatus (e.g., polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74), ninth nucleotide sequences encode a ferredoxin or flavodoxin also derived from R. capsulatus (e.g., polypeptides comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:75-81), and tenth nucleotide sequencesencode a flavodoxin / ferredoxin-NADP reductase also derived from R. capsulatus. An exemplary flavodoxin / ferredoxin-NADP reductase derived from R. capsulatus is fpr, UniProt Accession No. D5ATP7 (SEQ ID NO:82). In some embodiments, the tenth nucleotide sequences encode flavodoxin / ferredoxin-NADP reductases comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:82. In some embodiments, the tenth nucleotide sequences encode flavodoxin / ferredoxin-NADP reductases comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:82. In some embodiments, the tenth nucleotide sequences encode flavodoxin / ferredoxin-NADP reductases comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO:82. In some embodiments, the tenth nucleotide sequences encode flavodoxin / ferredoxin-NADP reductases comprising the amino acid sequence of SEQ ID NO:82.6.4. Increasing Flux into DXP Pathway

[0201] In addition to comprising first through eighth nucleotide sequences encoding polypeptides (1)-(8), recombinant microorganisms of the present disclosure can further be engineered to increase flux into the DXP pathway. As seen in FIG. 1 , various enzymatic pathways lead from glucose (A) or xylose (R) to DXP (H), the first intermediate in the DXP pathway. Increasing the activity of one or more polypeptides catalyzing one or more reactions leading to DXP (H), such as RibB*, YajO, XylB, and / or polypeptides active in the conversion of glucose (A) to GAP (E) and pyruvate (F), can increase flux into the DXP pathway and production rate and / or yield of isoprene and / or isoprenoids.

[0202] Increasing the activity of one or more polypeptides to increase flux into the DXP pathway can include one or more of increasing the copy number of genes comprising coding sequences for the polypeptides; operably linking coding sequences to promoters with higher activity and / or that are inducible at desired stages of the cell cycle compared to the promoters of parental microorganisms; operably linking coding sequences to other regulatory sequences such that expression is increased relative to endogenous expression in parental microorganisms; codon optimization; modifying the amino acid sequences of polypeptides to have higher activity than wild-type; replacing endogenous polypeptide coding sequences with heterologous polypeptide coding sequences having higher activity; and adding genes comprising coding sequences for polypeptides to a parentalmicroorganism lacking those genes, among other approaches that persons of ordinary skill in the art will be able to implement with the benefit of the present disclosure.

[0203] In some embodiments, flux into the DXP pathway can be increased by engineering recombinant microorganisms to have improved assimilation of 2-keto-3-deoxy-gluconate (KDG) relative to a parental strain. Improved KDG assimilation is described in Section 6.4.1.6.4.1. Improving Assimilation of 2-keto-3-deoxy-gluconate (KDG)

[0204] As can be seen in FIG. 1 , glucose (A) can be converted by multiple steps to gluconate (B), with subsequent reactions leading to KDG (C), KDGP (D), and GAP (E) and pyruvate (F). Dxs polypeptides (1) can act on GAP (E) and pyruvate (F) as described elsewhere herein to produce DXP (H). Accordingly, recombinant microorganisms of the present disclosure can be engineered to assimilate KDG at increased levels relative to a parental strain. Recombinant microorganisms of the present disclosure can also be engineered to increase production of KDG by the recombinant microorganism. In some embodiments, a microorganism that lacks the ability to produce KDG (e.g., an E. coli strain) is engineered in a way that imparts an ability to produce KDG. This can be accomplished, for example, by introducing gluconate dehydratase, as discussed further below.

[0205] The parental strain can be a wild-type strain, or a recombinant microorganism comprising coding sequences for DXP pathway polypeptides as described herein. The order in which organisms are engineered to assimilate (e.g., produce) and / or phosphorylate KDG at increased levels and to comprise coding sequences for DXP pathway polypeptides is not crucial.

[0206] In some embodiments, recombinant microorganisms comprise nucleotide sequences encoding gluconate dehydratases (EC 4.2.1.39 or EC 4.2.1.140), which catalyze the dehydration of gluconate (B in FIG. 1) to KDG (C).

[0207] Exemplary gluconate dehydratases include those from Achromobacter. For example, gluconate dehydratases can comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1. For example, gluconate dehydratases can comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1. For example, gluconate dehydratases can comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQID NO:1. For example, gluconate dehydratases can comprise the amino acid sequence of SEQ ID NO:1.

[0208] In some embodiments, recombinant microorganisms comprise nucleotide sequences encoding KDG kinases (EC 2.7.1.45), which catalyze the phosphorylation of KDG (C) to KDGP (D).

[0209] Exemplary KDG kinases include those from E. coli, such as E. coli K-12. For example, KDG kinases can comprise amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2. For example, KDG kinases can comprise amino acid sequences having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2. For example, KDG kinases can comprise amino acid sequences having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2. For example, KDG kinases can comprise the amino acid sequence of SEQ ID NO:2. Additional exemplary KDG kinases include those having any one of the following gene ID nos.: AAU42643.1 (Bacillus licheniformis ATCC 14570), CAQ33844.1 (E. coli), NP_279296.1 (Halobacterium sp. NRC-1), ZP_02184941.1 (Carnobacterium sp. AT7), CAE11231.1 (Bacillus amyloliquefaciens FZB42), AAK88038.2 (Agro bacterium tumefaciens), ZP_00991972.1 (Vibrio splendidus), NP_172158.1 (Arabidopsis thaliana), PDB: 1V1 B_D (Erwinia chrysanthemi), NP_347035.1 (Clostridium acetobutylicum ATCC 824), YP_528747.1 (Saccharophagus degradans 2-40), ABI72944.1 (Shewanella frigidimarina NCI MB 400).

[0210] In recombinant microorganisms comprising nucleotide sequences encoding gluconate dehydratases and / or KDG kinases, the sequences encoding these polypeptides can be operably linked to (which may be termed “under the control of”) a strong promoter. Strong promoters active in given recombinant microorganisms are generally known to persons of ordinary skill in the art. Strong promoters can be used alone (e.g., as nucleotide sequences comprising -35 regions and -10 regions) or with one or more regulatory components. The regulatory components can be those with which the strong promoters are typically found in a parental microorganism or can be heterologous to the strong promoters. Such regulatory components can include, but are not limited to, anti-termination sites, gt10 sequences, ribosome binding sites / Shine-Dalgarno sequences, or spacer sequences, among others.

[0211] For example, in E. coli, strong promoters operably linked to nucleotide sequences encoding gluconate dehydratases and / or KDG kinases can include pTrc promoters, such as pTrc promoters in a regulatory sequence comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:3.

[0212] Flux from glucose (A) to DXP (H) via KDG (C) in recombinant microorganisms can be increased by disrupting coding sequences and / or regulatory sequences for polypeptides catalyzing reactions that divert intermediates in or precursors of members of the (A)-(H) pathway away from production of DXP (H), and / or disrupting coding sequences and / or regulatory sequences for polypeptides that are irrelevant, unnecessary, and / or may have negative effects on other nucleotide sequences and / or polypeptides.

[0213] For example, gluconate-6-phosphate dehydratases (edd; EC 4.2.1.12) catalyzes the conversion of gluconate-6-phosphate to KDGP. In strains incapable of producing gluconate- 6-phosphate, there is no benefit in maintaining edd activity to produce KDGP. For another, in microorganisms and strains in which edd is co-cistronic with eda, the retention of coding sequences and / or regulatory sequences of edd may reduce expression of eda through transcription-translation coupling effects, such as polarity.

[0214] Disrupting gluconate-6-phosphate dehydratase (edd) coding sequences and / or regulatory sequences can involve insertions, deletions, and / or substitutions in such coding sequences and / or regulatory sequences. Particular insertions, deletions, and / or substitutions will depend on the recombinant microorganisms in which the disruption is desired. For example, in E. coli K12, disruptions of edd genes can comprise insertions, deletions, or substitutions within nucleotides 1-1966 of SEQ ID NO:4.

[0215] Other polypeptides catalyzing reactions that divert intermediates away from production of DXP (H) include gluconate kinases (gntK; EC 2.7.1.12) and idonate kinases (idnK; EC 2.7.1.12). Both gntK and idnK catalyze the phosphorylation of gluconate (B) to gluconate-6-P.

[0216] Disrupting of gntK genes (e.g., coding sequences encoding gntK polypeptides and operably linked regulatory sequences) or idnK genes can involve insertions, deletions, and / or substitutions. Particular insertions, deletions, and / or substitutions will depend on the recombinant microorganisms in which the disruption is desired. For example, in E. coli K12, disruption of gntK can comprise an insertion, deletion, or substitution within nucleotides1490-2155 of SEQ ID NO:6. Disruption of idnK can comprise an insertion, deletion, or substitution within nucleotides 1-561 of SEQ ID NO:7.

[0217] In E. coli, the idonate pathway degrades gluconate in addition to its role in idonate assimilation. Coding sequences and / or regulatory sequences for one or members of the idonate pathway can be disrupted to enhance flux from gluconate (B) to KDG (C) and ultimately DXP (H). Disruptions can be in any one or more of coding sequences and / or regulatory sequences for idonate dehydrogenase (idnD; EC 1.1.1.264), 5-keto-D-gluconate 5-reductase (idnO; EC 1.1.1.69), and idonate transporter (idnT; Transporter Classification Database: TCDB 2.A.8.1.2). Specifically, in E. coli, disruptions of idnD can comprise insertions, deletions, and / or substitutions within nucleotides 1-1029 of SEQ ID NO:8. Disruptions of idnO can comprise insertions, deletions, and / or substitutions within nucleotides 1-762 of SEQ ID NO:9. Disruptions of idnT can comprise insertions, deletions, and / or substitutions within nucleotides 1-1317 of SEQ ID NQ:10.

[0218] Additionally or alternatively, flux from glucose (A) to DXP (H) via KDG (C) in recombinant microorganisms can be increased by overexpressing polypeptides catalyzing reactions in the pathway relative to parental microorganisms. For example, KDG-phosphate aldolase (eda; EC 4.1.3.16) catalyzes the cleavage of KDGP (D) to GAP (E) and pyruvate (F).

[0219] Overexpression of eda can be affected by any known technique, such as regulatory sequence engineering (including promoter engineering), codon optimization, or increase of copy number by insertion of multiple copies of genes or portions thereon into the genome and / or inclusion in the cells of one or more plasmids and / or BACs containing one or more copies, among others. For example, the native eda promoter can be replaced by a synthetic promoter with higher activity. In a particular example, in E. coli k12, the native eda promoter, which is within nucleotides 1967-2002 of SEQ ID NO:4, can be replaced with a synthetic promoter in a regulatory sequence comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:5. For example, the synthetic promoter can be in a regulatory sequence comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:5. For example, the synthetic promoter can be in a regulatory sequence comprising the nucleotide sequence of SEQ ID NO:5.

[0220] Regulatory sequences, e.g., regulatory sequences comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:5, can comprise a RBS and at least one spacer sequences, such as two spacer sequences, e.g., a spacer sequence upstream of the promoter and a spacer sequence downstream of the promoter.6.5. Improving Isoprene and / or Isoprenoid Production

[0221] As can be seen in FIG. 1 , DMAPP (P) and / or IPP (Q) can be precursors for the production of isoprene and / or isoprenoids, such as limonene, beta-myrcene, nerol, geraniol, linalool, beta-ocimene,1 ,8-cineole, farnesene, and / or farnesol. Accordingly, recombinant microorganisms of the present disclosure can be engineered to increase output from the DXP pathway and / or increase isoprene and / or isoprenoid yields relative to parental strains.

[0222] The parental strain can be a wild-type strain, or a recombinant microorganism comprising coding sequences for DXP pathway polypeptides, and optionally able to assimilate, produce, and / or phosphorylate KDG at increased levels, as described herein. The order in which organisms are engineered to improve isoprene and / or isoprenoid production and to comprise coding sequences for DXP pathway polypeptides is not crucial.

[0223] Depending on which isoprenoid(s) are desired to be produced, one or more polypeptide activities can be increased. Increasing activity can comprise engineering recombinant microorganisms to overexpress endogenous polypeptides, and / or engineering recombinant microorganisms to express heterologous polypeptides. Some microorganisms may lack one or more desired endogenous polypeptide activities, in which case, engineering the recombinant microorganisms to express heterologous polypeptides may be desired.

[0224] Specific polypeptide activities that can be increased to convert DMAPP (P) and IPP (Q) to isoprene and / or desired isoprenoid(s) include, but are not limited to, those described in Sections 6.5.1 to 6.5.12.6.5.1. Isoprene Biosynthesis

[0225] Isoprene production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous isoprene biosynthesis activity and / or to express one or more heterologous isoprene biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologousisoprene biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous isoprene biosynthesis polypeptides.

[0226] In some embodiments, at least one heterologous isoprene and / or isoprenoid biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is an isoprene synthase (ispS; EC 4.2.3.27). IspS catalyzes the conversion of DMAPP into isoprene. The ispS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:67. The ispS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:67. The ispS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:67. The ispS can comprise the amino acid sequence of SEQ ID NO:67. Other enzymes known to produce isoprene are ispH polypeptides, such as those described in section 6.2.7 above. This is a side activity of ispH which normally catalyzes step 7 of the DXP pathway (see Fig. 1).6.5.2. Geranyl-Pyrophosphate Biosynthesis

[0227] Biosynthesis of isoprenoids with a chain length of 10 or more carbons starts with the condensation of one molecule of IPP and one molecule of DMAP to form geranylpyrophosphate (GPP) by geranyl-pyrophosphate synthases (gppS; EC 2.5.1.1). GPP is used by isoprenoid synthases to produce C10-isoprenoids, or further extended to form isoprenoids with a longer chain. The gppS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:68. The gppS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:68. The gppS can comprise the amino acid sequence of SEQ ID NO:68.

