Gluconate-regulated expression systems

Abstract

Disclosed are expression systems comprising sequences of interest operably linked to regulatory elements inducible by gluconate. Nucleic acids comprising the expression systems can be incorporated into recombinant microorganisms. The recombinant microorganisms can be used in methods for expressing sequences of interest and in methods for producing products by pathways comprising catalysis by proteins of interest encoded by sequences of interest.

Description

GLUCONATE-REGULATED EXPRESSION SYSTEMS1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63 / 581 ,791, filed September 11 , 2023, the contents of which are incorporated herein in their entireties by reference thereto.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 September 10, is named BPC-014WO_SL.xml and is 106,573 bytes in size.3. BACKGROUND

[0003] There is a need in the art for controllable methods of producing industrially important products by recombinant microorganisms. The present disclosure addresses this need and provides controllable expression systems for sequences of interest in prokaryotic cells.4. SUMMARY

[0004] To maximize output of expression systems by microorganisms in bioindustrial processes, it can be desirable for cells to grow without expressing sequences of interest until a desired cell biomass has been reached, at which time expression of the sequences of interest can be switched on. After switching on expression, cell biomass can be maintained while the amount of expression products, e.g., proteins of interest encoded by sequences of interest, can increase.

[0005] Switchable expression systems can be provided by operably linking sequences of interest to inducible promoters. Upon introduction of inducers or precursors thereof to media in which cells are growing, inducers can activate expression of sequences of interest. A number of inducible promoters and their inducers are known. In prokaryotes, one such inducer is gluconate, which can induce expression of GntK and GntU in E. coli by binding to the repressor protein GntR and allowing transcription to occur from the GntKU promoter. Gluconate can induce expression of GntK and GntU whether it is provided directly to E. colicells or arises from metabolic processes acting on gluconate precursors, e.g., glucose, which are relatively inexpensive, plentiful, and acted on in well-understood metabolic pathways.

[0006] Though inducible, the GntKU promoter and operator region in E. coli is relatively weak. The -35 sequence and the -10 sequence of the promoter are notably different from consensuses of these sequences. The ribosome binding site (RBS) of the operator region is also notably different from consensus sequences. The spacer between the RBS and the translation initiation codon has several cytosine nucleotides. Also, the translation initiation codon of GntK is TTG, which is uncommon in E. coli.

[0007] The present disclosure addresses the problem of gluconate-inducible but low-level expression of sequences of interest operably linked to the GntKU promoter and operator region by applying synthetic biology to engineer regulatory elements to provide high expression with induction by gluconate.

[0008] Accordingly, the present disclosure provides engineered gluconate-regulated expression systems. The engineered gluconate-regulated expression systems of the disclosure generally have improved expression yields as compared to expression systems utilizing native gluconate regulatory elements, such as the E. coli GntK regulatory element.

[0009] In various embodiments, the gluconate-regulated expression system incorporate modified regulatory elements, e.g., modified promoter elements (such as -35 sequences and / or -10 sequences), modified operator region elements (such as ribosome binding sites (RBSs) and spacers between RBSs and translation initiation codons), and / or modified GntR binding sites. The modified regulatory elements can all be derived from a gluconate- regulated gene, or can be combined with other regulatory elements to render them gluconate-inducible. For instances, promoters such as PH207 from bacteriophage T5 can be combined with GntR binding site(s) to render them gluconate-inducible.

[0010] Additionally or alternatively, when sequences of interest comprise open reading frames (ORFs) encoding proteins of interest, the translation initiation codons of the ORFs can be optimized based on the tRNA pool of the prokaryotic cells in which expression is to occur.

[0011] In various embodiments, gluconate-regulated expression systems incorporate enhancers, such as enhancers positioned upstream (5’-ward) of promoters.

[0012] In various embodiments, gluconate-regulated expression systems comprise only one GntR binding site.

[0013] Examples of nucleic acids comprising expression systems of the disclosure are described in Section 6.2 and numbered embodiments 1 to 155 and 222.

[0014] Nucleic acids comprising expression systems can be engineered into prokaryotic cells by various techniques. Examples of recombinant microorganisms comprising the nucleic acids are described in Section 6.3 and numbered embodiments 156 to 172 and 223.

[0015] Engineered cells comprising nucleic acids comprising expression systems can be used to express sequences of interest. The sequences of interest can be transcribed to yield RNAs of interest or translated to yield proteins of interest. Uses of engineered cells to express sequences of interest are described in Section 6.4.3 and numbered embodiments 173 to 193 and 224.

[0016] In some embodiments wherein sequences of interest are expressed to yield proteins of interest, proteins of interest can be used to produce a product. The production can be in vivo, in cells producing proteins of interest, or in vitro, after production of proteins of interest in cells and purification of the proteins of interest. Production can involve catalysis of substrates to yield products or precursors which can lead to the products by activity of other enzyme(s) and / or chemical catalysis. Use of proteins of interest in production of products are described in 6.4.4 and numbered embodiments 194 to 221 and 225.5. BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1 schematically represents certain features of the GntR regulon in E. coli. FIG. 1A: The regulon contains two GntR binding sites that partially overlap one another and the promoter region. FIG. 1 B: Binding of GntR to the binding sites blocks gene expression. FIG. 1C: Formation of gluconate and GntR complexes reduces binding of GntR to the binding sites, and expression can occur.

[0018] FIG. 2 provides a map and the sequence for the GntK promoter region in E. coli MG1655. In addition to the annotated mismatches with canonical -35 and -10 sequences,the GntK promoter region includes a suboptimal RBS, a relatively large number of cytosine nucleotides, and the uncommon initiation codon TTG (present in only about 3% of E. coli genes). FIG. 2 discloses SEQ ID NOS:3, 8, and 64, respectively, in order of appearance.

[0019] FIG. 3 is a schematic for a recombinant construct comprising an engineered gluconate-regulated promoter. In general, expression systems of the disclosure comprise an engineered gluconate-responsive regulatory element operably linked to a sequence of interest. Typically, the gluconate-responsive regulatory element comprises a promoter (9), comprising a -35 sequence (1) and a -10 sequence (2); an operator region (10), comprising a ribosome binding site (5) and a spacer (6); and two GntR binding sites (3) and (4). The sequence of interest (7) may comprise an open reading frame comprising an initiation codon (8). The components shown in FIG. 3 are not mutually exclusive; for example a GntR binding site (3) may overlap (11) with the -10 sequence (2), as shown by overlapping dotted portions of lines 2 and 3; or the GntR binding sites (3 and 4) may overlap, as shown by overlapping dotted portions of lines 3 and 4. Additionally, while the recombinant constructs depict two GntR binding sites, one of the two binding sites (either of 3 and 4) is optional, e.g., where leaky gluconate regulation is desirable.

[0020] FIG. 4A provides the map of the region of plasmid pBR-GntRK-LacZ 710 containing the modified region of E. coli MG1655, which encodes gntR and lacZ. It also contains the gntR and gntK regulatory regions. Other relevant features like restriction sites, binding sites for regulatory proteins, and location of primers used to amplify and create mutations of the gntK promoter are also shown. The map is not at scale.

[0021] FIG. 4B provides a map and the sequence for an engineered GntK promoter region, with modifications in the -35 sequence (Mut1) and the -10 sequence (Mut2) as compared to the GntK promoter region in E. coli MG1655. Exemplary constructs incorporating the -35 sequence (Mut1), the -10 sequence (Mut2) modifications, or both (Mut12) are described in Example 1. FIG. 4B discloses SEQ ID NO:64.

[0022] FIG. 5 provides a map and the sequence for an engineered GntK promoter region, with modifications of the RBS. Exemplary plasmids featuring the RBS modifications, pBR- GntRK-M1 lacZ, pBR-GntRK-M2 lacZ, and pBR-GntRK-Mut12 lacZ, in which the engineered GntK regulons are operably linked to a lacZ reporter gene, are described in Example 1 . FIG. 5 discloses SEQ ID NOS:65-67, respectively, in order of appearance.

[0023] FIG. 6 presents relative lacZ activity for engineered cells comprising the pBR-GntRK- M1 lacZ, pBR-GntRK-M2 lacZ, and pBR-GntRK-Mut12 lacZ plasmids, as described in Example 1.

[0024] FIG. 7 provides a map and the sequence for an engineered hybrid T5-GntR promoter combining the promoter region of bacteriophage T5 PH2o? promoter with an operator region comprising GntR binding sites. Exemplary plasmids, in which hybrid T5-GntR regulons are operably linked to mScarlet or lacZ reporter genes, are described in Example 3. FIG. 7 discloses SEQ ID NO:68 and SEQ ID NO:69 in order of appearance.

[0025] FIG. 8 provides a map and the sequences for engineered hybrid T5-GntR promoters (PT5-GntR- r, PT5-GntR-5o, Pi5-GntR-4o, PT5-GntR-i 5, and Pi5-GntR-s) for exemplary plasmids in which hybrid T5-GntR regulons are operably linked to mScarlet, as described in Example 2. FIG. 8 discloses SEQ ID NO:69.

[0026] FIG. 9 provides a map of some relevant features of plasmid pT5-GntR-LacZ-50 described in Example 2. The map depicts the engineered hybrid T5-GntR promoter operably linked to lacZ. Other relevant features, e.g., the location of the gntR gene, restriction sites, binding sites for regulatory proteins, and heterologous transcriptional terminators are also shown. The map is not at scale.

[0027] FIG. 10 provides a map and sequence of the hybrid T5-GntR promoter, Prs-GntR- r region operably linked to lacZ, present in pT5-GntR-LacZ-50, as described in Example 2. FIG. 10 discloses SEQ ID NQS:70-72, respectively, in order of appearance.

[0028] FIG. 11 presents relative lacZ activity for engineered cells comprising plasmid pT5- GntR-LacZ-50 in the absence or presence of gluconate inducer, as described in Example 2.

[0029] FIG. 12A depicts the original structure of the GntRKU region present in the chromosome of E. coli strain MG 1655.

[0030] FIG. 12B depicts the organization of the GntRKU region after integrating the Mut2 GntK promoter operably linked to the red fluorescent protein mScarlet, as described in Example 3.

[0031] FIG. 12C depicts the organization of the GntRKU region after integrating the Pys-GntR- wr promoter operably linked to the red fluorescent protein mScarlet, as described in Example 3.

[0032] FIG. 13 is a schematic depicting growth and production phases for production of products by recombinant microorganisms. In the production phase, cells do not divide but have a high capacity to produce desired products. GntR regulons provide switches by which gene expression in the production phase can be induced by gluconate. Engineered GntR regulons can impart greater expression of a sequence of interest than is observed for parental GntR regulons.

[0033] FIG. 14 schematically represents a pathway for the conversion of 2-keto-3-deoxy-D- gluconate (KDG) to 2-keto-3-deoxy-D-gluconate-phosphate (KDGP); KDGP to glyceraldehyde-3-phosphate and pyruvate; and glyceraldehyde-3-phosphate and pyruvate via the DXP pathway and the action of terpene synthases to isoprenoids. One or more of the enzymes catalyzing reactions of the pathway can be encoded by ORFs operably linked to engineered regulons in expression systems of the present disclosure.

[0034] FIG. 15A provides a map of some relevant features of the pT5-GntR-WT promoter studied in Example 4.

[0035] FIG. 15B provides a map of some relevant features of the UP enhancer+pT5-GntR- WT promoter studied in Example 4. The UP element from the upstream region of the PL promoter from lambda bacteriophage was integrated between Heterologous Transcriptional Terminator 1 and the -35 sequence of the PH207 promoter from T5 bacteriophage of the pT5- GntR-WT promoter shown in FIG. 15A to yield UP+pT5-GntR-WT promoter.

[0036] FIG. 16 shows the annotated sequence of UP+pT5-GntR-WT promoter linked with other regulatory elements, as described in Example 4. FIG. 16 discloses SEQ ID NO:73.

[0037] FIG. 17A shows time-course profiles of cell density (OD600) and relative mScarlet level (represented by fluorescence emission at 591 nm) of strains carrying mScarlet expression cassettes operably linked to the pT5-GntR-WT promoter (no enhancer, solid line) or UP+pT5-GntR-WT promoter (with enhancer, dashed line), where expression of mScarlet is induced by gluconate at the beginning of culture (t = 0 h), as described in Example 4.

[0038] FIG. 17B shows time-course profiles of cell density (OD600) and relative mScarlet level (represented by fluorescence emission at 591 nm) of strains carrying mScarlet expression cassettes operably linked to the pT5-GntR-WT promoter (no enhancer, solid line) or UP+pT5-GntR-WT promoter (with enhancer, dashed line), where expression of mScarletis induced by gluconate the effect of the UP element (enhancer) on cell growth (as OD600) and relative activity of pT5-GntR-WT promoter measured by mScarlet fluorescence, where expression of mScarlet is induced by gluconate during the exponential phase of growth (t=7 h), as described in Example 4.