[0228] Another strategy to overproduce GPP is by using variants of the enzyme geranyl diphosphate / farnesyl diphosphate synthase ispA (EC 2.5.1.10), e.g., an ispA comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11. It has been shown (Reiling, et al. 2004. Biotech Bioeng. 87: 200-212: and Zhou, et al. 2014. J. Biotech. 169: 42-50) that mutated enzyme variants of E. coli ispA, can catalyze a single condensation of IPP and DMAPP to form GPP only; unlike wild-type IspA that normally catalyzes two sequential condensations of two IPPs with DMAPP to form farnesyl diphosphate (FPP). One of those ispA variants has a mutation of serine tophenylalanine at residue 80 (S80F; SEQ ID NO:69), however mutation(s) in other residue(s) may also favor the production of GPP instead of FPP.6.5.3. Neryl-Pyrophosphate Biosynthesis

[0229] Biosynthesis of isoprenoids with a chain length of 10 or more carbons can also start with the condensation of one molecule of IPP and one molecule of DMAP to form nerylpyrophosphate (NPP) by neryl-pyrophosphate synthases (nppS; EC 2.5.1.28), which is used by isoprenoid synthases to produce C10-isoprenoids, or further extended to form isoprenoids with a longer chain. The nppS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:70. The nppS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:70. The nppS can comprise the amino acid sequence of SEQ ID NQ:70.6.5.4. Limonene Biosynthesis

[0230] Limonene production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous limonene biosynthesis activity and / or to express one or more heterologous limonene biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous limonene biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous limonene biosynthesis polypeptides.

[0231] In some embodiments, at least one heterologous limonene biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a limonene synthase (limS; EC 4.2.3.16 and EC 4.2.3.20). The limS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12. The limS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:12. The limS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 12. The limS can comprise the amino acid sequence of SEQ ID NO:12.6.5.5. Beta-Myrcene Biosynthesis

[0232] Beta-myrcene production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous beta-myrcene biosynthesis activity and / or to express one or more heterologous beta-myrcene biosynthesis polypeptides. Forexample, recombinant E. coli can be engineered to express two or more heterologous betamyrcene biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous beta-myrcene biosynthesis polypeptides.

[0233] In some embodiments, at least one heterologous beta-myrcene biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a myrcene synthase (myrS; EC 4.2.3.15). The myrS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13. The myrS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:13. The myrS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 13. The myrS can comprise the amino acid sequence of SEQ ID NO: 13.6.5.6. Nerol Biosynthesis

[0234] Nerol production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous nerol biosynthesis activity and / or to express one or more heterologous nerol biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous nerol biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous nerol biosynthesis polypeptides.

[0235] In some embodiments, at least one heterologous nerol biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a nerol synthase, (nerS; EC 3.1.7.13). This enzyme utilizes NPP as a substrate to produce nerol. The nerS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:57. The nerS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:57. The nerS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:57. The nerS can comprise the amino acid sequence of SEQ ID NO:57.

[0236] Another strategy to produce nerol from NPP is by the use of endogenous, or heterologous non-specific phosphatases. For example, E. coli has around 13 non-specific phosphatases that belong to the Nudix hydrolase family, and many others of differentfamilies. Some of them, like ppa, cdh, IpxH, mutT, nudC, nudF, and nudJ have shown to be able to convert NPP into nerol (Lei, et al., 2021. ACS Synth. Biol. 10: 1531-1544).6.5.7. Geraniol Biosynthesis

[0237] Geraniol production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous geraniol biosynthesis activity and / or to express one or more heterologous geraniol biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous geraniol biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous geraniol biosynthesis polypeptides.

[0238] In some embodiments, at least one heterologous geraniol biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a geraniol synthase (gerS; EC 3.1.7.11). The gerS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:58. The gerS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:58. The gerS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:58. The gerS can comprise the amino acid sequence of SEQ ID NO:58.

[0239] Another strategy to produce geraniol from GPP is by the use of endogenous or heterologous non-specific phosphatases. For example, E. coli has around 13 non-specific phosphatases that belong to the Nudix hydrolase family, and many others of different families. Some of them, such as phoA, yfcD (nudl), nudB, aphA and ybjG, have been shown to be able to convert GPP into geraniol (Liu, et al., 2015. Bioengineered 6: 288-293; Bessman, 2019. Prot. Sci. 28: 1494-1500; Zada, et al., 2018. Biotechnol. Biofuels 11 : 210.6.5.8. Linalool Biosynthesis

[0240] Linalool production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous linalool biosynthesis activity and / or to express one or more heterologous linalool biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous linalool biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous linalool biosynthesis polypeptides.

[0241] In some embodiments, at least one heterologous linalool biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a linalool synthase (linS; EC 4.2.3.25). The linS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:59. The linS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:59. The linS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:59. The linS can comprise the amino acid sequence of SEQ ID NO:59.6.5.9. Beta-Ocimene Biosynthesis

[0242] Beta-ocimene production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous beta-ocimene biosynthesis activity and / or to express one or more heterologous beta-ocimene biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous beta- ocimene biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous beta-ocimene biosynthesis polypeptides.

[0243] In some embodiments, at least one heterologous beta-ocimene biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a beta- ocimene synthase (ociS; EC 4.2.3.106). The ociS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:60. The ociS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:60. The ociS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:60. The ociS can comprise the amino acid sequence of SEQ ID NQ:60.6.5.10. 1,8-Cineole Biosynthesis

[0244] 1 ,8-cineole production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous 1 ,8-cineole biosynthesis activity and / or to express one or more heterologous 1 ,8-cineole biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous 1 ,8- cineole biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous 1 ,8-cineole biosynthesis polypeptides.

[0245] In some embodiments, at least one heterologous 1 ,8-cineole biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a 1,8- cineole synthase (cinS; EC 4.2.3.108). The cinS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:61. The cinS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:61. The cinS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:61. The cinS can comprise the amino acid sequence of SEQ ID NO:61 .6.5.11. Farnesene Biosynthesis

[0246] Production of both alpha-farnesene and beta-farnesene can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous farnesene biosynthesis activity and / or to express one or more heterologous farnesene biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous farnesene biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous farnesene biosynthesis polypeptides.

[0247] In some embodiments, at least one heterologous farnesene biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is an alpha-farnesene synthase (fnsS; EC 4.2.3.46 and / or a beta-farnesene synthase (aaFS; EC 4.2.3.47).

[0248] The fnsS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:62. The fnsS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:62. The fnsS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:62. The fnsS can comprise the amino acid sequence of SEQ ID NO:62.

[0249] The aaFS can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:71 . The aaFS can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:71. The aaFS can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:71 . The aaFS can comprise the amino acid sequence of SEQ ID NO:71.6.5.12. Farnesol Biosynthesis

[0250] Farnesol can be produced from farnesyl-diphosphate (FPP), a common intermediate of the isoprenoid biosynthetic pathways. Farnesol production can be increased by engineering recombinant cells, e.g., recombinant E. coli cells, to have increased endogenous farnesol biosynthesis activity and / or to express one or more heterologous farnesol biosynthesis polypeptides. For example, recombinant E. coli can be engineered to express two or more heterologous farnesol biosynthesis polypeptides. For another example, recombinant E. coli can be engineered to express three or more heterologous farnesol biosynthesis polypeptides.

[0251] Farnesol can be produced from FPP by the use of endogenous or heterologous nonspecific phosphatases. For example, E. coli has around 13 non-specific phosphatases that belong to the Nudix hydrolase family, and many other phosphatases of different families. Some of these phosphatases like pgpA, pgpB, aphA, phoA, ybjG and bacA have shown to be able to convert FPP into farnesol (Wang, 2010. Biotechnol. Bioeng. 107: 421-429; Wang, 2016. Biotechnol. J. 11 : 1291-1297).

[0252] Farnesol can also be produced via a farnesol / terpene synthase enzyme. It has been shown that the gene OsTPS13 product from Oryza sativa subsp. japonica (Rice) (SEQ ID NO:72), when expressed in E. coli, can convert FPP into Farnesol at 84.2% efficiency (i.e., other products comprised 15.8%).

[0253] In some embodiments, at least one heterologous farnesol biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a farnesol / terpene synthase. The farnesol / terpene synthase can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:72. The farnesol / terpene synthase can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:72. The farnesol / terpene synthase can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:72. The farnesol / terpene synthase can comprise the amino acid sequence of SEQ ID NO:72.

[0254] In some embodiments, at least one heterologous farnesol biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a farnesol synthase. The farnesol synthase can comprise an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:63. The farnesol synthase can comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:63. The farnesol synthase can comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:63. The farnesol synthase can comprise the amino acid sequence of SEQ ID NO:63.

[0255] In some embodiments, at least one endogenous or heterologous farnesyl- diphosphate biosynthesis polypeptide engineered into recombinant cells, e.g., recombinant E. coli cells, is a farnesyl-diphosphate synthase. One example of an endogenous FPP synthase is the product of E.coli gene ispA, known to catalyze two sequential reactions to convert two molecules IPP and one molecule of DMAPP into one molecule of FPP. IspA polypeptides include those described in Section 6.5.4.6.6. Parental Microorganisms

[0256] Any prokaryotes can be parental microorganisms engineered to yield recombinant microorganisms described herein. The parental microorganisms include, but are not limited to, bacteria, e.g., E. coli.

[0257] 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.

[0258] Other E. coli strains from which recombinant microorganisms of the present disclosure can be engineered include, but are not limited to, E. coli K12 W3110, E. coli K12 DH5alpha, and non-K12 strains, such as E. coli BL21 and E. coli \N.

[0259] 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.6.6.1. Engineering Methods

[0260] Parental microorganisms can be engineered using techniques known in the art. For example, reductions or increases of activities of one or more enzymes as described herein can be engineered into parental microorganism via techniques known in the art.

[0261] In some embodiments, activities are reduced or increased by introducing into parental microorganisms nucleic acids comprising modified coding sequences and / or modified regulatory sequences into cells of the parental microorganism.

[0262] 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.

[0263] 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 regulatory sequences 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.

[0264] With particular reference to coding sequences for polypeptides (1)-(8) of the DXP pathway, the coding sequences can be organized into a first expression cassette and a second expression cassette. At least two of the coding sequences for polypeptides (1)-(8) can be in the first expression cassette. For example, two of the coding sequences for polypeptides (1)-(8) can be in the first expression cassette, e.g., the coding sequences for polypeptides (1)-(2) can be in the first expression cassette. For another example, three of the coding sequences for polypeptides (1)-(8) can be in the first expression cassette. For another example, four of the coding sequences for polypeptides (1)-(8) can be in the first expression cassette.

[0265] The first expression cassette can be an operon. The operon can comprise coding sequences operably linked to a constitutive promoter or an inducible promoter. Inducible promoters can be those activated by any desired inducer. For example, the inducible promoter can be inducible by gluconate. An exemplary gluconate-inducible promoter comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:64.

[0266] If integrated into the cell genome, the first expression cassette can be integrated at any desired location. For example, the first expression cassette can be integrated into the E. coli genome downstream of the gluconate repressor gene gntR.

[0267] At least three of the coding sequences for polypeptides (1)-(8) can be in a second expression cassette. For example, three of the coding sequences for polypeptides (1)-(8) can be in the second expression cassette. For another example, four of the coding sequences for polypeptides (1)-(8) can be in the second expression cassette. For yet another example, five of the coding sequences for polypeptides (1)-(8) can be in the second expression cassette. For a further example, six of the coding sequences for polypeptides (1)-(8) can be in the second expression cassette, e.g., the coding sequences for polypeptides (3)-(8) can be in the second expression cassette.

[0268] The second expression cassette can be an operon, which can comprise coding sequences operably linked to a constitutive promoter or an inducible promoter. For example, the second expression cassette can be under the control of a constitutive promoter, e.g., a bacteriophage T7 A3 promoter.

[0269] If integrated into the cell genome, the second expression cassette can be integrated at any desired location. For example, the second expression cassette can be integrated into the E. coli genome at the insH10 locus. Optionally, the second expression cassette can replace the yhcE gene and the insH10 insertion element.

[0270] In some embodiments, nucleic acids comprising coding sequences for polypeptides described herein are integrated into bacterial genomes (e.g., E. coli genomes) at the lac operon locus. For example, the nucleic acids can comprise coding sequences for one or more isoprene and / or isoprenoid biosynthesis polypeptides described in Section 6.5. Optionally, all or part of lacZ (SEQ ID NO :14), lacY (SEQ ID NO:15), and / or lacA (SEQ ID NO: 16) can be deleted.

[0271] Various genome editing techniques, including but not limited to homologous recombination, CRISPR-Cas, 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.

[0272] 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.

[0273] 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.6.7. Methods of Use6.7.1. Culture Media

[0274] Generally, methods disclosed herein comprise growing cells of a recombinant microorganism (i.e. , cells of a prokaryotic species into which nucleic acids disclosed herein have been introduced) 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 and / or isoprenoids. Culturing can be in a batch mode or a continuous mode.

[0275] 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: sodiumphosphate 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).

[0276] 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).

[0277] 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).

[0278] 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). Gluconate can both provide a carbon source for recombinant microorganisms and induce expression of gluconate-inducible promoters.

[0279] 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-derived sugars (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.

[0280] 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 or precursors thereof 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. Alternatively or additionally, induction of expression from gluconate- inducible promoters can occur if the cells produce gluconate from other sugars during culturing.

[0281] In some embodiments, a production medium comprises an inducer other than gluconate, i.e., a molecule other than gluconate which induces translation of a coding sequence regulated by an inducible promoter.

[0282] 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. Aculture 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).

[0283] 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. A growth 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).

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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 tosustain 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 / L6.7.2. Culture Conditions

[0288] Recombinant cells comprising expression systems of the disclosure may be cultured under suitable conditions in a medium, such as a medium described in Section 6.7.1. 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.

[0289] 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 mesophilictemperature. In some embodiments, the mesophilic temperature is selected from any of the foregoing ranges.

[0290] 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.6.8. Methods for Producing Isoprene and / or Isoprenoids

[0291] The present disclosure also relates to methods for producing isoprene and / or isoprenoids. In some embodiments, the methods comprising culturing recombinant microorganisms as described in Section 6.7.2 under conditions in which isoprene and / or an isoprenoid is produced.

[0292] The conditions can include culturing recombinant microorganisms in appropriate media, e.g., media comprising glucose.

[0293] Cells can be grown to desired cell concentrations in appropriate media. After growth to desired cell concentrations, cells in which one or more genes encoding enzymes for which increased activity are operably linked to gluconate-inducible promoters can be cultured in media comprising gluconate and / or in which the cells produce gluconate during a production phase.

[0294] In some embodiments, methods of producing isoprene and / or isoprenoids comprise culturing recombinant cells of the disclosure in at least two phases: (1) a growth phase in which cells divide and cell density increases and in which one or more heterologous enzymes (e.g., Dxs and / or Dxr and optionally IspG and / or IspH and / or their redox partners) under control of an inducible promoter are not expressed or are expressed at a non-induced level; and (2) a production phase in which cell density of the culture does not increase or increases more slowly than during the growth phase and in which one or more heterologous enzymes under control of an inducible promoter are expressed at their induced level. A production phase may begin by changing culture conditions such that expression of one or more heterologous enzymes (e.g., Dxs and / or Dxr and optionally IspG and / or IspH and / or their redox partners) from an inducible promoter is induced, and the cells begin producing the products of the biosynthetic pathway of which the one or more heterologous enzymesare a part. Transition to a growth phase may be made when a particular biomass concentration or cell density is achieved. For example, in some embodiments, a growth phase continues until the recombinant cells reach a biomass concentration of at least about 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml of dry cell mass. In some embodiments, a production phase is initiated (e.g., by the induction of an inducible promoter) after the recombinant cells reach a biomass concentration of at least about 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml of dry cell mass. In some embodiments, a growth phase continues until the recombinant cells reach a cell density of at least about 5x106cells / ml, 6x106cells / ml, 7x106cells / ml, 8x106cells / ml, 9x106cells / ml, 1x107cells / ml, 2x107cells / ml, 3x107cells / ml, 4x107cells / ml, 5x107cells / ml, 6x107cells / ml, 7x107cells / ml, 8x107cells / ml, 9x107cells / ml, 1x108cells / ml, 2x108cells / ml, or 3x108cells / ml. In some embodiments, a production phase is initiated after the recombinant cells reach a cell density of at least about 5x106cells / ml, 6x106cells / ml, 7x106cells / ml, 8x106cells / ml, 9x106cells / ml, 1x107cells / ml, 2x107cells / ml, 3x107cells / ml, 4x107cells / ml, 5x107cells / ml, 6x107cells / ml, 7x107cells / ml, 8x107cells / ml, 9x107cells / ml, 1x108cells / ml, 2x108cells / ml, or 3x108cells / ml. In some embodiments, a production phase is initiated after the recombinant cells in the culture reach a stationary growth phase. In some embodiments, a production phase is initiated after the recombinant cells in the culture exit an exponential growth phase.

[0295] Isoprene and / or isoprenoids produced in the methods of the disclosure can be recovered from media or from head spaces above media in a fermenter or other vessel. Recovery can comprise recovery of isoprene and / or isoprenoids secreted by recombinant microorganisms into the media; recovery of isoprene and / or volatile isoprenoids in head spaces; lysis of cells in the media to release isoprene and / or isoprenoids, followed by recovery from media and / or head spaces; separation of cells from media, followed by lysis and isolation of isoprene and / or isoprenoids; or a combination thereof.

[0296] In some embodiments, limonene is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a limonene synthase (limS; EC 4.2.3.16 and EC 4.2.3.20). Exemplary limS polypeptides are set forth in Section 6.5.4.

[0297] In some embodiments, p-myrcene is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a myrcene synthase (myrS; EC 4.2.3.15). Exemplary myrS polypeptides are set forht in Section 6.5.5.

[0298] In some embodiments, nerol is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a nerol synthase, (nerS; EC 3.1.7.13). Exemplary nerS polypeptides are set forth in Section 6.5.6.

[0299] In some embodiments, geraniol is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a geraniol synthase (gerS; EC 3.1.7.11). Exemplary gerS polypeptides are set forth in Section 6.5.7.

[0300] In some embodiments, linalool is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a linalool synthase (linS; EC 4.2.3.25). Exemplary linS polypeptides are set forth in Section 6.5.8.

[0301] In some embodiments, p-ocimene is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a beta-ocimene synthase (ociS; EC 4.2.3.106). Exemplary ociS polypeptides are set forth in Section 6.5.9.

[0302] In some embodiments, 1 ,8-cineole is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express a 1,8-cineole synthase (cinS; EC 4.2.3.108). Exemplary cinS polypeptides are set forht in Section 6.5.10.

[0303] In some embodiments, farnesene (e.g., a-farnesene and / or p-farnesene) is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express an alpha-farnesene synthase (fnsS; EC 4.2.3.46) and / or a beta- farnesene synthase (aaFS; EC 4.2.3.47). Exemplary fnsS and aaFS polypeptides are set froth in Section 6.5.11.

[0304] In some embodiments, farnesol is produced and recovered by the methods of the disclosure, e.g., by engineering the recombinant microorganisms to express endogenous or heterologous non-specific phosphatases and / or farnesol / terpene synthase enzymes. Exemplary phosphatases and farnesol / terpene synthases are set forth in Section 6.5.12.

[0305] The recombinant microorganisms can be E. coH, including but not limited to E. coli described in Section 6.2.9.6.9. Numbered Embodiments