[0039] FIG. 18A provides a map of some relevant features of the pT5-GntR-WT promoter studied in Example 5.

[0040] FIG. 18B provides a map of some relevant features of the pT5-GntR-leaky promoter studied in Example 5. GntR-binding site 2 was eliminated from the pT5-GntR-WT promoter shown in FIG. 18A to yield the pT5-GntR-leaky promoter.

[0041] FIG. 19 shows the annotated sequence of the pT5-GntR-leaky promoter linked with other regulatory elements, as described in Example 5. FIG. 19 discloses SEQ ID NO:74.6. DETAILED DESCRIPTION6.1. Definitions

[0042] 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.

[0043] Operator region: An “operator region” as used herein refers to a nucleic acid sequence between a promoter and a translation initiation codon of a sequence of interest that encodes a protein of interest, or a corresponding position in a sequence of interest that is transcribed to a non-coding RNA. An operator region includes a from 5’ to 3’ a ribosome binding site (RBS) and a spacer. An operator region may include at least a portion of a repressor binding site and / or an activator binding site. An operator region having 100%sequence identity to the operator region of a gene “Xxx” in a given organism may be referred to herein as “the Xxx operator region.”

[0044] Promoter: A “promoter” as used herein refers to a nucleic acid sequence which is capable of interacting with an RNA polymerase such that transcription of a sequence of interest begins. A typical prokaryotic promoter includes a -35 sequence (a region of about 6 nucleotides, the 5’ end of which is located from 30 to 40 nucleotides, such as 35 nucleotides, upstream ( / .e., 5’-ward) of the transcription start site) and a -10 sequence, also known as a Pribnow box (a region of about 6 nucleotides, the 5’ end of which is located from 5 to 15 nucleotides, such as 10 nucleotides, upstream of the transcription initiation site). A prokaryotic promoter typically has from 12 to 22 nucleotides, and in some embodiments 17 ± 3 (e.g., 14, 15, 16, 17, 18, 19, or 20 nucleotides), intervening between the -35 sequence and the -10 sequence. A promoter having 100% sequence identity to the promoter of a gene “Xxx” in a given organism may be referred to herein as “the Xxx promoter.” A promoter may include at least a portion of a repressor binding site and / or an activator binding site.

[0045] Protein of interest: A “protein of interest” as used herein refers to a peptide, polypeptide, or protein encoded by ( / .e., translated from a transcript of) a sequence of interest. In other words, coding regions encoding proteins of interest are a subset of sequences of interest.

[0046] Repressor binding site: A “repressor binding site” as used herein is a nucleic acid sequence to which a repressor protein can bind and thereby block transcription of a sequence of interest. A repressor binding site can overlap with a promoter or region thereof, such as a -35 sequence, a -10 sequence, or both; can overlap with an RBS, a spacer, or both of an operator region; can overlap with an open reading frame of a sequence of interest that is translated to a protein of interest (or corresponding nucleotides that are transcribed in a non-coding RNA); and / or, if multiple repressor binding sites are included in a nucleic acid sequence, two or more repressor binding sites can overlap each other. In some embodiments herein, a repressor is GntR, in which circumstance a repressor binding site may be termed a “GntR binding site.”

[0047] RNA of interest: An “RNA of interest” as used herein refers to an RNA molecule transcribed from a sequence of interest. In other words, sequences transcribed to yieldRNAs of interest are a subset of sequences of interest RNAs of interest include mRNAs from which translation of proteins of interest is desired. Herein, mRNAs may be referred to as “coding RNAs”. RNAs of interest also include RNAs for which translation is not expected and / or desired, such as RNAs performing regulatory, structural, or similar functions. RNAs for which translation is not expected and / or desired may be referred to herein as “noncoding RNAs”.

[0048] 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 sequence. 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 acidsequence (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.

[0049] Sequence of interest: A “sequence of interest” as used herein refers to a nucleic acid sequence which is operably linked to a promoter for transcription thereof. The term “sequence of interest” includes sequences encoding “proteins of interest” and sequences encoding “RNAs of interest.”6.2. Expression Systems

[0050] The present disclosure provides gluconate-inducible expression systems, suitable for expression in prokaryotic cells such as E. coli. The expression systems typically include promoters, operator regions, first GntR binding sites and optionally second GntR binding sites, and sequences of interest, operably linked to provide gluconate-inducible expression of the sequences of interest. An example of a suitable configuration of these components is set forth in FIG. 3.

[0051] Examples of promoters that can be incorporated into the gluconate-inducible expression systems of the disclosure are described in Section 6.2.1.

[0052] Examples of operator regions that can be incorporated into the gluconate-inducible expression systems of the disclosure are described in Section 6.2.2.

[0053] Examples of GntR binding sites that can be incorporated into the gluconate-inducible expression systems of the disclosure are described in Section 6.2.3.

[0054] In some embodiments, the expression systems further include enhancers.

[0055] Examples of enhancers that can be incorporated into the gluconate-inducible expression systems of the disclosure are described in Section 6.2.4.

[0056] Examples of sequences of interest that can be incorporated into the gluconate- inducible expression systems of the disclosure are described in Section 6.2.5.6.2.1. Promoters

[0057] Promoters useful in the expression systems of the present disclosure include any promoters capable of operable linkage to sequences of interest such that the sequences of interest can be transcribed in a selected prokaryotic species in vivo.

[0058] Promoters useful in the expression systems of the present disclosure typically comprise, from 5’ to 3’, a -35 sequence and a -10 sequence. When selecting -35 sequences and -10 sequences for use in an expression system, nucleotide sequences are utilized that allow for sufficient binding by an RNA polymerase of the prokaryotic species for which the expression system is intended.

[0059] The consensus sequence of prokaryotic -35 sequences is TTGACA. In some embodiments, the -35 sequences of the promoters the present disclosure comprise the nucleotide sequence TTGACA or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof.

[0060] In various aspects, a -35 sequence in an expression system of the disclosure comprises any one of the nucleotide sequences TTGACA, TTGATA, TTGATA, TTGATC, TTTATA, TTGAGA, and TTGCTT.

[0061] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGACA.

[0062] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGATA.

[0063] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGATA.

[0064] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGATC.

[0065] In some embodiments, a -35 sequence comprises the nucleotide sequence TTTATA.

[0066] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGAGA.

[0067] In some embodiments, a -35 sequence comprises the nucleotide sequence TTGCTT, which has 100% sequence identity to the -35 sequence of the GntK promoter of E. con MG1655.

[0068] The consensus sequence of prokaryotic -10 sequences is TATAAT. In some embodiments, the -10 sequences of the promoters comprise the nucleotide sequence TATAAT or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof.

[0069] In various aspects, a -10 sequence in an expression system of the disclosure comprises any one of the nucleotide sequences TACAAT, TATAAT, and GACAAT.

[0070] In some embodiments, a -10 sequence comprises the nucleotide sequence TACAAT.

[0071] In some embodiments, a -10 sequence comprises the nucleotide sequence TATAAT.

[0072] In some embodiments, - a -10 sequence comprises the nucleotide sequence GACAAT, which has 100% sequence identity to the -10 sequence of the GntK promoter of E. con MG1655.

[0073] The -35 sequence and -10 sequences in an expression system of the disclosure can be independently selected from:(a) -35 sequences: TTGACA, TTGATA, TTGATA, TTGATC, TTTATA, TTGAGA, and TTGCTT; and(b) -10 sequences: TACAAT, TATAAT, and GACAAT.6.2.2. Operator Regions

[0074] Operator regions useful in the expression systems of the present disclosure include any nucleic acid sequences which operably link promoters and sequences of interest.

[0075] In some embodiments, e.g., where the sequence of interest comprises an open reading frame that encodes a protein, operator regions useful in the expression systems of the present disclosure include a ribosome binding site (RBS) and a spacer.

[0076] Prokaryotic RBSs are sequences downstream of a transcription initiation codon ( / .e., are transcribed). RBSs base-pair with complementary sequences in the ribosome to bind the transcript to the ribosome and allow translation to occur. In prokaryotes, the 5’ ends ofRBSs are generally 5 to 20 nucleotides, (e.g., 10 to 15 nucleotides) upstream of the initiation codon of open reading frames.

[0077] The consensus RBS in prokaryotes, also known as the Shine-Dalgarno sequence, comprises AGGAGG, which is fully complementary to the CCUCCU sequence of the 30S ribosomal subunit. For transcripts for which translation may be desired, e.g., if the sequence of interest encodes a protein of interest, RBSs can have relatively high sequence identity to AGGAGG.

[0078] In some embodiments, an RBS comprises the nucleotide sequence TAAGGAGG or a sequence having at least 4, at least 5, at least 6, or at least 7 nucleotides thereof.

[0079] In some embodiments, an RBS comprises the nucleotide sequence GGAGG.

[0080] In some embodiments, an RBS comprises the nucleotide sequence AAGGA.

[0081] In some embodiments, an RBS comprises the nucleotide sequence GGGGG, which has 100% sequence identity to the RBS or a portion thereof of the GntK operator region of E. con MG1655.

[0082] In some embodiments, an RBS comprises a nucleotide sequence having no more than 3, no more than 2, no more than 1 , or 0 nucleotides of the nucleotide sequence TAAGGAGG.

[0083] Spacers, i.e., nucleotides between the RBS and the translation initiation codon, are involved in interactions between mRNAs and ribosomes. Spacer sequences can influence the rate of expression of sequences of interest operably linked thereto. In some embodiments, expression systems of the present disclosure comprise spacers located downstream of the RBSs and upstream of the initiation codon of an ORF encoding the protein of interest (or corresponding nucleotides of a non-coding RNA of interest). Spacers can have three to ten (e.g., three, four, five, six, seven, eight, nine, or ten) nucleotides. In some embodiments, the spacers have seven nucleotides.

[0084] The presence of cytosine nucleotides in spacers can lead to the formation of secondary structures in the spacer. Secondary structures in the spacer can reduce the rate of translation of an ORF encoding a protein of interest. In some embodiments, spacers used in the expression systems comprise three or fewer (e.g., three, two, one, or zero) cytosine nucleotides.

[0085] In some embodiments, spacers comprise two or fewer (e.g., two, one, or zero) cytosine nucleotides.

[0086] For transcripts for which translation may not be desired, e.g., if the sequence of interest is transcribed to yield a non-coding RNA of interest, in some embodiments, the operator regions can be absent or present. Operator regions may be present to provide all or part of one or more GntR binding sites and thus prevent or reduce the extent of overlap of GntR binding sites with sequences of interest.

[0087] If operator regions are present, RBSs can have relatively low sequence identity to the nucleotide sequence AGGAGG and / or can have less than five or more than eight nucleotides, or RBSs can be absent. If operator regions are present, spacers can have less than two or more than ten nucleotides and / or can have four or more cytosine nucleotides, or spacers can be absent.6.2.3. GntR Binding Sites

[0088] Expression systems of the present disclosure comprise at least one GntR binding site to confer gluconate regulation of expression. In some embodiments, the expression systems comprise one, two or more GntR binding sites.

[0089] In some embodiments, the GntR binding sites are derived from the E. coli GntK operon. The E. coli GntK operon, comprising two GntR binding sites, is schematically depicted in FIG. 1A. When GntR binds to the GntR binding sites, transcription of GntK and GntU is blocked (FIG. 1 B). When gluconate binds to GntR, a conformational change in GntR occurs that prevents GntR binding to the GntR binding sites, and transcription of GntK and GntU takes place (FIG. 1C).

[0090] The affinity of GntR for a GntR binding site in the absence of gluconate may be low enough that a GntR molecule may disassociate from the GntR binding site from time to time, thereby exposing the GntR binding site. Hence, regulation by gluconate of an expression system comprising only one GntR binding site may be “leaky,” i.e., some amount of transcription may occur in the absence of gluconate.

[0091] Leaky regulation can be advantageous in certain applications, for example when low level constitutive expression of a sequence of interest is desirable. Thus, when gluconate is used to induce maximal transcription of a sequence of interest from a leaky promoter in theproduction phase of a culture, the absence of gluconate induction in the growth phase can result in expression of the sequence of interest at a reduced level.

[0092] Alternatively or additionally, leaky regulation can increase the total amount of a protein of interest (encoded by a sequence of interest) produced as of any given timepoint in the production phase, relative to the total amount produced when a non-leaky promoter is used.