[0306] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below.1. A recombinant E. coli cell comprising:(a) a first nucleotide sequence encoding a dxs polypeptide (1) (EC 2.2.1.7);(b) a second nucleotide sequence encoding a dxr polypeptide (2) (EC 1.1.1.267);(c) a third nucleotide sequence encoding an ispD polypeptide (3) (EC 2.7.7.60);(d) a fourth nucleotide sequence encoding an ispE polypeptide (4) (EC 2.7.1.148);(e) a fifth nucleotide sequence encoding an ispF polypeptide (5) (EC 4.6.1.12);(f) a sixth nucleotide sequence encoding an ispG polypeptide (6) (EC 1.17.7.1 or EC 1.17.7.3);(g) a seventh nucleotide sequence encoding an ispH polypeptide (7) (EC 1.17.7.4); and(h) an eighth nucleotide sequence encoding an idi polypeptide (8) (EC 5.3.3.2).2. The recombinant E. coli cell of embodiment 1, wherein at least 1 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.3. The recombinant E. coli cell of embodiment 1, wherein at least 2 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.4. The recombinant E. coli cell of embodiment 1, wherein at least 3 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.5. The recombinant E. coli cell of embodiment 1, wherein at least 4 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.6. The recombinant E. coli cell of embodiment 1, wherein at least 5 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.7. The recombinant E. coli cell of embodiment 1 , wherein at least 6 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.8. The recombinant E. coli cell of embodiment 1, wherein at least 7 of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.9. The recombinant E. coli cell of embodiment 1 , wherein all of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are non-native to E. coli.10. The recombinant E. coli cell of any one of embodiments 1 to 9, wherein the coding sequences are organized into at least a first expression cassette and a second expression cassette.11 . The recombinant E. coli cell of any one of embodiments 1 to 10, wherein the coding sequences are organized into a first expression cassette and a second expression cassette.12. The recombinant E. coli cell of embodiment 10 or embodiment 11 , wherein the first expression cassette is integrated into the E. coli genome.13. The recombinant E. coli cell of embodiment 10 or embodiment 11 , wherein the second expression cassette is integrated into the E. coli genome.14. The recombinant E. coli cell of embodiment 10 or embodiment 11 , wherein the first expression cassette and the second expression cassette are integrated into the E. coli genome.15. The recombinant E. coli cell of any one of embodiments 10 to 14, wherein each of the first expression cassette and the second expression cassette is an operon.16. The recombinant E. coli cell of any one of embodiments 10 to 15, wherein at least two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the first expression cassette.17. The recombinant E. coli cell of any one of embodiments 10 to 16, wherein two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the first expression cassette.18. The recombinant E. coli cell of any one of embodiments 10 to 16, wherein three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the first expression cassette.19. The recombinant E. coli cell of any one of embodiments 10 to 16, wherein four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the first expression cassette.20. The recombinant E. coli cell of any one of embodiments 10 to 19, wherein the first and the second coding sequences are in the first expression cassette.21 . The recombinant E. coli cell of any one of embodiments 10 to 20, wherein the first expression cassette is under the control of an inducible promoter.22. The recombinant E. coli cell of embodiment 21 , wherein the inducible promoter is inducible by gluconate.23. The recombinant E. coli cell of embodiment 21 , wherein the gluconate- inducible promoter comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:64.24. The recombinant E. coli cell of any one of embodiments 10 to 23, wherein the first expression cassette is integrated into the E. coli genome downstream of the gluconate repressor gene gntR.25. The recombinant E. coli cell of any one of embodiments 10 to 24 wherein at least three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.26. The recombinant E. coli cell of any one of embodiments 10 to 25, wherein three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.27. The recombinant E. coli cell of any one of embodiments 10 to 25, wherein four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.28. The recombinant E. coli cell of any one of embodiments 10 to 25, wherein five of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.29. The recombinant E. coli cell of any one of embodiments 10 to 25, wherein six of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.30. The recombinant E. coli cell of embodiment 29, wherein the third, fourth, fifth, sixth, seventh and eighth coding sequences are in the second expression cassette.31 . The recombinant E. coli cell of any one of embodiments 10 to 30, wherein the second expression cassette is under the control of a constitutive promoter.32. The recombinant E. coli cell of embodiment 31 , wherein the constitutive promoter is a bacteriophage T7 A3 promoter.33. The recombinant E. coli cell of any one of embodiments 10 to 32, wherein the second expression cassette is integrated into the E. coli genome at the insHIO locus.34. The recombinant E. coli cell of embodiment 33, wherein the second expression cassette replaces the yhcE gene and the insHIO insertion element.35. The recombinant E. coli cell of any one of embodiments 1 to 35, wherein the third nucleotide sequence and fifth nucleotide sequence are operably linked so as to encode a fusion protein comprising the ispD polypeptide (3) and the ispF polypeptide (5).36. The recombinant E. coli cell of any one of embodiments 1 to 35, which is engineered to assimilate and / or produce 2-keto-3-deoxy-gluconate (KDG) at increased levels relative to a parental strain.37. The recombinant E. coli cell of embodiment 36, wherein the parental strain is a wild-type strain.38. The recombinant E. coli cell of embodiment 36, wherein the parental strain is a recombinant organism as defined in any one of embodiments 1 to 35.39. The recombinant E. coli cell of any one of embodiments 36 to 38, which comprises a nucleotide sequence encoding a gluconate dehydratase (EC 4.2.1.39 or EC 4.2.1.140).40. The recombinant E. coli cell of embodiment 39, wherein the gluconate dehydratase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ I D NO: 1.41 . The recombinant E. coli cell of embodiment 39, wherein the gluconate dehydratase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ I D NO: 1.42. The recombinant E. coli cell of embodiment 39, wherein the gluconate dehydratase comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ I D NO: 1.43. The recombinant E. coli cell of embodiment 39, wherein the gluconate dehydratase comprises the amino acid sequence of SEQ ID NO:1.44. The recombinant E. coli cell of any one of embodiments 36 to 43, which comprises a nucleotide sequence encoding a KDG kinase (EC 2.7.1.45).45. The recombinant E. coli cell of embodiment 44, wherein the KDG kinase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2.46. The recombinant E. coli cell of embodiment 44, wherein the KDG kinase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.47. The recombinant E. coli cell of embodiment 44, wherein the KDG kinase comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2.48. The recombinant E. coli cell of embodiment 44, wherein the KDG kinase comprises the amino acid sequence of SEQ ID NO:2.49. The recombinant E. coli cell of any one of embodiments 39 to 48, wherein the nucleotide sequence encoding the gluconate dehydratase and / or the nucleotide sequence encoding the KDG kinase are under the control of a constitutive promoter.50. The recombinant E. coli cell of embodiment 49, wherein the promoter is a pTrc promoter in a regulatory sequence comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:351 . The recombinant E. coli cell of any one of embodiments 36 to 50, which comprises a disruption of a gluconate-6-phosphate dehydratase (edd; EC 4.2.1.12) gene.52. The recombinant E. coli cell of embodiment 51 , wherein the disruption of the edd gene is an insertion, deletion, or substitution within nucleotides 1-1966 of SEQ ID NO:4.53. The recombinant E. coli cell of any one of embodiments 36 to 52, in which a native KDG-phosphate aldolase (eda; EC 4.1.3.16) promoter is replaced by a synthetic promoter with higher activity.54. The recombinant E. coli cell of embodiment 53, wherein the native eda promoter is within nucleotides 1967-2002 of SEQ ID NO:4.55. The recombinant E. coli cell of embodiment 53 or embodiment 54, wherein the synthetic promoter is in a regulatory sequence comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:5.56. The recombinant E. coli cell of embodiment 53 or embodiment 54, wherein the synthetic promoter is in a regulatory sequence comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:5.57. The recombinant E. coli cell of embodiment 53 or embodiment 54, wherein the synthetic promoter is in a regulatory sequence comprising the nucleotide sequence of SEQ ID NO:5.58. The recombinant E. coli cell of any one of embodiments 36 to 57, which comprises a disruption of a gluconate kinase (gntK EC 2.7.1.12) gene.59. The recombinant E. coli cell of embodiment 58, wherein the disruption of the gntK gene is an insertion, deletion, or substitution within nucleotides 1490-2155 of SEQ ID NO:6.60. The recombinant E. coli cell of any one of embodiments 36 to 59, which comprises a disruption of an idonate kinase ( dnK', EC 2.7.1.12) gene.61 . The recombinant E. coli cell of embodiment 60, wherein the disruption of the idnK gene is an insertion, deletion, or substitution within nucleotides 1-561 of SEQ ID NO:7.62. The recombinant E. coli cell of any one of embodiments 36 to 61 , which comprises a disruption of an idonate dehydrogenase (idnD EC 1.1.1.264) gene.63. The recombinant E. coli cell of embodiment 62, wherein the disruption of the idnD gene is an insertion, deletion, or substitution within nucleotides 1-1029 of SEQ ID NO:8.64. The recombinant E. coli cell of any one of embodiments 36 to 63, which comprises a disruption of a 5-keto-D-gluconate 5-reductase (idnO', EC 1.1.1.69) gene.65. The recombinant E. coli cell of embodiment 64, wherein the disruption of the idnO gene is an insertion, deletion, or substitution within nucleotides 1-762 of SEQ ID NO:9.66. The recombinant E. coli cell of any one of embodiments 36 to 65, which comprises a disruption of an idonate transporter (idnT, Transporter Classification Database: TCDB 2.A.8.1.2) gene.67. The recombinant E. coli cell of embodiment 66, wherein the disruption of the idnT gene is an insertion, deletion, or substitution within nucleotides 1-1317 of SEQ ID NQ:10.68. The recombinant E. coli cell of any one of embodiments 1 to 67, which is engineered to express one or more heterologous isoprene biosynthesis polypeptides.69. The recombinant E. coli cell of embodiment 68, which is engineered to express two or more heterologous isoprene biosynthesis polypeptides.70. The recombinant E. coli cell of embodiment 68, which is engineered to express three or more heterologous isoprene biosynthesis polypeptides.71 . The recombinant E. coli cell of any one of embodiments 68 to 70, wherein at least one heterologous isoprene biosynthesis polypeptide is an isoprene synthase (ispS; EC 4.2.3.27).72. The recombinant E. coli cell of embodiment 71 , wherein the ispS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:67.73. The recombinant E. coli cell of embodiment 71 , wherein the ispS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:67.74. The recombinant E. coli cell of embodiment 71 , wherein the ispSA comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:67.75. The recombinant E. coli cell of embodiment 71 , wherein the ispS comprises the amino acid sequence of SEQ ID NO:67.76. The recombinant E. coli cell of any one of embodiments 1 to 75, which is engineered to express one or more heterologous geranyl-pyrophosphate (GPP) biosynthesis polypeptides.77. The recombinant E. coli cell of embodiment 76, wherein at least one heterologous GPP biosynthesis polypeptide is a geranyl-pyrophosphate synthase (gppS; EC 2.5.1.1).78. The recombinant E. coli cell of embodiment 77, wherein the gppS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:68.79. The recombinant E. coli cell of embodiment 77, wherein the gppS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:68.80. The recombinant E. coli cell of embodiment 77, wherein the gppS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:68.81 . The recombinant E. coli cell of embodiment 77, wherein the gppS comprises the amino acid sequence of SEQ ID NO:68.82. The recombinant E. coli cell of embodiment 76, wherein at least one heterologous GPP biosynthesis polypeptide is an ispA variant comprising a mutation of a serine to a phenylalanine at the position corresponding to amino acid 80 of SEQ ID NO:11.83. The recombinant E. coli cell of embodiment 82, wherein the ispA variant comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:69, provided the residue corresponding to amino acid 80 of SEQ ID NO:69 is a phenylalanine.84. The recombinant E. coli cell of embodiment 82, wherein the ispA variant comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:69, provided the residue corresponding to amino acid 80 of SEQ ID NO:69 is a phenylalanine.85. The recombinant E. coli cell of embodiment 82, wherein the ispA variant comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:69, provided the residue corresponding to amino acid 80 of SEQ ID NO:69 is a phenylalanine.86. The recombinant E. coli cell of embodiment 82, wherein the ispA variant comprises the amino acid sequence of SEQ ID NO:68.87. The recombinant E. coli cell of any one of embodiments 1 to 86, which is engineered to express one or more heterologous neryl-pyrophosphate (NPP) biosynthesis polypeptides.88. The recombinant E. coli cell of embodiment 87, wherein at least one heterologous NPP biosynthesis polypeptide is a neryl-pyrophosphate synthases (nppS; EC 2.5.1.28),89. The recombinant E. coli cell of embodiment 87, wherein the nppS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOTO.90. The recombinant E. coli cell of embodiment 87, wherein the nppS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOTO.91 . The recombinant E. coli cell of embodiment 87, wherein the nppS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NOTO.92. The recombinant E. coli cell of embodiment 87, wherein the nppS comprises the amino acid sequence of SEQ ID NOTO.93. The recombinant E. coli cell of any one of embodiments 1 to 92, which is engineered to express one or more heterologous limonene biosynthesis polypeptides.94. The recombinant E. coli cell of embodiment 93, which is engineered to express two or more heterologous limonene biosynthesis polypeptides.95. The recombinant E. coli cell of embodiment 93, which is engineered to express three or more heterologous limonene biosynthesis polypeptides.96. The recombinant E. coli cell of any one of embodiments 93 to 95, wherein at least one heterologous limonene biosynthesis polypeptide is a limonene synthase (limS; EC 4.2.3.16 and EC 4.2.3.20).97. The recombinant E. coli cell of embodiment 96, wherein the limS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12.98. The recombinant E. coli cell of embodiment 96, wherein the limS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:12.99. The recombinant E. coli cell of embodiment 96, wherein the limS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:12.100. The recombinant E. coli cell of embodiment 96, wherein the limS comprises the amino acid sequence of SEQ ID NO:12.101. The recombinant E. coli cell of any one of embodiments 1 to 100, which is engineered to express one or more heterologous beta-myrcene biosynthesis polypeptides.102. The recombinant E. coli cell of embodiment 101 , which is engineered to express two or more heterologous beta-myrcene biosynthesis polypeptides.103. The recombinant E. coli cell of embodiment 101 , which is engineered to express three or more heterologous beta-myrcene biosynthesis polypeptides.104. The recombinant E. coli cell of any one of embodiments 101 to 103, wherein at least one heterologous beta-myrcene biosynthesis polypeptide is a myrcene synthase (myrS; EC 4.2.3.15).105. The recombinant E. coli cell of embodiment 104, wherein the myrS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:13.106. The recombinant E. coli cell of embodiment 104, wherein the myrS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:13.107. The recombinant E. coli cell of embodiment 104, wherein the myrS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:13.108. The recombinant E. coli cell of embodiment 104, wherein the myrS comprises the amino acid sequence of SEQ ID NO: 13.109. The recombinant E. coli cell of any one of embodiments 1 to 108, which is engineered to express one or more heterologous nerol biosynthesis polypeptides.110. The recombinant E. coli cell of embodiment 109, which is engineered to express two or more heterologous nerol biosynthesis polypeptides.111. The recombinant E. coli cell of embodiment 109, which is engineered to express three or more heterologous nerol biosynthesis polypeptides.112. The recombinant E. coli cell of any one of embodiments 109 to 111 , wherein at least one heterologous nerol biosynthesis polypeptide is a neryl-pyrophosphate synthase (nerS; EC 3.1.7.13).113. The recombinant E. coli cell of embodiment 112, wherein the nerS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:57.114. The recombinant E. coli cell of embodiment 112, wherein the nerS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:57.115. The recombinant E. co / / cell of embodiment 112, wherein the nerS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:57.116. The recombinant E. coli cell of embodiment 112, wherein the nerS comprises the amino acid sequence of SEQ ID NO:57.117. The recombinant E. coli cell of any one of embodiments 1 to 116, which is engineered to express one or more heterologous geraniol biosynthesis polypeptides.118. The recombinant E. coli cell of embodiment 117, which is engineered to express two or more heterologous geraniol biosynthesis polypeptides.119. The recombinant E. coli cell of embodiment 117, which is engineered to express three or more heterologous geraniol biosynthesis polypeptides.120. The recombinant E. coli cell of any one of embodiments 117 to 119, wherein at least one heterologous geraniol biosynthesis polypeptide is a geraniol synthase (gerS; EC 3.1.7.11).121. The recombinant E. coli cell of embodiment 120, wherein the gerS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:58.122. The recombinant E. coli cell of embodiment 120, wherein the gerS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:58.123. The recombinant E. coli cell of embodiment 120, wherein the gerS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:58.124. The recombinant E. coli cell of embodiment 120, wherein the gerS comprises the amino acid sequence of SEQ ID NO:58.125. The recombinant E. coli cell of any one of embodiments 1 to 124, which is engineered to express one or more heterologous linalool biosynthesis polypeptides.126. The recombinant E. coli cell of embodiment 125, which is engineered to express two or more heterologous linalool biosynthesis polypeptides.127. The recombinant E. coli cell of embodiment 125, which is engineered to express three or more heterologous linalool biosynthesis polypeptides.128. The recombinant E. coli cell of any one of embodiments 125 to 127, wherein at least one heterologous linalool biosynthesis polypeptide is a linalool synthase (linS; EC 4.2.3.25).129. The recombinant E. coli cell of embodiment 128, wherein the linS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:59.130. The recombinant E. coli cell of embodiment 128, wherein the linS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:59.131. The recombinant E. coli cell of embodiment 128, wherein the linS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:59.132. The recombinant E. coli cell of embodiment 128, wherein the linS comprises the amino acid sequence of SEQ ID NO:59.133. The recombinant E. coli cell of any one of embodiments 1 to 132, which is engineered to express one or more heterologous beta-ocimene biosynthesis polypeptides.134. The recombinant E. coli cell of embodiment 133, which is engineered to express two or more heterologous beta-ocimene biosynthesis polypeptides.135. The recombinant E. coli cell of embodiment 133, which is engineered to express three or more heterologous beta-ocimene biosynthesis polypeptides.136. The recombinant E. coli cell of any one of embodiments 133 to 135, wherein at least one heterologous beta-ocimene biosynthesis polypeptide is a beta-ocimene synthase (ociS) (EC 4.2.3.106).137. The recombinant E. coli cell of embodiment 136, wherein the ociS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:60.138. The recombinant E. coli cell of embodiment 136, wherein the ociS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:60.139. The recombinant E. coli cell of embodiment 136, wherein the ociS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:60.140. The recombinant E. coli cell of embodiment 136, wherein the ociS comprises the amino acid sequence of SEQ ID NO:60.141. The recombinant E. coli cell of any one of embodiments 1 to 140, which is engineered to express one or more heterologous 1 ,8-cineole biosynthesis polypeptides.142. The recombinant E. coli cell of embodiment 141 , which is engineered to express two or more heterologous 1 ,8-cineole biosynthesis polypeptides.143. The recombinant E. coli cell of embodiment 141 , which is engineered to express three or more heterologous 1 ,8-cineole biosynthesis polypeptides.144. The recombinant E. coli cell of any one of embodiments 141 to 143, wherein at least one heterologous 1,8-cineole biosynthesis polypeptide is a 1 ,8-cineole synthase (cinS; EC 4.2.3.108).145. The recombinant E. coli cell of embodiment 144, wherein the cinS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:61.146. The recombinant E. coli cell of embodiment 144, wherein the cinS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:61.147. The recombinant E. coli cell of embodiment 144, wherein the cinS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:61.148. The recombinant E. coli cell of embodiment 144, wherein the cinS comprises the amino acid sequence of SEQ ID NO:61.149. The recombinant E. coli cell of any one of embodiments 1 to 148, which is engineered to express one or more heterologous farnesene biosynthesis polypeptides.150. The recombinant E. coli cell of embodiment 149, which is engineered to express two or more heterologous farnesene biosynthesis polypeptides.151. The recombinant E. coli cell of embodiment 149, which is engineered to express three or more heterologous farnesene biosynthesis polypeptides.152. The recombinant E. coli cell of any one of embodiments 149 to 151 , wherein at least one heterologous farnesene biosynthesis polypeptide is an alpha-farnesene synthase (fnsS; EC 4.2.3.46).153. The recombinant E. coli cell of embodiment 152, wherein the fnsS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:62.154. The recombinant E. coli cell of embodiment 152, wherein the fnsS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:62.155. The recombinant E. coli cell of embodiment 152, wherein the fnsS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:62.156. The recombinant E. coli cell of embodiment 152, wherein the fnsS comprises the amino acid sequence of SEQ ID NO:62.157. The recombinant E. coli cell of any one of embodiments 149 to 151 , wherein at least one heterologous farnesene biosynthesis polypeptide is a beta-farnesene synthase (aaFS; EC 4.2.3.47).158. The recombinant E. coli cell of embodiment 157, wherein the aaFS comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:71.159. The recombinant E. coli cell of embodiment 157, wherein the aaFS comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:71.160. The recombinant E. coli cell of embodiment 157, wherein the aaFS comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:71.161. The recombinant E. coli cell of embodiment 157, wherein the aaFS comprises the amino acid sequence of SEQ I D NO:71.162. The recombinant E. coli cell of any one of embodiments 1 to 161 , which is engineered