[0093] Additionally or alternatively, leaky regulation can be used to select for cells of recombinant microorganisms that maintain expression systems of the disclosure. For example, if a sequence of interest encodes a protein of interest that provides an activity required for cell survival or growth is included in an extrachromosomal expression system, an endogenous chromosomal sequence encoding a protein providing the required activity can be deleted or disrupted. In this scenario, setting aside integration of the expression system into the chromosome, cells would only survive and grow if they maintained the extrachromosomal expression system. In some embodiments, the protein of interest is a 1- deoxy-D-xylulose 5-phosphate (DXP) pathway (also known as the MEP pathway) protein. Examples of DXP pathway proteins are described in Section 6.2.5.1 and include 4-hydroxy- 3-methylbut-2-en-1-yl diphosphate synthase (IspG; EC 1.17.7.1 or EC 1.17.7.3) or a 4- hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH; EC 1.17.7.4).

[0094] Accordingly, in some embodiments, expression systems of the present disclosure comprise a single GntR binding site.

[0095] In some embodiments, expression systems of the present disclosure comprise at least two (and in some embodiments only two) GntR binding sites.

[0096] A number of gluconate-regulated operons of prokaryotes comprise two GntR binding sites. For one example, E. coli MG1655 comprises two GntR binding sites, as shown in FIG. 2. The first GntR binding site of E. coli MG1655 has the nucleotide sequence CAATGTTACCGATAACAGTT (SEQ ID NO:3). The 5’ end of the first GntR binding site is at the -8 nucleotide of the promoter and the 3’ end is at the +13 nucleotide of the transcribed region. The second GntR binding site of E. coli MG1655 has the nucleotide sequence AACAGTTACCCGTAACATTT (SEQ ID NO:11), with a reverse complement of AAATGTTACGGGTAACTGTT (SEQ ID NO:8). The 5’ end of the second GntR binding siteis at the +7 nucleotide of the transcribed region and the 3’ end is at the +26 nucleotide of the transcribed region.

[0097] As depicted in FIG. 3, and as the example of E. coli MG1655 indicates, a GntR binding site can at least partially overlap with the promoter and / or another GntR binding site.

[0098] In some embodiments, the 3’ ends of promoters and the 5’ ends of GntR binding sites (e.g., first GntR binding sites) overlap. In some embodiments, the 3’ ends of -10 sequences and the 5’ ends of GntR binding sites (e.g., first GntR binding sites) overlap.

[0099] In some embodiments, the 3’ ends of first GntR binding sites and the 5’ ends of second GntR binding sites overlap.

[0100] In some embodiments, the 3’ ends of GntR binding sites (e.g., second GntR binding sites) and the 5’ ends of operator regions overlap. In some embodiments, the 3’ ends of GntR binding sites (e.g., second GntR binding sites) and the 5’ ends of RBSs overlap.

[0101] In some embodiments, the 3’ ends of GntR binding sites (e.g., second GntR binding sites) and the 5’ ends of sequences of interest overlap. For example, in some embodiments, when sequences of interest are transcribed to yield non-coding RNAs, RBSs, spacers, and / or operator regions are truncated relative to consensus sequences or omitted entirely, the 3’ ends of GntR binding sites and the 5’ ends of sequences of interest overlap.

[0102] Embodiments in which GntR binding sites overlap with promoters, operator regions, sequences of interest, or other GntR binding sites are not mutually exclusive. In some embodiments, expression systems comprise zero, any one, any two, any three, or all four of binding site-promoter overlap, binding site-operator region overlap, binding site-sequence of interest overlap, and / or binding site-binding site overlap.

[0103] Sequences that can be considered a consensus sequence for GntR binding sites are the nucleotide sequence TAATGTTACCSGTAACATTT (SEQ ID NO:1), wherein S is C or G, or its reverse TTTACAATGSCCATTGTAAT (SEQ ID NO:52), wherein S is C or G.

[0104] In some embodiments, first GntR binding sites and, if present, second GntR binding sites each independently comprise nucleotide sequences having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TAATGTTACCSGTAACATTT (SEQ ID NO:1), wherein S is C or G or the nucleotide sequence TTTACAATGSCCATTGTAAT (SEQ ID NO:52), wherein S is C orG. Thus, in some embodiments, a GntR binding site comprises the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:52, or a nucleotide sequence that differs from SEQ ID NO:1 or SEQ ID NO:52 by up 1 , 2 or 3 nucleotides.

[0105] In some embodiments, first GntR binding sites comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.

[0106] In some embodiments, first GntR binding sites comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.

[0107] In some embodiments, first GntR binding sites comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.

[0108] In some embodiments, first GntR binding sites comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.

[0109] In some embodiments, first GntR binding sites comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.

[0110] In some embodiments, second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TTATTTTACCGGTAACATGA (SEQ ID NO:7) or a sequence reverse to SEQ ID NO:7.

[0111] In some embodiments, second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence AACAGTTACCCGTAACATTT(SEQ ID N0:11) or a reverse complement to SEQ ID NO:11. Accordingly, in some embodiments second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence AAATGTTACGGGTAACTGTT (SEQ ID NO:8).

[0112] In some embodiments, second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.

[0113] In some embodiments, second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NO: 10.

[0114] In some embodiments, second GntR binding sites, if present, comprise a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the nucleotide sequence AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.6.2.4. Enhancers

[0115] In some embodiments, the expression systems of the disclosure include enhancers. Enhancers can increase expression from a nearby promoter on the same nucleic acid (relative to expression when the enhancer is absent), even when they are not adjacent to the promoter. Enhancers can increase expression when positioned upstream (5’-ward) or downstream (3’-ward) of the promoter.

[0116] In some embodiments, expression systems of the disclosure (e.g., an expression system comprising a first GntR binding site and optionally a second GntR binding site) include an enhancer 5’-ward of the promoter.

[0117] In some embodiments, the 5’ end of the -35 sequence of the promoter and the 3’ end of the enhancer are on the same face of the double helix. This typically involves the 3’ end of the enhancer and the 5’ end of the -35 sequence being separated by a number of nucleotides in the range of 5 nt to 35 nt. In some embodiments, enhancers and -35 sequences are separated by a number of nucleotides in the range of 25 nt to 35 nt or in therange of 26 nt to 32 nt or 33 nt In some embodiments, enhancers and -35 sequences are separate by a number of nucleotides in the range of 5 nt to 10 nt, 10 nt to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, or 30 nt to 35 nt. In some embodiments, enhancers and -35 sequences are separated by 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt or 35 nt.

[0118] In some embodiments, the enhancer is derived from the lambda bacteriophage P upstream (UP) element. Exemplary enhancers derived from the lambda bacteriophage P upstream element include the enhancer having the nucleotide sequence of SEQ ID NO:62 and truncated enhancers comprising the nucleotides sequences of SEQ ID NOS:75-77. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO:62. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO:75. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO:76. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO:77.6.2.5. Sequences of Interest

[0119] The expression systems of the disclosure include sequences of interest that are operably linked to regulatory elements to allow transcription of the sequences of interest under gluconate regulation.

[0120] Sequences of interest can be open reading frames (ORFs) encoding proteins of interest, or the transcript itself (e.g., a non-coding RNA) can be of interest.

[0121] Expression systems of the present disclosure can be used in the production of any proteins of interest for which regulation by gluconate is desired. Expression systems of the present disclosure may have higher rates of transcription of sequences of interest compared to the GntK operon of E. coli MG1655.6.2.5.1. Proteins of Interest

[0122] In some embodiments, proteins of interest are any one of or all proteins encoded by coding regions under the control of the GntK operon of E. coli MG 1655 (e.g., GntK, GntU, or both).

[0123] In other embodiments, proteins of interest are those not naturally associated with gluconate regulation, e.g., proteins not encoded by an E. coli GntK operon.

[0124] For example, in some embodiments, proteins of interest are 2-keto-3-deoxy- gluconate (KDG) aldolases. KDG aldolases catalyze the conversion of KDG to pyruvate and glyceraldehyde (and vice versa). In some embodiments, KDG aldolases comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:48.

[0125] For example, in some embodiments, proteins of interest are 2-dehydro-3-deoxy-6- phospho-D-gluconate (KDGP) aldolases. KDGP aldolases catalyze the conversion of KDGP to pyruvate and glyceraldehyde-3-phosphate (and vice versa). In some embodiments, KDGP aldolases comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:60.

[0126] In some embodiments, proteins of interest are DXP pathway enzymes. DXP pathway enzymes include 1-deoxy-D-xylulose-5-phosphate synthases (Dxs; EC 2.2.1.7), which catalyze the condensation of pyruvate to glyceraldehyde-3-phosphate to yield 1-deoxy-D- xylulose-5-phosphate (DXP). In some embodiments, the Dxs is an E. coli Dxs, e.g., a Dxs identified by UniProt ID number P77488 (SEQ ID NO:49). Other exemplary Dxs enzymes include those identified by UniProt ID numbers A0AIG6 (DXS_LISW6), A0AYZ0 (DXS_BURCH), A0JVG9 (DXS_ARTS2), A0KNF9 (DXS_AERHH), A0KZA9 (DXS_SHESA), A0L6H3 (DXS_MAGMM), A0PT40 (DXS_MYCUA), A0Q0A4 (DXS_CLONN), A0Q6B9 (DXS_FRATN), A0QIL6 (DXS_MYCA1), A0QW19 (DXS_MYCS2), A0RMN5 (DXS_CAMFF), A1A890 (DXS_ECOK1), A1JNR7 (DXS_YERE8), A1 K4R0 (DXS_AZOSB), A1 KM20 (DXS_MYCBP), A1 KS32 (DXS_NEIMF), A1 R5N7 (DXS_PAEAT), A1 RLV3 (DXS_SHESW), A1S8D4 (DXS_SHEAM), A1SKM6 (DXS_NOCSJ), A1SWW6 (DXS_PSYIN), A1T7Z0 (DXS_MYCVP), A1TNR1 (DXS_ACIAC), A1 UF44 (DXS_MYCSK), A1 URW6 (DXS_BARBK), A1VE69 (DXS_DESVV), A1VMD7 (DXS_POLNA), A1VY40 (DXS_CAMJJ), A1W4U9 (DXS_ACISJ), A1WN06 (DXS_VEREI), A2BR27 (DXS_PROMS), A2BWN6 (DXS_PROM5), A2C220 (DXS_PROM1), A2C9X1 (DXS_PROM3), A2SJ46 (DXS_METPP), A3D2B2 (DXS_SHEB5), A3MYS9 (DXS_ACTP2), A3NMF6 (DXS_BURP6), A3P7W4 (DXS_BURP0), A3PCV0 (DXS_PRQM0), A3PYK6 (DXS_MYCSJ), A3QGN9 (DXS_SHELP), A4FAQ5 (DXS_SACEN), A4IQR7 (DXS_GEOTN), A4IXW5 (DXS_FRATW),A4QEQ9 (DXS_CORGB), A4SDG1 (DXS_CHLPM), A4SJP9 (DXS_AERS4), A4TCS5 (DXS_MYCGI), A4TPG2 (DXS_YERPP), A4VQS8 (DXS_STUS1), A4W791 (DXS_ENT38), A4XZ25 (DXS_PSEMY), A4Y4X0 (DXS_SHEPC), A4YQ36 (DXS_BRASO), A5D2Z6 (DXS_PELTS), A5EEQ0 (DXS_BRASB), A5F331 (DXS_VIBC3), A5FRB9 (DXS_DEHMB), A5GL34 (DXS_SYNPW), A5GTT4 (DXS_SYNR3), A5ILK2 (DXS_THEP1), A5N7J2 (DXS_CL0K5), A5U634 (DXS_MYCTA), A5UC51 (DXS_HAEIE), A5UEV6 (DXS_HAEIG), A5V6A9 (DXS_RHIWR), A5VP09 (DXS_BRU02), A5VXW9 (DXS_PSEP1), A6L175 (DXS_PH0V8), A6LFB9 (DXS_PARD8), A6LU48 (DXS_CL0B8), A6Q1Z6 (DXS_NITSB), A6T5F3 (DXS_KLEP7), A6V058 (DXS_PSEA7), A6VUE5 (DXS_MARMS), A6WL04 (DXS_SHEB8), A6WWC4 (DXS_BRUA4), A7FLE4 (DXS_YERP3), A7GSJ5 (DXS_ BACON), A7H552 (DXS_CAMJD), A7H9E8 (DXS_ANADF), A7I2V7 (DXS_CAMHC), A7IPK6 (DXS_XANP2), A7MFG0 (DXS_CROS8), A7MYC6 (DXS_VIBC1), A7NCA4 (DXS_FRATF), A7Z6J5 (DXS_BACVZ), A7ZIH3 (DXS_ECO24), A7ZX72 (DXS_ECOHS), A8AK34 (DXS_CITK8), A8EWN0 (DXS_ALIB4), A8FF11 (DXS_BACP2), A8FKB0 (DXS_CAMJ8), A8FYL0 (DXS_SHESH), A8G4R9 (DXS_PROM2), A8GAP2 (DXS_SERP5), A8H6X1 (DXS_SHEPA), A8IBS1 (DXS_AZOC5), A8L0K9 (DXS_FRASN), A8MFI7 (DXS_ALKOO), A9BAC1 (DXS_PROM4), A9IQP2 (DXS_BART1), A9ITB0 (DXS_BORPD), A9KMB8 (DXS_LACP7), A9KTL5 (DXS_SHEB9), A9M1G3 (DXS_NEIM0), A9M8W0 (DXS_BRUC2), A9MM42 (DXS_SALAR), A9MX09 (DXS_SALPB), A9QZS3 (DXS_YERPG), A9VGD1 (DXS_BACMK), A9WRA9 (DXS_RENSM), B0B7P9 (DXS_CHLT2), B0BBW4 (DXS_CHLTB), BOBSLO (DXS_ACTPJ), B0C8J3 (DXS_ACAM1), BOCKCO (DXS_BRUSI), B0JL88 (DXS_MICAN), B0KL79 (DXS_PSEPG), B0RC26 (DXS_CLAMS), B0T3X7 (DXSJDAUSK), B0TEJ5 (DXS_HELMI), B0TQ36 (DXS_SHEHH), B0U0B3 (DXS_FRAP2), B0U3E1 (DXS_XYLFM), B0UUA4 (DXS_HISS2), B0V710 (DXS_ACIBY), B0VQB8 (DXS_ACIBS), B1 HRX4 (DXS_LYSSC), B1I3J6 (DXS_DESAP), B1J029 (DXS_ECOLC), B1J3G4 (DXS_PSEPW), B1JID8 (DXS_YERPY), B1K3S9 (DXS_BUROO), B1 KQY8 (DXS_SHEWM), B1 LAQ3 (DXS_THESQ), B1 LJHO (DXS_ECOSM), B1 MCU7 (DXS_MYCA9), B1VWJ8 (DXS_STRGG), B1WWM7 (DXS_CROS5), B1XF08 (DXS_ECODH), B1XKC5 (DXS_SYNP2), B1Y2X5 (DXS_LEPCP), B1YLQ5 (DXS_EXIS2), B1Z1G2 (DXS_BURA4), B2A526 (DXS_NATTJ), B2FN57 (DXS_STRMK), B2GJ56 (DXS_KOCRD), B2I607 (DXS_XYLF2), B2IDK3 (DXS_BEII9), B2J5P1 (DXS_NOSP7), B2JP68 (DXS_PARP8), B2K6T7 (DXS_YERPB), B2RMK4(DXS_P0RG3), B2S462 (DXS_TREPS), B2S9T6 (DXS_BRUA1), B2SGK5 (DXS_FRATM), B2SQV8 (DXS_XANOP), B2TRM5 (DXS_CLOBB), B2U4M3 (DXS_SHIB3), B2U930 (DXS_RALPJ), B2V4R3 (DXS_CLOBA), B2VHS3 (DXS_ERWT9), B3DW88 (DXS_METI4), B3H050 (DXS_ACTP7), B3PF22 (DXS_CELJU), B3PS68 (DXS_RHIE6), B3QFY7 (DXS_RHOPT), B3R5H4 (DXS_CUPTR), B4EN29 (DXS_BURCJ), B4EU31 (DXS_PROMH), B4RGW0 (DXS_PHEZH), B4RNW6 (DXS_NEIG2), B4RVY8 (DXS_ALTMD), B4SWR4 (DXS_SALNS), B4T8R3 (DXS_SALHS), B4TMA2 (DXS_SALSV), B4UHF5 (DXS_ANASK), B5BDB0 (DXS_SALPK), B5EXG3 (DXS_SALA4), B5FBG6 (DXS_ALIFM), B5FKS7 (DXS_SALDC), B5QTH0 (DXS_SALEP), B5R6S3 (DXS_SALG2), B5Y0X1 (DXS_KLEP3), B5YE06 (DXS_DICT6), B5Z3S5 (DXS_EC05E), B5ZAB7 (DXS_HELPG), B5ZS68 (DXS_RHILW), B6EIA7 (DXS_ALISL), B6HZM1 (DXS_ECOSE), B6IRB5 (DXS_RHOCS), B6YRV5 (DXS_AZOPC), B7HB48 (DXS_BACC4), B7HNU0 (DXS_BACC7), B7IXG8 (DXS_BACC2), B7JM28 (DXS_BACCO), B7JVJ6 (DXS_RIP01), B7KAF7 (DXS_GL0C7), B7L654 (DXS_ECO55), B7LMG7 (DXS_ESCF3), B7M3Q9 (DXS_EC08A), B7MD78 (DXS_ECO45), B7MQD5 (DXS_EC081), B7N8X3 (DXS_ECOLU), B7NJ77 (DXS_ECO7I), B7UJP3 (DXS_ECO27), B7V7R4 (DXS_PSEA8), B7VJA1 (DXS_VIBA3), B8CS19 (DXS_SHEPW), B8D2I3 (DXS_HALOH), B8D7Z3 (DXS_BUCAT), B8D9P1 (DXS_BUCA5), B8E247 (DXS_DICTD), B8EAU7 (DXS_SHEB2), B8F3A4 (DXS_GLAP5), B8FQ45 (DXS_DESHD), B8GN62 (DXS_THISH), B8GXC4 (DXS_CAUVN), B8HH36 (DXS_PSECP), B8HWL8 (DXS_CYAP4), B8JFY1 (DXS_ANAD2), B8ZQW9 (DXS_MYCLB), B9E104 (DXS_CL0K1), B9E6Q6 (DXS_MACCJ), B9JAL7 (DXS_AGRRK), B9JSL2 (DXS_ALLAM), B9L1 L6 (DXS_THERP), B9MEU8 (DXS_ACIET), C0Q7U7 (DXS_SALPC), C0RHE3 (DXS_BRUMB), C0ZC10 (DXS_BREBN), C0ZYV9 (DXS_RH0E4), C1AFE1 (DXS_MYCBT), C1 DAW8 (DXS_LARHH), C1 F3C4 (DXS_ACIC5), C1 L2S1 (DXS_LISMC), C3K2R1 (DXS_PSEFS), C3LJV1 (DXS_BACAC), C3LTD9 (DXS_VIBCM), C3P7V6 (DXS_BACAA), C4K6M7 (DXS_HAMD5), C4ZTH7 (DXS_ECOBW), C5BCH9 (DXS_EDWI9), C5D467 (DXS_GEOSW), C6DB37 (DXS_PECCP), 022567 (DXS1_0RYSJ), 