to express one or more heterologous farnesol biosynthesis polypeptides.163. The recombinant E. coli cell of embodiment 162, which is engineered to express two or more heterologous farnesol biosynthesis polypeptides.164. The recombinant E. coli cell of embodiment 162, which is engineered to express three or more heterologous farnesol biosynthesis polypeptides.165. The recombinant E. coli cell of any one of embodiments 162 to 164, wherein at least one heterologous farnesol biosynthesis polypeptide is a farnesol synthase.166. The recombinant E. coli cell of embodiment 165, wherein the farnesol synthase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:63.167. The recombinant E. coli cell of embodiment 165, wherein the farnesol synthase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:63.168. The recombinant E. coli cell of embodiment 165, wherein the farnesol synthase comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:63.169. The recombinant E. coli cell of embodiment 165, wherein the farnesol synthase comprises the amino acid sequence of SEQ ID NO:63.170. The recombinant E. coli cell of any one of embodiments 162 to 169, wherein at least one heterologous farnesol biosynthesis polypeptide is a farnesol / terpene synthase.171. The recombinant E. coli cell of embodiment 170, wherein the farnesol / terpene synthase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:72.172. The recombinant E. coli cell of embodiment 170, wherein the farnesol / terpene synthase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:72.173. The recombinant E. coli cell of embodiment 170, wherein the farnesol / terpene synthase comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:72.174. The recombinant E. coli cell of embodiment 170, wherein the farnesol / terpene synthase comprises the amino acid sequence of SEQ ID NO:72.175. The recombinant E. coli cell of any one of embodiments 68 to 174, wherein one or more (or all) heterologous isoprene and / or isoprenoid biosynthesis polypeptide coding sequences are integrated into the E. coli genome at the lac operon locus.176. The recombinant E. coli cell of embodiment 175, wherein all or part of lacZ (SEQ ID NO:14), lacY (SEQ ID NO:15), and / or lacA (SEQ ID NO:16) are deleted.177. The recombinant E. coli cell of any one of embodiments 1 to 176, wherein the first nucleotide sequence encodes a dxs polypeptide (1).178. The recombinant E. coli cell of any one of embodiments 1 to 177, wherein the dxs polypeptide comprises an amino acid sequence having less than 65% sequence identity to the amino acid sequence of SEQ ID NO: 19.179. The recombinant E. coli cell of embodiment 178, wherein the dxs polypeptide comprises an amino acid sequence having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:19.180. The recombinant E. coli cell of embodiment 178, wherein the dxs polypeptide comprises an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:19.181. The recombinant E. coli cell of embodiment 178, wherein the dxs polypeptide comprises an amino acid sequence having at least 45% sequence identity to the amino acid sequence of SEQ ID NO:19.182. The recombinant E. coli cell of any one of embodiments 1 to 181 , wherein the dxs polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.183. The recombinant E. coli cell of embodiment 182, wherein the dxs comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:17.184. The recombinant E. coli cell of embodiment 182, wherein the dxs comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:17.185. The recombinant E. coli cell of embodiment 182, wherein the dxs comprises the amino acid sequence of SEQ ID NO: 17.186. The recombinant E. coli cell of any one of embodiments 1 to 181 , wherein the dxs polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18.187. The recombinant E. coli cell of embodiment 186, wherein the dxs comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18.188. The recombinant E. coli cell of embodiment 186, wherein the dxs comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:18.189. The recombinant E. coli cell of embodiment 186, wherein the dxs comprises the amino acid sequence of SEQ ID NO: 18.190. The recombinant E. coli cell of any one of embodiments 1 to 181 , wherein the dxs polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:47.191. The recombinant E. coli cell of embodiment 190, wherein the dxs comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:47.192. The recombinant E. coli cell of embodiment 190, wherein the dxs comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:47.193. The recombinant E. coli cell of embodiment 190, wherein the dxs comprises the amino acid sequence of SEQ ID NO:47.194. The recombinant E. coli cell of any one of embodiments 1 to 193, wherein the second nucleotide sequence encodes a dxr polypeptide (2).195. The recombinant E. coli cell of any one of embodiments 1 to 194, wherein the dxr polypeptide comprises an amino acid sequence having less than 60% sequence identity to the amino acid sequence of SEQ ID NQ:20.196. The recombinant E. coli cell of embodiment 195, wherein the dxr polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NQ:20.197. The recombinant E. coli cell of embodiment 195, wherein the dxr polypeptide comprises an amino acid sequence having at least 35% sequence identity to the amino acid sequence of SEQ ID NQ:20.198. The recombinant E. coli cell of embodiment 195, wherein the dxr polypeptide comprises an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:20.199. The recombinant E. coli cell of any one of embodiments 1 to 198, wherein the dxr polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:21.200. The recombinant E. coli cell of embodiment 199, wherein the dxr polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:21.201. The recombinant E. coli cell of embodiment 199, wherein the dxr polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:21.202. The recombinant E. coli cell of embodiment 199, wherein the dxr polypeptide comprises the amino acid sequence of SEQ ID NO:21 .203. The recombinant E. coli cell of any one of embodiments 1 to 202, wherein the dxr polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:48.204. The recombinant E. coli cell of embodiment 203, wherein the dxr polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:48.205. The recombinant E. coli cell of embodiment 203, wherein the dxr polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:48.206. The recombinant E. coli cell of embodiment 203, wherein the dxr polypeptide comprises the amino acid sequence of SEQ ID NO:48.207. The recombinant E. coli cell of any one of embodiments 1 to 206, wherein the third nucleotide sequence encodes an ispD polypeptide (3).208. The recombinant E. coli cell of any one of embodiments 1 to 207, wherein the ispD polypeptide comprises an amino acid sequence having less than 50% sequence identity to the amino acid sequence of SEQ ID NO:22.209. The recombinant E. coli cell of embodiment 208, wherein the ispD polypeptide comprises an amino acid sequence having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:22.210. The recombinant E. coli cell of embodiment 208, wherein the ispD polypeptide comprises an amino acid sequence having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:22.211. The recombinant E. coli cell of embodiment 208, wherein the ispD polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:22.212. The recombinant E. coli cell of any one of embodiments 1 to 211 , wherein the ispD polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23.213. The recombinant E. coli cell of embodiment 212, wherein the ispD polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:23.214. The recombinant E. coli cell of embodiment 212, wherein the ispD polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:23.215. The recombinant E. coli cell of embodiment 212, wherein the ispD polypeptide comprises the amino acid sequence of SEQ ID NO:23.216. The recombinant E co / / cell of any one of embodiments 1 to 211 , wherein the ispD polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:65.217. The recombinant E. coli cell of embodiment 212, wherein the ispD polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:65.218. The recombinant E coli cell of embodiment 212, wherein the ispD polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:65.219. The recombinant E coli cell of embodiment 212, wherein the ispD polypeptide comprises the amino acid sequence of SEQ ID NO:65.220. The recombinant E coli cell of any one of embodiments 1 to 211 , wherein the ispD polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:49.221. The recombinant E coli cell of embodiment 220, wherein the ispD polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:49.222. The recombinant E coli cell of embodiment 220, wherein the ispD polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:49.223. The recombinant E coli cell of embodiment 220, wherein the ispD polypeptide comprises the amino acid sequence of SEQ ID NO:49.224. The recombinant E coli cell of any one of embodiments 1 to 223, wherein the fourth nucleotide sequence encodes an ispE polypeptide (4).225. The recombinant E. coli cell of any one of embodiments 1 to 224, wherein the ispE polypeptide comprises an amino acid sequence having less than 60% sequence identity to the amino acid sequence of SEQ ID NO:24.226. The recombinant E. coli cell of embodiment 225, wherein the ispE polypeptide comprises an amino acid sequence having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:24.227. The recombinant E. coli cell of embodiment 225, wherein the ispE polypeptide comprises an amino acid sequence having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:24.228. The recombinant E. coli cell of embodiment 225, wherein the ispE polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:24.229. The recombinant E. coli cell of any one of embodiments 1 to 228, wherein the ispE polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:25.230. The recombinant E. coli cell of embodiment 229, wherein the ispE polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:25.231. The recombinant E. coli cell of embodiment 229, wherein the ispE polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:25.232. The recombinant E. coli cell of embodiment 229, wherein the ispE polypeptide comprises the amino acid sequence of SEQ ID NO:25.233. The recombinant E. coli cell of any one of embodiments 1 to 228, wherein the ispE polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NQ:50.234. The recombinant E. coli cell of embodiment 233, wherein the ispE polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:50.235. The recombinant E. coli cell of embodiment 233, wherein the ispE polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:50.236. The recombinant E. coli cell of embodiment 233, wherein the ispE polypeptide comprises the amino acid sequence of SEQ ID NQ:50.237. The recombinant E. coli cell of any one of embodiments 1 to 236, wherein the fifth nucleotide sequence encodes an ispF polypeptide (5).238. The recombinant E. coli cell of any one of embodiments 1 to 237, wherein the ispF polypeptide comprises an amino acid sequence having less than 75% sequence identity to the amino acid sequence of SEQ ID NO:26.239. The recombinant E. coli cell of embodiment 238, wherein the ispF polypeptide comprises an amino acid sequence having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:26.240. The recombinant E. coli cell of embodiment 238, wherein the ispF polypeptide comprises an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:26.241. The recombinant E. coli cell of embodiment 238, wherein the ispF polypeptide comprises an amino acid sequence having at least 45% sequence identity to the amino acid sequence of SEQ ID NO:26.242. The recombinant E. coli cell of any one of embodiments 1 to 241 , wherein the ispF polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:23.243. The recombinant E. coli cell of embodiment 242, wherein the ispF polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:23.244. The recombinant E. coli cell of embodiment 242, wherein the ispF polypeptide comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO:23.245. The recombinant E. coli cell of embodiment 242, wherein the ispF polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO:23.246. The recombinant E. coli cell of any one of embodiments 1 to 241 , wherein the ispF polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:51.247. The recombinant E. coli cell of embodiment 246, wherein the ispF polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:51.248. The recombinant E. coli cell of embodiment 246, wherein the ispF polypeptide comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO:51.249. The recombinant E. coli cell of embodiment 246, wherein the ispF polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO:51.250. The recombinant E. coli cell of any one of embodiments 1 to 249, wherein the sixth nucleotide sequence encodes an ispG polypeptide (6).251. The recombinant E. coli cell of any one of embodiments 1 to 250, wherein the ispG polypeptide comprises an amino acid sequence having less than 100% sequence identity to the amino acid sequence of SEQ ID NO:27.252. The recombinant E. coli cell of embodiment 251 , wherein the ispG polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:27.253. The recombinant E. coli cell of embodiment 251 , wherein the ispG polypeptide comprises an amino acid sequence having at least 35% sequence identity to the amino acid sequence of SEQ ID NO:27.254. The recombinant E. coli cell of embodiment 251 , wherein the ispG polypeptide comprises an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:27.255. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28.256. The recombinant E. coli cell of embodiment 255, wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:28.257. The recombinant E. coli cell of embodiment 255, wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:28.258. The recombinant E. coli cell of embodiment 255, wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NO:28.259. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29.260. The recombinant E. coli cell of embodiment 259, wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:29.261. The recombinant E. coli cell of embodiment 259, wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:29.262. The recombinant E. coli cell of embodiment 259, wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NO:29.263. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:30.264. The recombinant E. coli cell of embodiment 263, wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:30.265. The recombinant E. coli cell of embodiment 263, wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:30.266. The recombinant E. coli cell of embodiment 263, wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NQ:30.267. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:31.268. The recombinant E. coli cell of embodiment 267, wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:31.269. The recombinant E. coli cell of embodiment 267, wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:31.270. The recombinant E. coli cell of embodiment 267, wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NO:31 .271. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:52.272. The recombinant E. coli cell of embodiment 271 , wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:52.273. The recombinant E. coli cell of embodiment 271 , wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:52.274. The recombinant E. coli cell of embodiment 271 , wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NO:52.275. The recombinant E. coli cell of any one of embodiments 1 to 254, wherein the ispG polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:73.276. The recombinant E. coli cell of embodiment 275, wherein the ispG polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:73.277. The recombinant E. coli cell of embodiment 275, wherein the ispG polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:73.278. The recombinant E. coli cell of embodiment 275, wherein the ispG polypeptide comprises the amino acid sequence of SEQ ID NO:73.279. The recombinant E. coli cell of any one of embodiments 250 to 278, wherein the ispG polypeptide comprises one, any combination of two or more, or all of: (i) anarginine at the position corresponding to R14 of SEQ ID NO:27; (ii) an aspartic acid at the position corresponding D24 of SEQ ID NO:27; (iii) a glycine at the position corresponding to G25 of SEQ ID NO:27; (iv) a cysteine at the position corresponding to C124 of SEQ ID NO:27; (v) an asparagine at the position corresponding to N129 of SEQ ID NO:27; (vi) a glutamine at the position corresponding to Q175 of SEQ ID NO:27; (vii) a serine at the position corresponding to S191 of SEQ ID NO:27; (viii) an alanine at the position corresponding to A213 of SEQ ID NO:27; (ix) an arginine at the position corresponding to R364 of SEQ ID NO:27; and (x) an isoleucine at the position corresponding to I365 of SEQ ID NO:27.280. The recombinant E. coli cell of embodiment 279, wherein the ispG polypeptide comprises an aspartic acid at the position corresponding D24 of SEQ ID NO:27 and a glycine at the position corresponding to G25 of SEQ ID NO:27, and optionally one, any combination of one or more, or all of: an arginine at the position corresponding to R14 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N 129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; a serine at the position corresponding to S191 of SEQ ID NO:27; an alanine at the position corresponding to A213 of SEQ ID NO:27; an arginine at the position corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to I365 of SEQ ID NO:27.281. The recombinant E. coli cell of embodiment 279 or embodiment 280, wherein the ispG polypeptide comprises comprise an arginine at the position corresponding to R364 of SEQ ID NO:27 and an isoleucine at the position corresponding to I365 of SEQ ID NO:27, an optionally one, any combination of two or more, or all of an arginine at the position corresponding to R14 of SEQ ID NO:27; an aspartic acid at the position corresponding D24 of SEQ ID NO:27; a glycine at the position corresponding to G25 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; a serine at the position corresponding to S191 of SEQ ID NO:27; an alanine at the position corresponding to A213 of SEQ ID NO:27; an arginine at the position corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to I365 of SEQ ID NO:27.282. The recombinant E. coli cell of any one of embodiments 279 to 281 , wherein the ispG polypeptide comprises an arginine at the position corresponding to R14 of SEQ ID NO:27; an aspartic acid at the position corresponding D24 of SEQ ID NO:27; a glycine at the position corresponding to G25 of SEQ ID NO:27; a cysteine at the position corresponding to C124 of SEQ ID NO:27; an asparagine at the position corresponding to N129 of SEQ ID NO:27; a glutamine at the position corresponding to Q175 of SEQ ID NO:27; an arginine at the position corresponding to R364 of SEQ ID NO:27; and an isoleucine at the position corresponding to I365 of SEQ ID NO:27, and optionally one or both of a serine at the position corresponding to S191 of SEQ ID NO:27 and an alanine at the position corresponding to A213 of SEQ ID NO:27.283. The recombinant E. coli cell of any one of embodiments 1 to 282, wherein the seventh nucleotide sequence encodes an ispH polypeptide (7).284. The recombinant E. coli cell of any one of embodiments 1 to 283, wherein the ispH polypeptide comprises an amino acid sequence having less than 100% sequence identity to the amino acid sequence of SEQ ID NO:32.285. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 10% sequence identity to the amino acid sequence of SEQ ID NO:32.286. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 15% sequence identity to the amino acid sequence of SEQ ID NO:32.287. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:32.288. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:32.289. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO:32.290. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence of SEQ ID NO:32.291. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:32.292. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:32.293. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:32.294. The recombinant E. coli cell of embodiment 284, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32.295. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:33.296. The recombinant E. coli cell of embodiment 295, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:33.297. The recombinant E. coli cell of embodiment 295, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:33.298. The recombinant E. coli cell of embodiment 295, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:33.299. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:34.300. The recombinant E. coli cell of embodiment 299, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:34.301. The recombinant E. coli cell of embodiment 299, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:34.302. The recombinant E. coli cell of embodiment 299, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:34.303. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:35.304. The recombinant E. coli cell of embodiment 303, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:35.305. The recombinant E. coli cell of embodiment 303, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:35.306. The recombinant E. coli cell of embodiment 303, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:35.307. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:36.308. The recombinant E. coli cell of embodiment 307, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:36.309. The recombinant E. coli cell of embodiment 307, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:36.310. The recombinant E. coli cell of embodiment 307, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:36.311. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:37.312. The recombinant E. coli cell of embodiment 311 , wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:37.313. The recombinant E. coli cell of embodiment 311 , wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:37.314. The recombinant E coli cell of embodiment 311 , wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:37.315. The recombinant E coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:38.316. The recombinant E. coli cell of embodiment 315, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:38.317. The recombinant E. coli cell of embodiment 315, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:38.318. The recombinant E. coli cell of embodiment 315, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:38.319. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:39.320. The recombinant E. coli cell of embodiment 319, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:39.321. The recombinant E. coli cell of embodiment 319, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:39.322. The recombinant E. coli cell of embodiment 319, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:39.323. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:40.324. The recombinant E. coli cell of embodiment 323, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:40.325. The recombinant E. coli cell of embodiment 323, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:40.326. The recombinant E. coli cell of embodiment 323, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NQ:40.327. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:41.328. The recombinant E. coli cell of embodiment 327, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:41.329. The recombinant E. coli cell of embodiment 327, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:41.330. The recombinant E. coli cell of embodiment 327, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:41.331. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:42.332. The recombinant E. coli cell of embodiment 331 , wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:42.333. The recombinant E. coli cell of embodiment 331 , wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:42.334. The recombinant E. coli cell of embodiment 331 , wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:42.335. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:43.336. The recombinant E. coli cell of embodiment 335, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:43.337. The recombinant E. coli cell of embodiment 335, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:43.338. The recombinant E. coli cell of embodiment 335, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:43.339. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:53.340. The recombinant E. coli cell of embodiment 339, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:53.341. The recombinant E. coli cell of embodiment 339, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:53.342. The recombinant E. coli cell of embodiment 339, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:53.343. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:54.344. The recombinant E. coli cell of embodiment 343, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:54.345. The recombinant E. coli cell of embodiment 343, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:54.346. The recombinant E. coli cell of embodiment 343, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:54.347. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:55.348. The recombinant E. coli cell of embodiment 347, wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:55.349. The recombinant E. coli cell of embodiment 347, wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:55.350. The recombinant E. coli cell of embodiment 347, wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:55.351. The recombinant E. coli cell of any one of embodiments 1 to 294, wherein the ispH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:74.352. The recombinant E. coli cell of embodiment 351 , wherein the ispH polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:74.353. The recombinant E. coli cell of embodiment 351 , wherein the ispH polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:74.354. The recombinant E. coli cell of embodiment 351 , wherein the ispH polypeptide comprises the amino acid sequence of SEQ ID NO:74.355. The recombinant E. coli cell of any one of embodiments 1 to 354, wherein the eighth nucleotide sequence encodes an idi polypeptide (8).356. The recombinant E. coli cell of any one of embodiments 1 to 355, wherein the idi polypeptide comprises an amino acid sequence having less than 40% sequence identity to the amino acid sequence of SEQ ID NO:44.357. The recombinant E. coli cell of embodiment 356, wherein the idi polypeptide comprises an amino acid sequence having at least 15% sequence identity to the amino acid sequence of SEQ ID NO:44.358. The recombinant E. coli cell of embodiment 356, wherein the idi polypeptide comprises an amino acid sequence having at least 20% sequence identity to the amino acid sequence of SEQ ID NO:44.359. The recombinant E. coli cell of embodiment 356, wherein the idi polypeptide comprises an amino acid sequence having at least 25% sequence identity to the amino acid sequence of SEQ ID NO:44.360. The recombinant E. coli cell of embodiment 356, wherein the idi polypeptide comprises an amino acid sequence having at least 30% sequence identity to the amino acid sequence of SEQ ID NO:44.361. The recombinant E. coli cell of any one of embodiments 1 to 360, wherein the idi polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:45.362. The recombinant E. coli cell of embodiment 361 , wherein the idi polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:45.363. The recombinant E. coli cell of embodiment 361 , wherein the idi polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:45.364. The recombinant E. coli cell of embodiment 361 , wherein the idi polypeptide comprises the amino acid sequence of SEQ ID NO:45.365. The recombinant E. coli cell of any one of embodiments 1 to 359, wherein the idi polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:46.366. The recombinant E. coli cell of embodiment 365, wherein the idi polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:46.367. The recombinant E. coli cell of embodiment 365, wherein the idi polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:46.368. The recombinant E. coli cell of embodiment 365, wherein the idi polypeptide comprises the amino acid sequence of SEQ ID NO:46.369. The recombinant E. coli cell of any one of embodiments 1 to 359, wherein the idi polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:56.370. The recombinant E. coli cell of embodiment 369, wherein the idi polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:56.371. The recombinant E. coli cell of embodiment 369, wherein the idi polypeptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:56.372. The recombinant E. coli cell of embodiment 369, wherein the idi polypeptide comprises the amino acid sequence of SEQ ID NO:56.373. The recombinant E. coli cell of any one of embodiments 1 to 372, wherein the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from at least two different organisms.374. The recombinant E. coli cell of any one of embodiments 1 to 373, wherein the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from at least three different organisms.375. The recombinant E. coli cell of any one of embodiments 1 to 374, wherein the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from at least four different organisms.376. The recombinant E. coli cell of any one of embodiments 1 to 375, wherein at least one of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.377. The recombinant E. coli cell of any one of embodiments 1 to 376, wherein at least two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.378. The recombinant E. coli cell of any one of embodiments 1 to 377, wherein at least three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.379. The recombinant E. coli cell of any one of embodiments 1 to 378, wherein at least four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.380. The recombinant E. coli cell of any one of embodiments 1 to 379, wherein no more than two of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.381. The recombinant E. coli cell of any one of embodiments 1 to 380, wherein no more than three of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.382. The recombinant E. coli cell of any one of embodiments 1 to 381 , wherein no more than four of the first, second, third, fourth, fifth, sixth, seventh and eighth coding sequences are from E. coli.383. The recombinant E. coli cell of any one of embodiments 1 to 382, wherein the first nucleotide sequence encodes a dxs2 polypeptide (1) comprising the amino acid sequence of SEQ ID NO: 18; and the second nucleotide sequence encodes a dxr polypeptide (2) comprising the amino acid sequence of SEQ ID NO:21 .384. The recombinant E. coli cell of embodiment 383, comprising an expression cassette comprising in order a promoter, the second nucleotide sequence, and the first nucleotide sequence.385. The recombinant E. coli cell of embodiment 383 or embodiment 384, wherein the promoter, the first nucleotide sequence, and the second nucleotide sequence are integrated in the cell’s genome downstream of the gluconate repressor gene gntR.386. The recombinant E. coli cell of any one of embodiments 383 to 385, wherein the first nucleotide sequence and the second nucleotide sequence are under the control of a synthetic T5 gluconate-inducible promoter.387. The recombinant E. coli cell of any one of embodiments 383 to 386, wherein the eighth nucleotide sequence encodes an idi 1 polypeptide (8) comprising the amino acid- HO -sequence of SEQ ID NO:45; the third and fifth nucleotide sequences encode an ispDF fusion polypeptide (3 + 5)) comprising the amino acid sequence of SEQ ID NO:23; and the fourth nucleotide sequence encodes an ispE polypeptide (4) comprising the amino acid sequence of SEQ ID NO:25.388. The recombinant E. coli cell of embodiment 387, comprising an expression cassette comprising in order a promoter, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence.389. The recombinant E. coli cell of embodiment 387 or embodiment 388, wherein the promoter, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence are integrated in the cell’s genome at the insHIO locus.390. The recombinant E. coli cell of any one of embodiments 387 to 389, wherein the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence are under the control of a T7 A3 constitutive promoter.391. The recombinant E. coli cell of any one of embodiments 387 to 390, wherein the sixth nucleotide sequence encodes an ispG polypeptide (6) comprising the amino acid sequence of SEQ ID NO:31 ; and the seventh nucleotide sequence encodes an ispH polypeptide (7) comprising the amino acid sequence of SEQ ID NO:42.392. The recombinant E. coli cell of embodiment 391 , comprising an expression cassette comprising in order a promoter, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence.393. The recombinant E. coli cell of embodiment 391 or embodiment 392, wherein the promoter, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence are integrated in the cell’s genome at the insHIO locus.394. The recombinant E. coli cell of any one of embodiments 391 to 393, wherein the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotidesequence, the third and fifth nucleotide sequences, and the fourth nucleotide sequence are under the control of the T7 A3 constitutive promoter.395. The recombinant E. coli cell of any one of embodiments 1 to 394, wherein the third nucleotide sequence and the fifth nucleotide sequence are derived from genes from the same organism.396. The recombinant E. coli cell of embodiment 395, wherein the fourth nucleotide sequence is derived from a gene from the same organism.397. The recombinant E. coli cell of embodiment 396, wherein the organism is Rhodobacter capsulatus.398. The recombinant E. coli cell of any one of embodiments 1 to 397, wherein the sixth nucleotide sequence and the seventh nucleotide sequence are derived from genes from the same organism.399. The recombinant E. coli cell of embodiment 398, wherein the organism is Shewanella oneidensis.400. The recombinant E. coli cell of any one of embodiments 1 to 399, wherein the first nucleotide sequence is derived from a gene from an organism that produces at least twice the amount of pigments as E. coli on an average pigment: biomass weight ratio.401. The recombinant E. coli cell of any one of embodiments 1 to 400, wherein the first nucleotide sequence is derived from a gene from an organism that is photosynthetic.402. The recombinant E. coli cell of any one of embodiments 1 to 401 , wherein the first nucleotide sequence is derived from a gene from an organism that is distantly related to E. coli.403. The recombinant E. coli cell of embodiment 402, wherein the distantly related organism is not a member of order Enterobacterales.404. The recombinant E. coli cell of embodiment 402 or embodiment 403, wherein the distantly related organism is not a member of class Gammaproteobacteria.405. The recombinant E. coli cell of any one of embodiments 402 to 404, wherein the distantly related organism is not a member of phylum Pseudomonadota.406. The recombinant E. coli cell of any one of embodiments 402 to 405, wherein the distantly related organism is not a member of domain Bacteria.407. The recombinant E. coli cell of any one of embodiments 1 to 406, further comprising one or more nucleotide sequences encoding one or more redox polypeptides.408. The recombinant E. coli cell of embodiment 407, wherein the one or more redox polypeptides comprise a ferrodoxin.409. The recombinant E. coli cell of embodiment 408, wherein the ferredoxin comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:75.410. The recombinant E. coli cell of embodiment 408, wherein the ferredoxin comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:75.411. The recombinant E. coli cell of embodiment 408, wherein the ferredoxin comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:75.412. The recombinant E. coli cell of embodiment 408, wherein the ferredoxin comprises the amino acid sequence of SEQ ID NO:75.413. The recombinant E. coli cell of any one of embodiments 407 to 412, wherein the one or more redox polypeptides comprise a flavodoxin.414. The recombinant E. coli cell of any one of embodiments 407 to 413, wherein the one or more redox polypeptides comprise a flavodoxin / ferredoxin-NADP reductase (EC 1.19.1.1 or EC 1.18.1.2).- US -415. The recombinant E. coli cell of embodiment 414, wherein the flavodoxin / ferredoxin-NADP reductase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:82.416. The recombinant E. coli cell of embodiment 414, wherein the flavodoxin / ferredoxin-NADP reductase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:82.417. The recombinant E. coli cell of embodiment 414, wherein the flavodoxin / ferredoxin-NADP reductase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:82.418. The recombinant E. coli cell of embodiment 414, wherein the flavodoxin / ferredoxin-NADP reductase comprises the amino acid sequence of SEQ ID NO:82.419. The recombinant E. coli of any one of embodiments 407 to 418, wherein one or more of the redox polypeptides are compatible with the polypeptide encoded by the sixth nucleotide sequence and / or the polypeptide encoded by the seventh nucleotide sequence.420. The recombinant E. coli cell of any one of embodiments 407 to 419, wherein one or more nucleotide sequences encoding one or more redox polypeptides are derived from an organism in the same genus as the organism from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.421. The recombinant E. coli cell of any one of embodiments 407 to 419, wherein one or more nucleotide sequences encoding one or more redox polypeptides are derived from an organism in the same species as the organism from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.422. The recombinant E. coli cell of any one of embodiments 407 to 419, wherein one or more nucleotide sequences encoding one or more redox polypeptides are derived from the same strain as the organism from which the sixth nucleotide sequence and / or the seventh nucleotide sequence are derived.423. The recombinant E. coli cell of any one of embodiments 407 to 422, wherein one or more of the one or more redox polypeptides cannot complement a native fldA gene.424. The recombinant E. coli cell of any one of embodiments 407 to 423, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, and the ninth nucleotide sequence are derived from genes from organisms in the same genus.425. The recombinant E. coli cell of any one of embodiments 407 to 424, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, and one or more nucleotide sequences encoding one or more redox polypeptides are derived from the same genus.426. The recombinant E. coli cell of any one of embodiments 407 to 424, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, and one or more nucleotide sequences encoding one or more redox polypeptides are derived from the same species.427. The recombinant E. coli cell of any one of embodiments 407 to 424, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, the eighth nucleotide sequence, and one or more nucleotide sequences encoding one or more redox polypeptides are derived from the same strain.428. The recombinant E. coli cell of any one of embodiments 1 to 427, which is not engineered to express a heterologous isoprene synthase (ispS; EC 4.2.3.27).- US -429. The recombinant E. coli cell of any one of embodiments 1 to 428, which is not engineered to express a heterologous pyruvate:flavodoxin oxidoreductase (PFOR; EC:1.2.7.-).430. The recombinant E. coli cell of any one of embodiments 1 to 429, which:(a) has regulated expression levels of the native isc operon;(b) has approximately native levels of production of iron-sulfur cluster polypeptides encoded by isc operon genes;(c) comprises an intact iscR gene;(d) expresses iscR at approximately native levels; or(e) any combination of two, three or all four of (a)-(d).431. The recombinant E. coli cell of any one of embodiments 1 to 430, which has native expression levels of native ryhB and / or which has an intact rhyB gene.432. The recombinant E. coli cell of any one of embodiments 1 to 431 , which:(a) has native expression levels of native aceE and / or which has an intact aceE gene;(b) has native expression levels of native aceF and / or which has an intact aceF gene;(c) has native expression levels of native IpdA and / or which has an intact IpdA gene;(d) any combination of two or all three of (a), (b) and (c).433. The recombinant E. coli cell of any one of embodiments 1 to 432, which has intact pyruvate dehydrogenase complex (PDC) genes and / or which expresses the PDC at approximately native levels.434. The recombinant E. coli cell of any one of embodiments 1 to 433, which lacks:(a) a nucleotide sequence encoding a heterologous acetoacetyl-CoA thiolase polypeptide,(b) a nucleotide sequence encoding a heterologous 3-hydroxy-3- methyl- glutaryl-CoA (HMG-CoA) synthase polypeptide,(c) a nucleotide sequence encoding a heterologous HMG-CoA reductase polypeptide,(d) a nucleotide sequence encoding a heterologous mevalonate kinase polypeptide,(e) a nucleotide sequence encoding a heterologous phosphomevalonate kinase, diphosphomevalonate decarboxylase polypeptide,(f) a nucleotide sequence encoding a heterologous phosphomevalonate decarboxylase polypeptide,(g) a combination of two, three, four, five, or all six of (a) through (f).435. The recombinant E. coli cell of any one of embodiments 1 to 433, which lacks any nucleotide sequences encoding a heterologous mevalonate pathway polypeptide.436. A recombinant E. coli cell comprising:(a) a first nucleotide sequence encoding a dxs polypeptide that is nonnative to E. coli',(b) a second nucleotide sequence encoding a dxr polypeptide that is nonnative to E. coli',(c) a third nucleotide sequence encoding an ispD polypeptide that is nonnative to E. coli',(d) a fourth nucleotide sequence encoding an ispE polypeptide that is non-native to E. coli',(e) a fifth nucleotide sequence encoding an ispF polypeptide that is nonnative to E. coli',(f) a sixth nucleotide sequence encoding an ispG polypeptide that is nonnative to E. coir,(g) a seventh nucleotide sequence encoding an ispH polypeptide that is non-native to E. coir, and(h) an eighth nucleotide sequence encoding an idi polypeptide that is non-native to E. coir, wherein the first nucleotide sequence and / or the second nucleotide sequence are under the control of a first promoter, wherein the first promoter is an inducible promoter; and wherein one or more of the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence is under the control of a second promoter that is different from the first promoter, optionally wherein the second promoter is a constitutive promoter.437. The recombinant E. coli cell of embodiment 436, wherein the sixth nucleotide sequence (encoding the ispG polypeptide) and / or the seventh nucleotide sequence (encoding the ispH polypeptide) are under the control of a third promoter, optionally wherein the third promoter is an inducible promoter.438. The recombinant E. coli cell of embodiment 437, wherein the first promoter and the third promoter are different.439. The recombinant E. coli cell of embodiment 436 or 437, wherein the third promoter is an inducible promoter.440. The recombinant E. coli cell of any one of embodiments 436 to 439, wherein the second promoter is a constitutive promoter.441. The recombinant E. coli cell of any one of embodiments 436 to 440, wherein the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, and the eighth nucleotide sequence are under the control of the second promoter.442. The recombinant E. coli cell of any one of embodiments 436 to 441 , wherein the first promoter is a gluconate-inducible promoter.443. The recombinant E. coli cell of any one of embodiments 436 to 442, wherein the first nucleotide sequence and the second nucleotide sequence are organized into a first expression cassette under the control of the first promoter.444. The recombinant E. coli cell of embodiment 443, wherein the first expression cassette is integrated into the E. coli genome.445. The recombinant E. coli cell of any one of embodiments 436 to 444, wherein at least two of the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence are organized into a second expression cassette under the control of the second promoter.446. The recombinant E. coli cell of embodiment 445, wherein the second expression cassette is integrated into the E. coli genome.447. The recombinant E. coli cell of any one of embodiments 436 to 446, further comprising one or more nucleotide sequences encoding one or more redox polypeptides that are non-native to E. coli, optionally wherein the one or more redox polypeptides are from the same organism as the ispG and / or ispH polypeptides.448. The recombinant E. coli cell of embodiment 447, wherein the one or more redox polypeptides comprise a ferrodoxin or a flavodoxin.449. The recombinant E. coli cell of embodiment 448, wherein the one or more redox polypeptides comprise a flavodoxin / ferredoxin — NADP reductase.450. The recombinant E. coli cell of any one of embodiments 436 to 449, which is engineered to produce 2-keto-3-deoxy-gluconate (KDG) at increased levels relative to a parental strain.451. The recombinant E. coli cell of embodiment 450, which comprises a nucleotide sequence encoding a heterologous KDG kinase.452. The recombinant E. coli cell of embodiment 450 or 451 , in which a native KDG-phosphate aldolase promoter is replaced by a heterologous promoter with higher activity.453. The recombinant E. coli cell of any one of embodiments 436 to 452, which comprises a disruption of a gluconate-6-phosphate dehydratase.454. The recombinant E. coli cell of any one of embodiments 436 to 453, which comprises a disruption of a gluconate kinase.455. The recombinant E. coli cell of any one of embodiments 436 to 454, which is engineered to express one or more heterologous isoprene or isoprenoid biosynthesis polypeptides.456. A method of producing isoprene or an isoprenoid, the method comprising culturing recombinant E. coli cells according to any one of embodiments 1 to 455 under conditions such that the isoprene or isoprenoid is produced.457. The method of embodiment 456, wherein(a) the first promoter is a gluconate-inducible promoter;(b) the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence are under the control of the second promoter, wherein the second promoter is a constitutive promoter.458. The method of embodiment 456 or 457, wherein the culturing comprises:(a) culturing the E. coli cells under growth conditions in which the first promoter is not induced; and subsequently(b) culturing the E. coli cells under conditions in which the first promoter is induced.459. The method of embodiment 458, further comprising inducing expression from the first promoter after step (a).460. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a stationary growth phase.461. The method of embodiment 459, wherein the inducing is done after the E. coli cells exit an exponential growth phase.462. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a cell density of at least about 5x106cells / ml.463. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a cell density of at least about 1x107cells / ml.464. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a biomass of at least about 3 mg / ml of dry cell mass.465. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a biomass of at least about 7 mg / ml of dry cell mass.466. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a biomass of at least about 10 mg / ml of dry cell mass.467. The method of embodiment 459, wherein the inducing is done after the E. coli cells reach a biomass of at least about 14 mg / ml of dry cell mass.468. The method of any one of embodiments 458 to 467, wherein the first promoter is induced by gluconate that is produced by the recombinant E. coli cell.469. The method of any one of embodiments 458 to 468, wherein the seventh nucleotide sequence and the eighth nucleotide sequence are under the control of a thirdpromoter, and wherein the culturing conditions of step (b) further result in induction of the third promoter.470. A method for producing isoprene and / or an isoprenoid, comprising culturing an E. coli cell of any one of embodiments 1 to 455 in a production medium.471. The method of embodiment 470, wherein the production medium comprises glucose.472. The method of embodiment 470 or embodiment 471 , further comprising recovering the isoprene and / or isoprenoid from the production medium.473. The method of any one of embodiments 470 to 472, further comprising recovering the isoprene and / or isoprenoid from the head space above the production medium.7. EXAMPLES7.1. Example 1. Complementation studies in E. coli DXP pathway deletant strains7.1.1. Materials and methods