025121 (DXS_HELPY), 067036 (DXS_AQUAE), 078328 (DXS_CAPAN), 083796 (DXS_TREPA), 084335 (DXS_CHLTR), P0A555 (DXS_MYCB0), P45205 (DXS_HAEIN), P54523 (DXS_BACSU), P57536 (DXS_BUCAI), P57848 (DXS_PASMU), P73067 (DXS_SYNY3), P9WNS2 (DXS_MYCT0), P9WNS3 (DXS_MYCTU), Q02SL1 (DXS_PSEAB), Q04U59 (DXS_LEPBJ), Q053M2 (DXS_LEPBL),Q07SR3 (DXS_RH0P5), Q07ZD4 (DXS_SHEFN), Q0A8V7 (DXS_ALKEH), Q0AFY6 (DXS_NITEC), Q0ARE5 (DXS_MARMM), Q0AZE2 (DXS_SYNWW), Q0BAL8 (DXS_BURCM), Q0BLU9 (DXS_FRATO), Q0C154 (DXS_HYPNA), Q0HGL5 (DXS_SHESM), Q0HSW6 (DXS_SHESR), Q0I3G1 (DXS_HISS1), Q0IAA6 (DXS_SYNS3), Q0K860 (DXS_CUPNH), Q0S1 H1 (DXS_RHOJR), Q0SS05 (DXS_CLOPS), Q0TKM1 (DXS_EC0L5), Q0TPD8 (DXS_CL0P1), Q0VMI4 (DXS_ALCBS), Q10ZY2 (DXS_TRIEI), Q11 KE0 (DXS_CHESB), Q11 NY7 (DXS_CYTH3), Q12CQ9 (DXS_POLSJ), Q12L26 (DXS_SHEDO), Q130G7 (DXS_RHOPS), Q13RX1 (DXS_PARXL), Q14HJ1 (DXS_FRAT1), Q15W93 (DXS_PSEA6), Q16CP0 (DXS2_R0SD0), Q16DV7 (DXS1_R0SD0), Q18B68 (DXS_CL0D6), Q1 B9W8 (DXS_MYCSS), Q1 BLY7 (DXS_BUR01), Q1C4I9 (DXS_YERPA), Q1CL87 (DXS_YERPN), Q1CUF6 (DXS_HELPH), Q1D3G4 (DXS_MYXXD), Q1GCG4 (DXS_RUEST), Q1GQK9 (DXS_SPHAL), Q1GZD7 (DXS_METFK), Q1 IFL1 (DXS_PSEE4), Q1 IZP0 (DXS_DEIGD), Q1 LK34 (DXS_CUPMC), Q1 LTI9 (DXS_BAUCH), Q1MKN4 (DXS_RHIL3), Q1 MRB3 (DXS_LAWIP), Q1QE74 (DXS_PSYCK), Q1QQ40 (DXS_NITHX), Q1 R1 E5 (DXS_CHRSD), Q1RFC0 (DXS_ECOUT), Q21A74 (DXS_RHOPB), Q21 F93 (DXS_SACD2), Q21 UG7 (DXS_ALBFT), Q24V05 (DXS_DESHY), Q253R7 (DXS_CHLFF), Q28W25 (DXS2_JANSC), Q28WA7 (DXS1_JANSC), Q2A3D3 (DXS_FRATH), Q2GC13 (DXS_NOVAD), Q2IPZ2 (DXS_ANADE), Q2IRL7 (DXS_RH0P2), Q2JDD9 (DXS_FRACC), Q2JK64 (DXS_SYNJB), Q2JTX2 (DXS_SYNJA), Q2KBR2 (DXS_RHIEC), Q2KZ15 (DXS_B0RA1), Q2LUA7 (DXS_SYNAS), Q2N6U5 (DXS_ERYLH), Q2NV94 (DXS_SODGM), Q2P472 (DXS_XANOM), Q2RIB9 (DXS_MOOTA), Q2RR29 (DXS2_RH0RT), Q2RYD6 (DXS1_RH0RT), Q2SA08 (DXSJHAHCH), Q2T7N5 (DXS_BURTA), Q2W367 (DXS_MAGSA), Q2YCH7 (DXS_NITMU), Q2YMF0 (DXS_BRUA2), Q30TC5 (DXS_SULDN), Q30Z99 (DXS_0LEA2), Q31AZ2 (DXS_PR0M9), Q325I1 (DXS_SHIBS), Q32JH8 (DXS_SHIDS), Q38854 (DXS_ARATH), Q393P4 (DXS_BURL3), Q39RT4 (DXS2_GE0MG), Q39UB1 (DXS1_GE0MG), Q3A3Z6 (DXS_SYNC1), Q3AAN0 (DXS_CARHZ), Q3AJP8 (DXS_SYNSC), Q3AXZ4 (DXS_SYNS9), Q3B5P3 (DXS_CHLL3), Q3BRW8 (DXS_XANC5), Q3II09 (DXS_PSET1), Q3IYR6 (DXS2_CERS4), Q3J1A8 (DXS1_CERS4), Q3JAD1 (DXS_NITOC), Q3JKA3 (DXS_BURP1), Q3K660 (DXS_PSEPF), Q3KM28 (DXS_CHLTA), Q3M4F6 (DXS_TRIV2), Q3SKF1 (DXS_THIDA), Q3SUZ1 (DXS_NITWN), Q3Z4Y9 (DXS_SHISS), Q3Z8G9 (DXS_DEHM1), Q3ZXC2 (DXS_DEHMC), Q46L36 (DXS_PROMT), Q474C2(DXS_CUPPJ), Q47BJ0 (DXS_DECAR), Q47NL9 (DXS_THEFY), Q487D3 (DXS_C0LP3), Q48NX0 (DXS_PSE14), Q493G7 (DXS_BLOPB), Q4FN07 (DXS_PELUB), Q4FV64 (DXS_PSYA2), Q4JVB5 (DXS_CORJK), Q4K5A5 (DXS_PSEF5), Q4QKG6 (DXS_HAEI8), Q4UW29 (DXS_XANC8), Q4ZYU8 (DXS_PSEU2), Q50000 (DXS_MYCLE), Q57ET1 (DXS_BRUAB), Q57SE2 (DXS_SALCH), Q5E6Z0 (DXS_ALIF1), Q5FAI2 (DXS_NEIG1), Q5FUB1 (DXS_GLUOX), Q5H1A0 (DXS_XANOR), Q5HWF0 (DXS_CAMJR), Q5L6H4 (DXS_CHLAB), Q5LH44 (DXS_BACFN), Q5LX42 (DXS_RUEPO), Q5NG39 (DXS_FRATT), Q5NM38 (DXS2_ZYMM0), Q5NN52 (DXS1_ZYMM0), Q5P228 (DXS_AROAE), Q5PFR6 (DXS_SALPA), Q5QVE8 (DXSJDILO), Q5SMD7 (DXS_THET8), Q5WF63 (DXS_ALKCK), Q5YTA2 (DXS_NOCFA), Q60AN1 (DXS_METCA), Q62DU1 (DXS_BURMA), Q635A7 (DXS_BACCZ), Q63JF4 (DXS_BURPS), Q64Y02 (DXS_BACFR), Q65HJ2 (DXS_BACLD), Q65TP4 (DXS_MANSM), Q66DV4 (DXS_YERPS), Q67NB6 (DXS_SYMTH), Q6A8V3 (DXS_CUTAK), Q6AFD5 (DXS_LEIXX), Q6AJQ1 (DXS_DESPS), Q6D844 (DXS_PECAS), Q6F7N5 (DXS_ACIAD), Q6G0D4 (DXS_BARQU), Q6G4D1 (DXS_BARHE), Q6HDY8 (DXS_BACHK), Q6LU07 (DXS_PHOPR), Q6MDK6 (DXS_PARUW), Q6NB76 (DXS_RHOPA), Q6NGV3 (DXS_CORDI), Q6YU51 (DXS2_ORYSJ), Q71ZV7 (DXS_LISMF), Q72CD3 (DXS_DESVH), Q72H81 (DXS_THET2), Q72U01 (DXS_LEPIC), Q731 B7 (DXS_BACC1), Q73LF4 (DXS_TREDE), Q73W57 (DXS_MYCPA), Q74CB0 (DXS2_GEOSL), Q74FC3 (DXS1_GEOSL), Q75TB7 (DXS_GEOKA), Q7M7Z0 (DXS_WOLSU), Q7MN49 (DXS_VIBVY), Q7MSZ3 (DXS_PORGI), Q7N0J7 (DXS_PHOLL), Q7NP63 (DXS_GLOVI), Q7NUK5 (DXS_CHRVO), Q7U6P6 (DXS_PARMW), Q7UWB7 (DXS_RHOBA), Q7V1G6 (DXS_PROMP), Q7V7Q3 (DXS_PROMM), Q7VC14 (DXS_PROMA), Q7VIJ7 (DXS_HELHP), Q7VNP7 (DXS_HAEDU), Q7VRH9 (DXS_BLOFL), Q7VV87 (DXS_BORPE), Q7W7Q0 (DXS_BORPA), Q7WL37 (DXS_BORBR), Q818R9 (DXS_BACCR), Q81 M54 (DXS_BACAN), Q823V1 (DXS_CHLCV), Q82KW8 (DXS2_STRAW), Q82ML4 (DXS1_STRAW), Q82VD3 (DXS_NITEU), Q83G46 (DXS_TROWT), Q83I20 (DXS_TR0W8), Q83SG2 (DXS_SHIFL), Q87C03 (DXS_XYLFT), Q87RU0 (DXS_VIBPA), Q889Q1 (DXS_PSESM), Q88QG7 (DXS_PSEPK), Q894H0 (DXSJDLOTE), Q89RW1 (DXS_BRADU), Q8A0C2 (DXS_BACTN), Q8CJP7 (DXS2_STRCO), Q8D357 (DXS_WIGBR), Q8DFA3 (DXS_VIBVU), Q8DL74 (DXS_THEVB), Q8EGR9 (DXS_SHEON), Q8EWX7 (DXS_MALP2), Q8F153 (DXS_LEPIN), Q8FKB9 (DXS_ECOL6), Q8FPI2 (DXS_COREF), Q8G292 (DXS_BRUSU), Q8GAA0(DXS_SYNE7), Q8K9A1 (DXS_BUCAP), Q8KFI9 (DXS_CHLTE), Q8NPB2 (DXS_CORGL), Q8P815 (DXS_XANCP), Q8PJG7 (DXS_XANAC), Q8R639 (DXS_FUSNN), Q8RAC5 (DXS_CALS4), Q8UHD7 (DXS_AGRFC), Q8VUR8 (DXS2_KITGR), Q8XE76 (DXS_ECO57), Q8XJE1 (DXS_CLOPE), Q8XX95 (DXS_RALSO), Q8Y7C1 (DXSJJSMO), Q8YFM2 (DXS_BRUME), Q8YZ80 (DXS_NOSS1), Q8Z8X3 (DXS_SALTI), Q8ZC45 (DXS_YERPE), Q8ZRD1 (DXS_SALTY), Q92BZ0 (DXS_LISIN), Q92RJ1 (DXS_RHIME), Q97HD5 (DXS_CLOAB), Q985Y3 (DXS_RHILO), Q9A6M5 (DXS_CAUVC), Q9F1V2 (DXS1_KITGR), Q9JW13 (DXS_NEIMA), Q9JXV7 (DXS_NEIMB), Q9K971 (DXS_HALH5), Q9KGU7 (DXS_PSEAE), Q9KTL3 (DXS_VIBCH), Q9PB95 (DXS_XYLFA), Q9PIH8 (DXS_CAMJE), Q9PK62 (DXS_CHLMU), Q9R6S7 (DXS_SYNP6), Q9RBN6 (DXS_STRC1), Q9RUB5 (DXS_DEIRA), Q9X291 (DXS_THEMA), Q9X7W3 (DXS1_STRCO), Q9Z6J9 (DXS_CHLPN), and Q9ZM94 (DXSJHELPJ).

[0127] DXP pathway enzymes also include 1-deoxy-D-xylulose-5-phosphate reductoisomerases (Dxr; EC 1.1.1.267), which catalyze the interconversion of 1-deoxy-D- xylulose 5-phosphate and 2-C-methyl-D-erythritol 4-phosphate. In some embodiments, the Dxr is an E. coli Dxr, e.g., a Dxr identified by UniProt ID number P45568. Other exemplary Dxr enzymes include those identified by UniProt ID numbers A0A1 U9X9N3 (DXR_THYVU), A0AIB8 (DXR_LISW6), A0JUS3 (DXR_ARTS2), A0K8D7 (DXR_BURCH), A0KHG9 (DXR_AERHH), A0KZ17 (DXR_SHESA), A0PQ95 (DXR_MYCUA), A0Q0R4 (DXR_CLONN), A0Q7Y5 (DXR_FRATN), A0QJ14 (DXR_MYCA1), A0QVH7 (DXR_MYCS2), A0RMI1 (DXR_CAMFF), A1AQN8 (DXR_PELPD), A1AV71 (DXR_RUTMC), A1 B969 (DXR_PARDP), A1 BJT7 (DXR_CHLPD), A1JP78 (DXR_YERE8), A1 KML5 (DXR_MYCBP), A1 R4Z8 (DXR_PAEAT), A1S4P6 (DXR_SHEAM), A1SYV7 (DXR_PSYIN), A1V563 (DXR_BURMS), A1VFB6 (DXR_DESVV), A1W0W2 (DXR_CAMJJ), A2BXK2 (DXR_PROM5), A2C3T8 (DXR_PROM1), A2SB78 (DXR_BURM9), A3D2L1 (DXR_SHEB5), A3DE52 (DXR_ACET2), A3M654 (DXR_ACIBT), A3MKT7 (DXR_BURM7), A3MZC4 (DXR_ACTP2), A3NAU2 (DXR_BURP6), A3NWM5 (DXR_BURP0), A3PJG0 (DXR_CERS1), A3QG97 (DXR_SHELP), A4G4S7 (DXR_HERAR), A4IWJ1 (DXR_FRATW), A4J5Y5 (DXR_DESRM), A4JF69 (DXR_BURVG), A4QF22 (DXR_CORGB), A4SCF3 (DXR_CHLPM), A4SQH6 (DXR_AERS4), A4TL87 (DXR_YERPP), A4VJS7 (DXR_STUS1), A4WUH5 (DXR_CERS5), A4XWT5 (DXR_PSEMY), A4YVG0 (DXR_BRASO), A5CY29 (DXR_VESOH), A5D2U3(DXR_PELTS), A5EK49 (DXR_BRASB), A5F615 (DXR_VIBC3), A5G7W2 (DXR_GEOUR), A5I4K2 (DXR_CLOBH), A5IIP4 (DXR_THEP1), A5N834 (DXR_CL0K5), A5U6M4 (DXR_MYCTA), A5UDR7 (DXR_HAEIE), A5UHV9 (DXR_HAEIG), A5W844 (DXR_PSEP1), A5WGE7 (DXR_PSYWF), A6L0W8 (DXR_PH0V8), A6LSP7 (DXR_CL0B8), A6SZP5 (DXR_JANMA), A6UDH9 (DXR_SINMW), A6V1 D8 (DXR_PSEA7), A7FFH3 (DXR_YERP3), A7FW01 (DXR_CL0B1), A7GG14 (DXR_CLOBL), A7H0K4 (DXR_CAMC5), A7H243 (DXR_CAMJD), A7HLL7 (DXR_FERNB), A7HZP3 (DXR_CAMHC), A7MXZ4 (DXR_VIBC1), A7NAP0 (DXR_FRATF), A7Z4S6 (DXR_BACVZ), A8F4Z0 (DXR_PSELT), A8FDB9 (DXR_BACP2), A8FN24 (DXR_CAMJ8), A8FY36 (DXR_SHESH), A8GIE2 (DXR_SERP5), A8H6L0 (DXR_SHEPA), A8MHH5 (DXR_ALKOO), A8ZTM6 (DXR_DESOH), A9AIM0 (DXR_BURM1), A9INU2 (DXR_BORPD), A9KMX2 (DXR_LACP7), A9KUL1 (DXR_SHEB9), A9R392 (DXR_YERPG), B0B9H9 (DXR_CHLT2), B0BB58 (DXR_CHLTB), BOBTRO (DXR_ACTPJ), B0CD21 (DXR_ACAM1), B0JWW6 (DXR_MICAN), B0K1 E5 (DXR_THEPX), B0K9Q9 (DXR_THEP3), B0KSA5 (DXR_PSEPG), B0RW72 (DXR_XANCB), BOSDN1 (DXR_LEPBA), B0SM60 (DXR_LEPBP), B0THN7 (DXR_HELMI), B0TP77 (DXR_SHEHH), B0U242 (DXR_XYLFM), B0V6F3 (DXR_ACIBY), B0VMU8 (DXR_ACIBS), B1H060 (DXR_ENDTX), B1 HQZ7 (DXR_LYSSC), B1 I2I8 (DXR_DESAP), B1 II57 (DXR_CLOBK), B1JBQ4 (DXR_PSEPW), B1JQG4 (DXR_YERPY), B1JUE4 (DXR_BUROO), B1 KNT7 (DXR_SHEWM), B1 KWL5 (DXR_CLOBM), B1WNP5 (DXR_CROS5), B1YI70 (DXR_EXIS2), B1YS68 (DXR_BURA4), B2HJP9 (DXR_MYCMM), B2I325 (DXR_ACIBC), B2I7P4 (DXR_XYLF2), B2ITX3 (DXR_NOSP7), B2JIC0 (DXR_PARP8), B2JZ30 (DXR_YERPB), B2KCJ9 (DXR_ELUMP), B2RJX5 (DXR_PORG3), B2SFW7 (DXR_FRATM), B2SR05 (DXR_XANOP), B2T5I8 (DXR_PARPJ), B2TJ47 (DXR_CLOBB), B2UB12 (DXR_RALPJ), B2US45 (DXR_HELPS), B2V4G1 (DXR_CLOBA), B2V847 (DXR_SULSY), B2VE12 (DXR_ERWT9), B3CN89 (DXR_WOLPP), B3DQ62 (DXR_BIFLD), B3E7B0 (DXR_TRIL1), B3EE57 (DXR_CHLL2), B3EK13 (DXR_CHLPB), B3PBQ4 (DXR_CELJU), B3PQ37 (DXR_RHIE6), B3Q7J8 (DXR_RHOPT), B3QXY0 (DXR_CHLT3), B3R2B1 (DXR_CUPTR), B4ECM5 (DXR_BURCJ), B4F2C9 (DXR_PROMH), B4S387 (DXR_PROA2), B4SAR7 (DXR_PELPB), B4UD63 (DXR_ANASK), B5EHV5 (DXR_CITBB), B5EQQ4 (DXR_ACIF5), B5F9X0 (DXR_ALIFM), B5YI09 (DXR_THEYD), B5Z9Y5 (DXR_HELPG), B6EK51 (DXR_ALISL), B6ISU5 (DXR_RHOCS), B6JH72 (DXR_AFIC5), B6JKE8 (DXR_HELP2), B7GQ02 (DXR_BIFLS), B7H1 U5 (DXR_ACIB3), B7HLF4(DXR_BACC7), B7I9U9 (DXR_ACIB5), B7J9P7 (DXR_ACIF2), B7K5G6 (DXR_RIP01), B7KAT5 (DXR_GL0C7), B7V7G3 (DXR_PSEA8), B8CQ79 (DXR_SHEPW), B8CW61 (DXR_HALOH), B8D7D6 (DXR_BUCAT), B8D932 (DXR_BUCA5), B8DG10 (DXR_LISMH), B8E7R0 (DXR_SHEB2), B8F4X2 (DXR_GLAP5), B8FR31 (DXR_DESHD), B8GWR7 (DXR_CAUVN), B8HFR2 (DXR_PSECP), B8HVK1 (DXR_CYAP4), B8I6D9 (DXR_RUMCH), B8J6S7 (DXR_ANAD2), B8ZRU7 (DXRJVIYCLB), B9E1J4 (DXR_CL0K1), B9EBE2 (DXR_MACCJ), B9IVB0 (DXR_BACCQ), B9JBW0 (DXR_AGRRK), B9JTP4 (DXR_ALLAM), B9KA80 (DXR_THENN), B9KE50 (DXR_CAMLR), B9KIT5 (DXR_ANAMF), B9KRU3 (DXR_CERSK), B9L006 (DXR_THERP), B9L7Q2 (DXR_NAUPA), B9M5C0 (DXR_GEODF), C0QTC4 (DXR_PERMH), C0R414 (DXR_WOLWR), C0ZF60 (DXR_BREBN), C1AFY6 (DXR_MYCBT), C1D9F0 (DXR_LARHH), C1 EP45 (DXR_BACC3), C1 FS67 (DXR_CLOBJ), C1 L2M3 (DXR_LISMC), C3K601 (DXR_PSEFS), C3L0C4 (DXR_CL0B6), C3LQ26 (DXR_VIBCM), C3M9M8 (DXR_SINFN), C4L656 (DXR_EXISA), C4L858 (DXR_TOLAT), C4Z5K1 (DXR_LACE2), C4ZA70 (DXR_AGARV), C5BHC1 (DXR_EDWI9), C5BWT4 (DXR_BEUC1), C5D9C1 (DXR_GEOSW), C6DAI7 (DXR_PECCP), C6E518 (DXR_GEOSM), H8ZW44 (DXR_ORIVU), 031753 (DXR_BACSU), 066722 (DXR_AQUAE), 083610 (DXR_TREPA), 084074 (DXR_CHLTR), 096693 (DXR_PLAFX), P44055 (DXR_HAEIN), P56139 (DXR_HELPY), P57329 (DXR_BUCAI), P57985 (DXR_PASMU), P64013 (DXR_MYCB0), P9WNS0 (DXR_MYCT0), P9WNS1 (DXR_MYCTU), Q01QM3 (DXR_S0LUE), Q02RC2 (DXR_PSEAB), Q04U66 (DXR_LEPBJ), Q053M9 (DXR_LEPBL), Q07NI7 (DXR_RH0P5), Q085D6 (DXR_SHEFN), Q0AEI0 (DXR_NITEC), Q0BE21 (DXR_BURCM), Q0BN25 (DXR_FRATO), Q0HGW1 (DXR_SHESM), Q0HT68 (DXR_SHESR), Q0IBN9 (DXR_SYNS3), Q0KA22 (DXR_CUPNH), Q0S275 (DXR_RHOJR), Q0SSC6 (DXR_CLOPS), Q0TPQ9 (DXR_CL0P1), Q136B0 (DXR_RHOPS), Q13XC2(DXR_PARXL), Q14G49 (DXR_FRAT1), Q185R8 (DXR_CL0D6), Q1 BHH6 (DXR_BUR01), Q1CAN0 (DXR_YERPA), Q1CFF1 (DXR_YERPN), Q1CUT8 (DXR_HELPH), Q1GGS3 (DXR_RUEST), Q1 H145 (DXR_METFK), Q1 LNF2 (DXR_CUPMC), Q1 LSV3 (DXR_BAUCH), Q1 MB26 (DXR_RHIL3), Q1MRD4 (DXR_LAWIP), Q1QA16 (DXR_PSYCK), Q1QMN1 (DXR_NITHX), Q215E3 (DXR_RHOPB), Q21HH8 (DXR_SACD2), Q24UG4 (DXR_DESHY), Q254F0 (DXR_CHLFF), Q28PJ0 (DXR_JANSC), Q2A4P9 (DXR_FRATH), Q2GGR4 (DXR_EHRCR), Q2GKQ8 (DXR_ANAPZ), Q2IW86(DXR_RH0P2), Q2JM39 (DXR_SYNJB), Q2JXQ9 (DXR_SYNJA), Q2K3K0 (DXR_RHIEC), Q2L155 (DXR_B0RA1), Q2NRL1 (DXR_SODGM), Q2P4B2 (DXR_XANOM), Q2RJN4 (DXR_MOOTA), Q2SBQ3 (DXR_HAHCH), Q2SWZ2 (DXR_BURTA), Q2YBA1 (DXR_NITMU), Q313H2 (DXR_0LEA2), Q319Z8 (DXR_PR0M9), Q31 N26 (DXR_SYNE7), Q39F49 (DXR_BURL3), Q39W82 (DXR_GEOMG), Q3AB86 (DXR_CARHZ), Q3AI71 (DXR_SYNSC), Q3AUE4 (DXR_CHLCH), Q3AZ20 (DXR_SYNS9), Q3B6R4 (DXR_CHLL3), Q3BVL0 (DXR_XANC5), Q3J2X1 (DXR_CERS4), Q3JCW9 (DXR_NITOC), Q3JR34 (DXR_BURP1), Q3KHA6 (DXR_PSEPF), Q3KMV0 (DXR_CHLTA), Q3MDL2 (DXR_TRIV2), Q3SRH8 (DXR_NITWN), Q3YRZ5 (DXR_EHRCJ), Q46JT6 (DXR_PROMT), Q470E3 (DXR_CUPPJ), Q47F86 (DXR_DECAR), Q485G4 (DXR_C0LP3), Q48F65 (DXR_PSE14), Q493C8 (DXR_BLOPB), Q4FM64 (DXR_PELUB), Q4FRH9 (DXR_PSYA2), Q4JV26 (DXR_CORJK), Q4KHH0 (DXR_PSEF5), Q4QM93 (DXR_HAEI8), Q4USQ4 (DXR_XANC8), Q4ZWS2 (DXR_PSEU2), Q55663 (DXR_SYNY3), Q57T35 (DXR_SALCH), Q5E3E5 (DXR_ALIF1), Q5F5X0 (DXR_NEIG1), Q5FHA4 (DXR_EHRRG), Q5FPZ1 (DXR_GLUOX), Q5GTA4 (DXR_WOLTR), Q5H1 E7 (DXR_XANOR), Q5HB55 (DXR_EHRRW), Q5HT65 (DXR_CAMJR), Q5L0J6 (DXR_GEOKA), Q5L651 (DXR_CHLAB), Q5L9P6 (DXR_BACFN), Q5LSU9 (DXR_RUEPO), Q5NEP6 (DXR_FRATT), Q5NZG9 (DXR_AROAE), Q5PAI9 (DXR_ANAMM), Q5PD59(DXR_SALPA), Q5QUF4 (DXRJDILO), Q5SJZ6 (DXR_THET8), Q5WFT4 (DXR_ALKCK), Q5YS72 (DXR_NOCFA), Q60BA4 (DXR_METCA), Q62JD0 (DXR_BURMA), Q636K5 (DXR2_BACCZ), Q638M6 (DXR1_BACCZ), Q63T18 (DXR_BURPS), Q64PY9 (DXR_BACFR), Q65JJ3 (DXR_BACLD), Q65R75 (DXR_MANSM), Q667J3 (DXR_YERPS), Q67PA9 (DXR_SYMTH), Q6A7K8 (DXR_CUTAK), Q6AEY1 (DXR_LEIXX), Q6AP35 (DXR_DESPS), Q6D8D9 (DXR_PECAS), Q6FCG9 (DXR_ACIAD), Q6HEZ4 (DXR2_BACHK), Q6HG59 (DXR1_BACHK), Q6LN30 (DXR_PHOPR), Q6MEL5 (DXR_PARUW), Q6N5Q6 (DXR_RHOPA), Q6NGL1 (DXR_CORDI), Q720A5 (DXR_LISMF), Q72DR3 (DXR_DESVH), Q72KE2 (DXR_THET2), Q72U08 (DXR_LEPIC), Q732P8 (DXR_BACC1), Q73GG3 (DXR_WOLPM), Q73K78 (DXR_TREDE), Q73VS1 (DXR_MYCPA), Q74BW4 (DXR_GEOSL), Q7M9M7 (DXR_WOLSU), Q7MIG6 (DXR_VIBVY), Q7MUW3 (DXR_PORGI), Q7N8P3 (DXR_PHOLL), Q7NC17 (DXR_MYCGA), Q7NID1 (DXR_GLOVI), Q7NVY8 (DXR_CHRVO), Q7U8C3 (DXR_PARMW), Q7URM5 (DXR_RHOBA), Q7V0W0 (DXR_PROMP), Q7V6J8(DXR_PROMM), Q7VB62 (DXR_PROMA), Q7VIT0 (DXR_HELHP), Q7VM27 (DXR_HAEDU), Q7VRE2 (DXR_BLOFL), Q7VYC4 (DXR_BORPE), Q7WA54 (DXR_BORPA), Q7WJ88 (DXR_BORBR), Q819Y3 (DXR2_BACCR), Q81B49 (DXR1_BACCR), Q81 N10 (DXR1_BACAN), Q81WL4 (DXR2_BACAN), Q823G9 (DXR_CHLCV), Q82K41 (DXR_STRAW), Q82U01 (DXR_NITEU), Q83GY8 (DXR_TROWT), Q83IC8 (DXR_TR0W8), Q83MD3 (DXR_SHIFL), Q87EH9 (DXR_XYLFT), Q87ME3 (DXR_VIBPA), Q886N7 (DXR_PSESM), Q88MH4 (DXR_PSEPK), Q895K5 (DXR_CLOTE), Q89KP9 (DXR_BRADU), Q8A684 (DXR_BACTN), Q8D2G6 (DXR_WIGBR), Q8DBF5 (DXR_VIBVU), Q8DK30 (DXR_THEVB), Q8EGG9 (DXR_SHEON), Q8EWQ6 (DXR_MALP2), Q8F146 (DXR_LEPIN), Q8FP80 (DXR_COREF), Q8G7Y7 (DXR_BIFLO), Q8IKG4 (DXR_PLAF7), Q8K9S7 (DXR_BUCAP), Q8KG43 (DXR_CHLTE), Q8NP10 (DXR_CORGL), Q8PAV9 (DXR_XANCP), Q8PML1 (DXR_XANAC), Q8R622 (DXR_FUSNN), Q8RA28 (DXR_CALS4), Q8UC86 (DXR_AGRFC), Q8W250 (DXR_ORYSJ), Q8X8Y1 (DXR_ECO57), Q8XJR1 (DXR_CLOPE), Q8XZI5 (DXR_RALSO), Q8Y7G4 (DXR_LISMO), Q8YP49 (DXR_N0SS1), Q8Z9A6 (DXR_SALTI), Q8ZH62 (DXR_YERPE), Q8ZRP3 (DXR_SALTY), Q92C37 (DXR_LISIN), Q92LP6 (DXR_RHIME), Q97I58 (DXR_CLOAB), Q9A709 (DXR_CAUVC), Q9AJD7 (DXR_KITGR), Q9CBU3 (DXR_MYCLE), Q9JX33 (DXR_NEIMA), Q9K1G8 (DXR_NEIMB), Q9KA69 (DXR_HALH5), Q9KGU6 (DXR_PSEAE), Q9KPV8 (DXR_VIBCH), Q9KYS1 (DXR_STRCO), Q9PEI0 (DXR_XYLFA), Q9PKW8 (DXR_CHLMU), Q9PMV3 (DXR_CAMJE), Q9RCT1 (DXR_SYNP6), Q9RU84 (DXR_DEIRA), Q9WZZ1 (DXR_THEMA), Q9X5F2 (DXR_ZYMMO), Q9XES0 (DXR_MENPI), Q9XFS9 (DXR_ARATH), Q9Z8J8 (DXR_CHLPN), and Q9ZML6 (DXR_HELPJ).