[0307] Six E. coli strains were engineered in which the activity of one DXP pathway polypeptide ((1), (2), (4), (6), or (7)), or two DXP pathway polypeptides ((3) and (5)) was knocked out. The resulting strains had greatly decreased growth rates. In each of these strains, the ability of heterologous polypeptides to complement the knockout was tested.7.1.2. Results

[0308] Table 1 identifies by step (1 )-(8), gene, enzyme name, EC number, and source organism of the heterologous polypeptides tested.

[0309] In Table 1 : *, amino acid sequences were aligned with E. coli homologs using Clustal Omega 1.2.2; ", identity between homolog 1 and homolog 2 of Rhodobacter polypeptides;***, identity between Rhodobacter IspDF and combined E. coli IspD and IspF; protein length expressed as the number of amino acids. Numbers in parentheses are the length of E. coli homologs.

[0310] Surprisingly, heterologous polypeptides with sequence identity to E. coli homologs as low as 22.4% were found to complement E. coli knockout strains.7.2. Example 2. Isoprenoid production studies in E. coli expressing heterologous DXP pathway polypeptides7.2.1. Materials and Methods

[0311] Three E. coli strains were engineered to assess isoprenoid production using lycopene as a reporter. First, a base strain SA60 was engineered to possess the following characteristics:

[0312] 1) Overexpression of Achromobacter sp. gluconate dehydratase (Achrom DHT) and KDG kinase (KdgK) using a synthetic constitutive promoter. The cassette Promoter + Achrom DHT was integrated in the chromosome, at the kdgK locus, creating a Achrom DHT- kdgK operon, where both genes are expressed from the same synthetic constitutive promoter derived from the promoter known as PTrc.

[0313] 2) Knockout of Gluconate-6-phosphate dehydratase (edd), and deregulation of KDG- phosphate aldolase by modifying its regulatory sequence. All changes were made in the chromosome, at the edd-eda locus. The promoter driving eda overexpression is synthetic.

[0314] 3) Reduction of other relevant competing reactions by knocking out the following activities / genes: Enzymes known to phosphorylate Gluconate: gluconate kinase gntK, and idonate kinase idnK. The idonate pathway is known to participate in gluconate degradation in E. coli. Because of this, three other genes of the idonate pathway were also knocked out: idnD, idnO, and idnT.

[0315] 4) The three genes ctrE, Ctrl, and ctrB of the lycopene biosynthetic pathway from Erwinia uredovora were synthesized and inserted into the chromosome at the Lac operon locus. In this process, the three E. coli genes of the lac operon, lacZ, lacY, and lacA weredeleted, and expression of the lycopene genes was put under control of the native lac promoter, inducible by IPTG.

[0316] From base strain SA60, three test strains were further engineered:

[0317] 1) Strain SA160: Regulated overexpression of Dxs2 and Dxr (reactions 1 and 2 in Fig. 1) from the bacterium Rhodobacter capsulatus SB1003, by using a synthetic T5- gluconate-regulated promoter. A native anti-termination sequence, gt10 sequence, and an optimized RBS were engineered between the promoter region and the start codon of dxs2. The cassette Promoter-dxs2-dxrwas integrated in the chromosome, downstream of the gluconate repressor gene gntR. A CRP binding site downstream of the gntR gene was swapped with a terminator from Bacillus subtilis to block readthrough from zwf transcription.