[0128] DXP pathway enzymes also include 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferases (IspD; EC 2.7.7.60), which catalyze the cytidylation of 2-C-methyl-D- erythritol 4-phosphate to 4-CDP-2-C-methyl-D-erythritol. In some embodiments, the IspD is an E. coli IspD, e.g., an IspD identified by UniProt ID number Q46893. Other exemplary IspD enzymes include those identified by UniProt ID numbers A0K867 (ISPD_BURCH), A0KGH5 (ISPD_AERHH), A0LIS2 (ISPD_SYNFM), A0PV29 (ISPD_MYCUA), A0PXS4 (ISPD_CLONN), A0R8F7 (ISPD_BACAH), A0RN28 (ISPDF_CAMFF), A1AEU1 (ISPD_ECOK1), A1AX25 (ISPD_RUTMC), A1 B890 (ISPDF_PARDP), A1 K644 (ISPD_AZOSB), A1 KPR8 (ISPD_MYCBP), A1 KUV0 (ISPD_NEIMF), A1S4D9(ISPD_SHEAM), A1USA2 (ISPDF_BARBK), A1V500 (ISPD_BURMS), A1VDX6 (ISPDF_DESW), A1W1 K9 (ISPDF_CAMJJ), A1WWZ0 (ISPD_HALHL), A2BPT7 (ISPD_PROMS), A2BVB5 (ISPD_PROM5), A2SBD6 (ISPD_BURM9), A3M5X8 (ISPD_ACIBT), A3MKM4 (ISPD_BURM7), A3N0G2 (ISPD_ACTP2), A3NAL9 (ISPD_BURP6), A3NWE0 (ISPD_BURP0), A3PBH7 (ISPD_PROMO), A3PJQ3 (ISPD_CERS1), A3QC79 (ISPD_SHELP), A4F6W3 (ISPD_SACEN), A4IJG4 (ISPD_GEOTN), A4J0Y3 (ISPD_DESRM), A4JF20 (ISPD_BURVG), A4QH60 (ISPD_CORGB), A4SF04 (ISPD_CHLPM), A4TPZ7 (ISPD_YERPP), A4WDV2 (ISPD_ENT38), A4WSL5 (ISPD_CERS5), A4XLJ3 (ISPD_CALS8), A4XWR9 (ISPD_PSEMY), A4YUQ7 (ISPDF_BRASO), A5CTY4 (ISPDF_CLAM3), A5CW51 (ISPD_VESOH), A5D5L4 (ISPD_PELTS), A5EIY9 (ISPDF_BRASB), A5FP88 (ISPD_DEHMB), A5G938 (ISPD_GEOUR), A5GMW9 (ISPD_SYNPW), A5I7N1 (ISPD_CLOBH), A5IMI1 (ISPD_THEP1), A5N4M5 (ISPD_CLOK5), A5U8Q7 (ISPD_MYCTA), A5UE59 (ISPD_HAEIE), A5UHG1 (ISPD_HAEIG), A5V2U9 (ISPDF_RHIWR), A5VQP4 (ISPDF_BRUO2), A5W827 (ISPD_PSEP1), A6KXL8 (ISPD_PHOV8), A6LPA6 (ISPD_THEM4), A6LPN9 (ISPD_CLOB8), A6Q2K0 (ISPDF_NITSB), A6Q7M3 (ISPDF_SULNB), A6TD39 (ISPD_KLEP7), A6TWL0 (ISPD_ALKMQ), A6U8F8 (ISPDF_SINMW), A6V1 F5 (ISPD_PSEA7), A6VQY1 (ISPD_ACTSZ), A6X0N1 (ISPDF_BRUA4), A7FLX8 (ISPD_YERP3), A7FZ94 (ISPD_CLOB1), A7GJ99 (ISPD_CLOBL), A7GJZ7 (ISPD_BACCN), A7GZI1 (ISPDF_CAMC5), A7H5V8 (ISPDF_CAMJD), A7HDA2 (ISPDF_ANADF), A7HXV6 (ISPDF_PARL1), A7I1V2 (ISPDF_CAMHC), A7MJ57 (ISPD_CROS8), A7Z0L2 (ISPD_BACVZ), A7ZCQ2 (ISPDF_CAMC1), A7ZQJ1 (ISPD_ECO24), A8A3M6 (ISPD_ECOHS), A8ANW1 (ISPD_CITK8), A8ER37 (ISPDF_ALIB4), A8F958 (ISPD_BACP2), A8FNR8 (ISPDF_CAMJ8), A8FST0 (ISPD_SHESH), A8G3G9 (ISPD_PROM2), A8G9Z2 (ISPD_SERP5), A8H1S7 (ISPD_SHEPA), A8IBL6 (ISPDF_AZOC5), A8LKV5 (ISPDF_DINSH), A8MLB1 (ISPD_ALKOO), A9AIT5 (ISPD_BURM1), A9BE76 (ISPD_PROM4), A9BHR2 (ISPD_PETMO), A9II44 (ISPD_BORPD), A9IS87 (ISPDF_BART1), A9KSU6 (ISPD_LACP7), A9M0U7 (ISPD_NEIM0), A9MF28 (ISPD_SALAR), A9N2D3 (ISPD_SALPB), A9R119 (ISPD_YERPG), A9VN98 (ISPD_BACMK), B0B835 (ISPD_CHLT2), B0BCA0 (ISPD_CHLTB), B0BP82 (ISPD_ACTPJ), B0C9F6 (ISPD_ACAM1), B0JUF7(ISPD_MICAN), B0KSC1 (ISPD_PSEPG), B0RU03 (ISPD_XANCB), B0TK06 (ISPD_SHEHH), B0U660 (ISPD_XYLFM), B0V5E6 (ISPD_ACIBY), B0VQH8 (ISPD_ACIBS), B1GYT4 (ISPD_ENDTX), B1 HNM7 (ISPD_LYSSC), B1 I0S9 (ISPDF_DESAP), B1 IGH9 (ISPD_CLOBK), B1 IUT2 (ISPD_ECOLC), B1JB36 (ISPD_PSEPW), B1JJF8 (ISPD_YERPY), B1 KTA8 (ISPD_CLOBM), B1 LBT2 (ISPD_THESQ), B1 LQ67 (ISPD_ECOSM), B1WSY4 (ISPD_CR0S5), B1XCS3 (ISPD_ECODH), B2FK90 (ISPD_STRMK), B2HJ23 (ISPD_MYCMM), B2I2A2 (ISPD_ACIBC), B2I939 (ISPD_XYLF2), B2J1D2 (ISPD_N0SP7), B2JGK2 (ISPD_PARP8), B2K577 (ISPD_YERPB), B2RJ15 (ISPD_P0RG3), B2SUA8 (ISPD_XANOP), B2T3X2 (ISPD_PARPJ), B2TIF4 (ISPD_CLOBB), B2TZI4 (ISPD_SHIB3), B2USR1 (ISPDF_HELPS), B3DTQ9 (ISPD_BIFLD), B3EQ34 (ISPD_CHLPB), B3H1 E1 (ISPD_ACTP7), B3PYS4 (ISPDF_RHIE6), B3QIL5 (ISPDF_RHOPT), B3QP85 (ISPD_CHLP8), B3QXK8 (ISPD_CHLT3), B4EC23 (ISPD_BURCJ), B4RL14 (ISPD_NEIG2), B4RZG5 (ISPD_ALTMD), B4S8U7 (ISPD_PR0A2), B4SAG7 (ISPD_PELPB), B4SR88 (ISPD_STRM5), B4T457 (ISPD_SALNS), B4TFW7 (ISPD_SALHS), B4TTW1 (ISPD_SALSV), B5BEY4 (ISPD_SALPK), B5EMB5 (ISPD_ACIF5), B5F411 (ISPD_SALA4), B5FAF7 (ISPD_ALIFM), B5FTS5 (ISPD_SALDC), B5QW18 (ISPD_SALEP), B5RDQ3 (ISPD_SALG2), B5XV34 (ISPD_KLEP3), B5YF77 (ISPD_DICT6), B5Z3A9 (ISPD_EC05E), B5Z6H4 (ISPDF_HELPG), B5ZNB6 (ISPDF_RHILW), B6EKL6 (ISPD_ALISL), B6I6D7 (ISPD_ECOSE), B6JL03 (ISPDF_HELP2), B6YQA2 (ISPD_AZOPC), B7GMX1 (ISPD_BIFLS), B7IF61 (ISPD_THEAB), B7J4C0 (ISPD_ACIF2), B7LEG5 (ISPD_ECO55), B7LWK8 (ISPD_ESCF3), B7LXF9 (ISPD_EC08A), B7MKM1 (ISPD_ECO45), B7MYQ2 (ISPD_EC081), B7N6X7 (ISPD_ECOLU), B7NT91 (ISPD_ECO7I), B7UHG6 (ISPD_ECO27), B7VK66 (ISPD_VIBA3), B8CJP8 (ISPD_SHEPW), B8GW85 (ISPDF_CAUVN), B8ZUA7 (ISPD_MYCLB), B9DY87 (ISPD_CL0K1), B9JF01 (ISPDF_AGRRK), B9JW91 (ISPDF_ALLAM), B9K8U1 (ISPD_THENN), B9KEX3 (ISPDF_CAMLR), B9KSH8 (ISPD_CERSK), B9MJW2 (ISPD_CALBD), C0PXA7 (ISPD_SALPC), C0ZPR7 (ISPD_RH0E4), C1AI41 (ISPD_MYCBT), C1 BAC2 (ISPD_RHOOB), C1 D558 (ISPD_LARHH), C1 F763 (ISPD_ACIC5), C1 FMX6 (ISPD_CLOBJ), C3K6H8 (ISPD_PSEFS), C3KVS6 (ISPD_CL0B6), C4LBQ9 (ISPD_TOLAT), C4XSW4 (ISPDF_S0LM1), C4Z312 (ISPD_LACE2), C4ZZQ1 (ISPD_ECOBW), C6DDG2 (ISPD_PECCP), 005029 (ISPD_HAEIN), 025664(ISPDF_HELPY), 067343 (ISPD_AQUAE), 083525 (ISPDF_TREPA), 084468 (ISPD_CHLTR), P57495 (ISPD_BUCAI), P57707 (ISPD_PSEAE), P57953 (ISPD_PASMU), P69834 (ISPD_ARATH), P74323 (ISPD_SYNY3), P9WKG8 (ISPD_MYCT0), P9WKG9 (ISPD_MYCTU), Q027G1 (ISPD_S0LUE), Q02RA5 (ISPD_PSEAB), Q04VR0 (ISPD_LEPBJ), Q04XR1 (ISPD_LEPBL), Q06755 (ISPD_BACSU), Q07MZ2 (ISPDF_RH0P5), Q086A8 (ISPD_SHEFN), Q0AFW3 (ISPD_NITEC), Q0APQ6 (ISPDF_MARMM), Q0BED6 (ISPD_BURCM), Q0BTD5 (ISPDF_GRABC), Q0C0N0 (ISPDF_HYPNA), Q0I880 (ISPD_SYNS3), Q0S890 (ISPD_RHOJR), Q0SQB9 (ISPD_CLOPS), Q0T1 H8 (ISPD_SHIF8), Q0TEB1 (ISPD_EC0L5), Q0TMM2 (ISPD_CL0P1), Q118M1 (ISPD_TRIEI), Q11HV9 (ISPDF_CHESB), Q12PZ2 (ISPD_SHEDO), Q137C3 (ISPDF_RHOPS), Q13Z31 (ISPD_PARXL), Q15P31 (ISPD_PSEA6), Q165P6 (ISPD_ROSDO), Q17WU1 (ISPDF_HELAH), Q18CD1 (ISPD_CL0D6), Q1AU08 (ISPD_RUBXD), Q1 BHA5 (ISPD_BUR01), Q1CLR6 (ISPD_YERPN), Q1CU78 (ISPDF_HELPH), Q1GGW9 (ISPDF_RUEST), Q1GTN0 (ISPDF_SPHAL), Q1 I648 (ISPD_PSEE4), Q1J200 (ISPD_DEIGD), Q1 LTP8 (ISPD_BAUCH), Q1MH21 (ISPDF_RHIL3), Q1MR76 (ISPDF_LAWIP), Q1Q9K8 (ISPD_PSYCK), Q1QM99 (ISPDF_NITHX), Q1QU73 (ISPD_CHRSD), Q1R7U4 (ISPD_ECOUT), Q214R1 (ISPDF_RHOPB), Q21YT7 (ISPDF_ALBFT), Q253C1 (ISPD_CHLFF), Q28Q60 (ISPDF_JANSC), Q2G708 (ISPDF_NOVAD), Q2GEM3 (ISPD_NEOSM), Q2IQG8 (ISPDF_ANADE), Q2IW23 (ISPDF_RH0P2), Q2J542 (ISPD_FRACC), Q2JUE5 (ISPD_SYNJA), Q2K8V5 (ISPDF_RHIEC), Q2LUS9 (ISPD_SYNAS), Q2NAE1 (ISPDF_ERYLH), Q2NVM4 (ISPD_SODGM), Q2P1 L0 (ISPD_XANOM), Q2RFM0 (ISPD_MOOTA), Q2RTS1 (ISPDF_RHORT), Q2S210 (ISPD_SALRD), Q2SKW7 (ISPD_HAHCH), Q2SWT6 (ISPD_BURTA), Q2W4Q8 (ISPDF_MAGSA), Q2Y751 (ISPD_NITMU), Q2YPW1 (ISPDF_BRUA2), Q30QG7 (ISPDF_SULDN), Q310X3 (ISPDF_0LEA2), Q31C80 (ISPD_PR0M9), Q31QF6 (ISPD_SYNE7), Q31XA9 (ISPD_SHIBS), Q32CI3 (ISPD_SHIDS), Q39FB8 (ISPD_BURL3), Q39ZL5 (ISPD_GEOMG), Q3A8C6 (ISPD_SYNC1), Q3A9N7 (ISPD_CARHZ), Q3ALY8 (ISPD_SYNSC), Q3AS33 (ISPD_CHLCH), Q3AWK9 (ISPD_SYNS9), Q3B3A7 (ISPD_CHLL3), Q3BUS8 (ISPD_XANC5), Q3IDQ6 (ISPD_PSET1), Q3J2K9 (ISPD_CERS4), Q3JCS9 (ISPD_NITOC), Q3JR99 (ISPD_BURP1), Q3KH90 (ISPD_PSEPF), Q3KLN6 (ISPD_CHLTA), Q3MAF5 (ISPD_TRIV2), Q3SK38(ISPD_THIDA), Q3SSN8 (ISPDF_NITWN), Q3YYB5 (ISPD_SHISS), Q3ZAD7 (ISPD_DEHM1), Q3ZWE1 (ISPD_DEHMC), Q46GW4 (ISPD_PROMT), Q472F2 (ISPD_CUPPJ), Q47EL2 (ISPD_DECAR), Q47LV0 (ISPD_THEFY), Q487E9 (ISPD_C0LP3), Q48F81 (ISPD_PSE14), Q494E8 (ISPD_BLOPB), Q4FM31 (ISPDF_PELUB), Q4FR76 (ISPD_PSYA2), Q4JXJ7 (ISPD_CORJK), Q4KHF4 (ISPD_PSEF5), Q4QMP4 (ISPD_HAEI8), Q4UTP4 (ISPD_XANC8), Q4ZWQ6 (ISPD_PSEU2), Q57D18 (ISPDF_BRUAB), Q57KJ4 (ISPD_SALCH), Q5E328 (ISPD_ALIF1), Q5F829 (ISPD_NEIG1), Q5FQD6 (ISPDF_GLUOX), Q5GSM7 (ISPDF_WOLTR), Q5GYK6 (ISPD_XANOR), Q5HSI4 (ISPDF_CAMJR), Q5L433 (ISPD_GEOKA), Q5L6V2 (ISPD_CHLAB), Q5L917 (ISPD_BACFN), Q5LRN5 (ISPDF_RUEPO), Q5N3T2 (ISPD_SYNP6), Q5N8G1 (ISPD_ORYSJ), Q5NYJ9 (ISPD_AROAE), Q5PEG1 (ISPD_SALPA), Q5QUC3 (ISPDJDILO), Q5SLX2 (ISPD_THET8), Q5WLT7 (ISPD_ALKCK), Q5Z2R3 (ISPD_NOCFA), Q604M2 (ISPD_METCA), Q62JI5 (ISPD_BURMA), Q63HB4 (ISPD_BACCZ), Q63T70 (ISPD_BURPS), Q64P77 (ISPD_BACFR), Q65PD2 (ISPD_BACLD), Q65Q78 (ISPD_MANSM), Q66EC3 (ISPD_YERPS), Q67JP5 (ISPD_SYMTH), Q6AAV8 (ISPD_CUTAK), Q6ADI0 (ISPDF_LEIXX), Q6ARN9 (ISPDF_DESPS), Q6D1 B3 (ISPD_PECAS), Q6FAU1 (ISPD_ACIAD), Q6G164 (ISPDF_BARQU), Q6G3Z8 (ISPDF_BARHE), Q6HPT2 (ISPD_BACHK), Q6LMT3 (ISPD_PHOPR), Q6MEE8 (ISPD_PARUW), Q6N6M5 (ISPDF_RHOPA), Q6NFC1 (ISPD_CORDI), Q724H7 (ISPD_LISMF), Q72C30 (ISPDF_DESVH), Q72GN3 (ISPD_THET2), Q72P59 (ISPD_LEPIC), Q73FC1 (ISPD_BACC1), Q73G24 (ISPDF_WOLPM), Q743W5 (ISPD_MYCPA), Q746Z9 (ISPD_GEOSL), Q7C093 (ISPD_SHIFL), Q7MHQ4 (ISPD_VIBVY), Q7MQW9 (ISPDF_WOLSU), Q7MUQ9 (ISPD_PORGI), Q7N8K7 (ISPD_PHOLL), Q7NGU6 (ISPD_GLOVI), Q7NYL6 (ISPD_CHRVO), Q7TW54 (ISPD_MYCBO), Q7U559 (ISPD_PARMW), Q7UM15 (ISPD_RHOBA), Q7V2M1 (ISPD_PROMP), Q7V647 (ISPD_PROMM), Q7VDC7 (ISPD_PROMA), Q7VFU3 (ISPDF_HELHP), Q7VLT5 (ISPD_HAEDU), Q7VZN2 (ISPD_BORPE), Q7W5C9 (ISPD_BORPA), Q7WCW3 (ISPD_BORBR), Q81J63 (ISPD_BACCR), Q81VV5 (ISPD_BACAN), Q824I4 (ISPD_CHLCV), Q82GC8 (ISPD_STRAW), Q82UR9 (ISPD_NITEU), Q83MX3 (ISPDF_TROWT), Q83NK3 (ISPDF_TROW8), Q87DY4 (ISPD_XYLFT), Q87LQ2 (ISPD_VIBPA), Q886M1 (ISPD_PSESM), Q88MF7(ISPD_PSEPK), Q890M1 (ISPD_CLOTE), Q89LQ8 (ISPDF_BRADU), Q8A0U8 (ISPD_BACTN), Q8D223 (ISPD_WIGBR), Q8DC60 (ISPD_VIBVU), Q8DL91 (ISPD_THEVB), Q8EBR2 (ISPD_SHEON), Q8F7A0 (ISPD_LEPIN), Q8FEJ5 (ISPD_EC0L6), Q8FMI3 (ISPD_COREF), Q8G0H4 (ISPDF_BRUSU), Q8G7E2 (ISPD_BIFLO), Q8K9D6 (ISPD_BUCAP), Q8KCU3 (ISPD_CHLTE), Q8NMB8 (ISPD_CORGL), Q8P9Z1 (ISPD_XANCP), Q8PLR8 (ISPD_XANAC), Q8R6H2 (ISPD_FUSNN), Q8R7S6 (ISPD_CALS4), Q8UFF4 (ISPDF_AGRFC), Q8X7Y4 (ISPD_ECO57), Q8XHQ3 (ISPD_CLOPE), Q8XYW3 (ISPD_RALSO), Q8YAB5 (ISPD_LISMO), Q8YHD8 (ISPDF_BRUME), Q8YLX9 (ISPD_N0SS1), Q8Z471 (ISPD_SALTI), Q8ZBP6 (ISPD_YERPE), Q8ZMF6 (ISPD_SALTY), Q92F40 (ISPD_LISIN), Q92Q90 (ISPDF_RHIME), Q97EC9 (ISPD_CLOAB), Q98MX9 (ISPDF_RHILO), Q9A7I5 (ISPDF_CAUVC), Q9CCW6 (ISPD_MYCLE), Q9JTM3 (ISPD_NEIMA), Q9JYM4 (ISPD_NEIMB), Q9KGF8 (ISPD_HALH5), Q9KUJ2 (ISPD_VIBCH), Q9L0Q8 (ISPD_STRCO), Q9PDT6 (ISPD_XYLFA), Q9PJT1 (ISPD_CHLMU), Q9PM68 (ISPDF_CAMJE), Q9RNZ1 (ISPDF_ZYMMO), Q9RR90 (ISPD_DEIRA), Q9X1 B3 (ISPD_THEMA), Q9Z7X5 (ISPD_CHLPN), and Q9ZM19 (ISPDF_HELPJ).