[0318] 2) Strain SA172: Regulated overexpression of Dxs2 and Dxr as described above, plus constitutive overexpression of R. capsulatus SB1003 Idi1, R. capsulatus SB1003 ispDF, and R. capsulatus SB1003 IspE. Constitutive overexpression was achieved using a T7 promoter known as A3. A native anti-termination sequence, gt10 sequence, and an optimized RBS were engineered between the promoter region and the start codon of idi1. The cassette T7A3p-idi1-ispDF-ispE was integrated at the insHIO locus, replacing the yhcE gene and the whole insHIO insertion element.

[0319] 3) Strain SA179: Regulated overexpression of Dxs2 and Dxr as described at (1), plus constitutive overexpression of the ispG and ispH genes from the bacterium Shewanella oneidensis MR-1 , as well as idi1-ispDF-ispE genes from R. capsulatus SB1003 from the T7A3 promoter. A native anti-termination sequence, gt10 sequence, and an optimized RBS were engineered between the promoter region and the start codon of ispG. The heterologous 5-gene operon, T7A3p-ispG-ispH-idi1-ispDF-ispE, was integrated at the insHIO locus, replacing the yhcE gene and the whole insHIO insertion element. This strain has a fully functional heterologous DXP pathway.7.2.2. Results

[0320] All three test strains, SA160, SA172, and SA179, produced lycopene.7.3. Example 3. Conserved sequences of ispGs complementing E. coli AispG strain7.3.1. Materials and Methods

[0321] As described in Example 1 , the ability of 13 ispG heterologous genes (see Table 1), to complement the deletion of the native-ispG E. coli gene was tested. It was found that only four were able to complement: Shewanella oneidensis MR-1 , Serratia marcescens ATCC13880, Pantoea ananatis PA13, and Proteus mirabilis HI4320.

[0322] To identify possible protein motifs common in the sequences that were able to complement, the amino acid sequences of the 13 ispG heterologous proteins were compared with the E. coli ispG protein sequence using Geneious Prime software (Boston, MA).7.3.2. Results

[0323] The alignment generated is shown in FIG. 2. From this alignment, residues that were common between E. coli and the complementing heterologous genes, and different in the non-complementing sequences, were identified. The following amino acid residues were found to be present only in all four complementing sequences and in the E. coli sequence (numbering based on the E. coli sequence, SEQ ID NO:27): R14, D24, G25, C124, N129, Q175, S191 , A213, R364, and I365.

[0324] The conservation of these ten residues in all four complementing sequences suggested that these residues may play an important role on the interaction of ispG with: (a) itself to form dimers, trimers, tetramers, etc.; (b) other isp protein(s) of the DXP pathway; (c) proteins involved in the formation of the iron-sulfur clusters in ispG; and / or (d) proteins involved in the oxidation-reduction reaction necessary for ispG’s enzymatic function.

[0325] The 3D structure of E. coli ispG has been predicted (UniProt Accession No. P62620), and it was used to map the positions of the ten conserved residues mentioned above (FIG. 3A-FIG. 3D). FIGS. 3A-3D show the 3D structure of E. coli ispG, the location of the ten conserved residues, and other relevant features. Eight of the conserved residues (R14, D24, G25, C124, N129, Q175, R364 and I365) are located in a discrete region of the protein, shown in closeup in FIG. 3B and FIG. 3D. Residues A213 and S191 are located on the opposite side of the protein. Without being bound by theory, the inventors believe that the region of the protein containing R14, D24, G25, C124, N129, Q175, R364 and I365 interactswith oxidoreductive proteins, such as flavodoxins, which are reduced in the conversion of MEcPP (M) to HMBPP (N).7.4. Example 4. Coexpression of R. capsulatus ispG, ispH, FdxA and / or Fpr in E. coli7.4.1. Materials and Methods

[0326] Two E. coli strains were engineered in which the activity of IspG or IspH polypeptide was knocked out. The resulting strains had no growth phenotype. In each of these strains, the ability of R. capsulatus IspG or IspH polypeptides to complement the knockout was tested. Specifically, the IspG and IspH polypeptides were tested in the presence and absence of R. capsulatus ferredoxin (FdxA) activity, alone or with R. capsulatus Fpr activity (provided using a plasmid).

[0327] Table 2. Plasmids and corresponding SEQ ID NOs7.4.2. Results

[0328] Complementation results demonstrate that R. capsulatus FdxA enzyme alone is sufficient for functional activity of both R. capsulatus IspG and IspH in E. coli. These results also suggest that the FdxA enzyme can functionally interact with native E. coli Fpr while R. capsulatus Fpr may show improved functional interaction with FdxA.

[0329] Table 3. Redox polypeptides tested and observed complementation by RcispG or RcispH of AEcispG strain or AEcispH stain, respectively7.5. Example 5: Effect of overexpression of R. capsulatus IspG and IsH enzymes along with their compatible reducing partners on growth and lycopene production.7.5.1. Materials and Methods

[0330] E. coli strain SA172 comprises a heterologous DXP pathway in which expression of enzymes Dxs and Dxr (both derived from R. capsulatus) is regulated by a gluconate- inducible promoter. To enhance overexpression of IPTG-regulated recombinant genes in E. coli strain SA172, the E. coli MG1655 native lactose / IPTG permease gene, lacY, and its upstream region containing lacY promoter and RBS was integrated downstream of crtEIB operon using the CRISPR / MAD7-associated Lambda-RED recombineering, to yield strain E. coli SA 194.

[0331] Recombinant genes encoding enzyme homolog sets (IspG, IspH, FdxA / FIdA) or (IspG, IspH, FdxA / FIdA, Fpr) derived from E. coli MG1655 or R. capsulatus SB1003 were assembled to single operons and separately cloned downstream of an IPTG-inducible TAC promoter on a low-copy plasmid, yielding four separate plasmids. Strain SA194 was then transformed with the resultant plasmids to yield four study strains.

[0332] Plasmids and corresponding sequence identifiers are set forth in Table 4 below.

[0333] Cell cultures were started by inoculating 5 mL defined media (3 g / L potassium phosphate dibasic; 2 g / L ammonium chloride; 0.2 g / L magnesium chloride hexahydrate; 1.5 g / L potassium sulfate; 1 g / L sodium citrate dihydrate; 1.92 g / L Drop-out Mix Synthetic Minus Uracil w / o Yeast Nitrogen Base, US Biological Life Sciences, Catalog No. D9535; 1X trace metals, Teknova, Catalog No. T1001 ; 1X Thomas Vitamins, Teknova, Catalog No. 2T1200; pH=7.0) comprising 0.5% (wt / vol) glycerol and 200 pg / mL spectinomycin (selective pressure for plasmid) in 50 mL gas-permeable tubes with the six study strains prepared from overnight seed cultures in the same media. After 5 h following inoculation, at which OD600 of all six strains are similar, cell cultures were spiked with IPTG (1 mM final concentration) to induce the expression of recombinant genes on plasmid. Concurrently, cell cultures were spiked with sodium gluconate (0.5% wt / vol final concentration) to induce expression of RcDxs and RcDxr of the heterologous DXP pathway. Cells were harvested after 24 h (29 h total fermentation run time), when all carbon sources were fully consumed, for OD600 measurement and lycopene quantification.

[0334] Specific lycopene levels were assessed by measuring lycopene concentration (wt / vol) and normalizing by OD600 at the given time.

[0335] Generally, lycopene is bound to cellular membranes. To measure lycopene concentration, briefly, cells harvested at the end of the fermentation were incubated in acetone at 55°C for 15 min to extract intracellular lycopene from heat-lysed cells in acetone. Lycopene level in acetone was then quantified by measuring OD475nm in a 284 QS 10mm quartz cuvette by a spectrophotometer. OD475nm values were then converted to wt / vol using a calibration curve.7.5.2. Results

[0336] Relative final OD600 and specific lycopene levels are shown in Table 5. Reported numbers are calculated from the mean of three biological replicates for each study strain and Fold Change (FC) values are calculated relative to values obtained from the E. coli EcispG-EcispH-EcfldA study strain (BSCPTS370). The results suggest that the enzyme sets (IspG, IspH, Fdx) and (IspG, IspH, Fdx, Fpr) from R. capsulatus SB1003 produced more lycopene than their counterparts from E. coli MG1655 (native) and S. oneidensis MR-1 and did not impair cell growth.

[0337] Fold Change in OD600 and specific lycopene relative to E. coli strain BSCPTS370 is shown in Table 5 below:8. SEQUENCES

[0338] Exemplary sequences referred to herein are provided in Table 6 below (where “SEQ” refers to the SEQ ID NO).9. INCORPORATION BY REFERENCE

[0339] 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.

[0340] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed anywhere before the priority date of this application.

Claims

WHAT IS CLAIMED IS:

1. A recombinant E. coli cell comprising:(a) a first nucleotide sequence encoding a dxs polypeptide that is non-native to E. coli',(b) a second nucleotide sequence encoding a dxr polypeptide that is non-native to E. coli',(c) a third nucleotide sequence encoding an ispD polypeptide that is non-native to E. coli',(d) a fourth nucleotide sequence encoding an ispE polypeptide that is non-native to E. coli',(e) a fifth nucleotide sequence encoding an ispF polypeptide that is non-native to E. coli;(f) a sixth nucleotide sequence encoding an ispG polypeptide that is non-native to E. coli;(g) a seventh nucleotide sequence encoding an ispH polypeptide that is non-native to E. coli; and(h) an eighth nucleotide sequence encoding an idi polypeptide that is non-native to E. coli; wherein the first nucleotide sequence and / or the second nucleotide sequence are under the control of a first promoter, wherein the first promoter is an inducible promoter; and wherein one or more of the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence is under the control of a second promoter that is different from the first promoter, optionally wherein the second promoter is a constitutive promoter.

2. The recombinant E. coli cell of claim 1 , wherein the sixth nucleotide sequence (encoding the ispG polypeptide) and / or the seventh nucleotide sequence (encoding the ispH polypeptide) are under the control of a third promoter, optionally wherein the third promoter is an inducible promoter.

3. The recombinant E. coli cell of claim 2, wherein the first promoter and the third promoter are different.

4. The recombinant E. coli cell of claim 2 or 3, wherein the third promoter is an inducible promoter.

5. The recombinant E. coli cell of any one of claims 1 to 4, wherein the second promoter is a constitutive promoter.

6. The recombinant E. coli cell of any one of claims 1 to 5, wherein the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, and the eighth nucleotide sequence are under the control of the second promoter.

7. The recombinant E. coli cell of any one of claims 1 to 6, wherein the first promoter is a gluconate-inducible promoter.

8. The recombinant E. coli cell of any one of claims 1 to 7, wherein the first nucleotide sequence and the second nucleotide sequence are organized into a first expression cassette under the control of the first promoter.

9. The recombinant E. coli cell of claim 8, wherein the first expression cassette is integrated into the E. coli genome.

10. The recombinant E. coli cell of any one of claims 1 to 9, wherein at least two of the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence are organized into a second expression cassette under the control of the second promoter.

11. The recombinant E. coli cell of claim 10, wherein the second expression cassette is integrated into the E. coli genome.

12. The recombinant E. coli cell of any one of claims 1 to 11 , further comprising one or more nucleotide sequences encoding one or more redox polypeptides that are non-native to E. coli, optionally wherein the one or more redox polypeptides are from the same organism as the ispG and / or ispH polypeptides.

13. The recombinant E. coli cell of claim 12, wherein the one or more redox polypeptides comprise a ferrodoxin or a flavodoxin.

14. The recombinant E. coli cell of claim 13, wherein the one or more redox polypeptides comprise a flavodoxin / ferredoxin — NADP reductase.

15. The recombinant E. coli cell of any one of claims 1 to 14, which is engineered to produce 2-keto-3-deoxy-gluconate (KDG) at increased levels relative to a parental strain.

16. The recombinant E. coli cell of claim 15, which comprises a nucleotide sequence encoding a heterologous KDG kinase.

17. The recombinant E. coli cell of claim 15, in which a native KDG-phosphate aldolase promoter is replaced by a heterologous promoter with higher activity.

18. The recombinant E. coli cell of any one of claims 1 to 17, which comprises a disruption of a gluconate-6-phosphate dehydratase.

19. The recombinant E. coli cell of any one of claims 1 to 18, which comprises a disruption of a gluconate kinase.

20. The recombinant E. coli cell of any one of claims 1 to 19, which is engineered to express one or more heterologous isoprene or isoprenoid biosynthesis polypeptides.

21. A method of producing isoprene or an isoprenoid, the method comprising culturing recombinant E. coli cells according to claim 20 under conditions such that the isoprene or isoprenoid is produced.

22. The method of claim 21 , wherein(a) the first promoter is a gluconate-inducible promoter;(b) the third nucleotide sequence, the fourth nucleotide sequence, the fifth nucleotide sequence, the sixth nucleotide sequence, the seventh nucleotide sequence, and the eighth nucleotide sequence are under the control of the second promoter, wherein the second promoter is a constitutive promoter.

23. The method of claim 21 or 22, wherein the culturing comprises:(a) culturing the E. coli cells under growth conditions in which the first promoter is not induced; and subsequently(b) culturing the E. coli cells under conditions in which the first promoter is induced.

24. The method of claim 23, wherein the first promoter is induced by gluconate that is produced by the recombinant E. coli cell.

25. The method of claim 23 or 24, wherein the seventh nucleotide sequence and the eighth nucleotide sequence are under the control of a third promoter, and wherein the culturing conditions of step (b) further result in induction of the third promoter.

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