[0129] DXP pathway enzymes also include 4-diphosphocytidyl-2-C-methyl-D-erythritol kinases (IspE; EC 2.7.1.148), which catalyze the phosphorylation of 4-CDP-2-C-methyl-D- erythritol to 4-CDP-2-C-methyl-D-erythritol 2-phosphate. In some embodiments, the IspE is an E. coli IspE, e.g., an IspE identified by UniProt ID number P62615. Other exemplary IspE enzymes include those identified by UniProt ID numbers A0AEZ5 (ISPE_LISW6), A0JU88 (ISPE_ARTS2), A0KAM4 (ISPE_BURCH), A0KN01 (ISPE_AERHH), A0KT85 (ISPE_SHESA), A0L5U5 (ISPE_MAGMM), A0LPG9 (ISPE_SYNFM), A0PW69 (ISPE_MYCUA), A0Q330 (ISPE_CLONN), A0Q492 (ISPE_FRATN), A0QBW1 (ISPE_MYCA1), A0R3D7 (ISPE_MYCS2), A0R8B7 (ISPE_BACAH), A1A3W4 (ISPE_BIFAA), A1AAD5 (ISPE_ECOK1), A1ALZ9 (ISPE_PELPD), A1AVE9 (ISPE_RUTMC), A1AZ43 (ISPE_PARDP), A1 BHM3 (ISPE_CHLPD), A1 K3G8 (ISPE_AZOSB), A1 KHE9 (ISPE_MYCBP), A1 KTB5 (ISPE_NEIMF), A1 R4F8 (ISPE_PAEAT), A1 RNE4 (ISPE_SHESW), A1S8R5 (ISPE_SHEAM), A1SNG8 (ISPE_NOCSJ), A1STD9 (ISPE_PSYIN), A1TEH2 (ISPE_MYCVP), A1TT72 (ISPE_ACIAC), A1 U371 (ISPE_MARN8), A1 URV3 (ISPE_BARBK), A1 UZN5 (ISPE_BURMS), A1VDQ8 (ISPE_DESVV), A1VKN9 (ISPE_POLNA), A1W091 (ISPE_CAMJJ), A1W4G3(ISPE_ACISJ), A1WGH3 (ISPE_VEREI), A1WVQ4 (ISPE_HALHL), A2BR01 (ISPE_PROMS), A2BWR1 (ISPE_PR0M5), A2C1Z6 (ISPE_PR0M1), A2CA52 (ISPE_PR0M3), A2S6B7 (ISPE_BURM9), A2SKU4 (ISPE_METPP), A3D0F8 (ISPE_SHEB5), A3DI26 (ISPE_ACET2), A3M2X4 (ISPE_ACIBT), A3MQB6 (ISPE_BURM7), A3N0D8 (ISPE_ACTP2), A3N5K2 (ISPE_BURP6), A3NR98 (ISPE_BURPO), A3PCS4 (ISPE_PROMO), A3PGS6 (ISPE_CERS1), A3QH33 (ISPE_SHELP), A4F7X3 (ISPE_SACEN), A4G919 (ISPEJHERAR), A4IJC2 (ISPE_GEOTN), A4IZZ4 (ISPE_FRATW), A4J0P1 (ISPE_DESRM), A4JHZ4 (ISPE_BURVG), A4QCP1 (ISPE_CORGB), A4SFS4 (ISPE_CHLPM), A4SK61 (ISPE_AERS4), A4T067 (ISPE_POLAQ), A4T6N7 (ISPE_MYCGI), A4TJP0 (ISPE_YERPP), A4VPC3 (ISPE_STUS1), A4WBC9 (ISPE_ENT38), A4WVE5 (ISPE_CERS5), A4X310 (ISPE_SALTO), A4XLL8 (ISPE_CALS8), A4XR57 (ISPE_PSEMY), A4Y3J5 (ISPE_SHEPC), A4YPQ3 (ISPE_BRASO), A5CTL3 (ISPE_CLAM3), A5CXR1(ISPE_VESOH), A5D654 (ISPE_PELTS), A5EEC6 (ISPE_BRASB), A5F684 (ISPE_VIBC3), A5FS53 (ISPE_DEHMB), A5G7R8 (ISPE_GEOUR), A5GLH6 (ISPE_SYNPW), A5GTK8 (ISPE_SYNR3), A5HY17 (ISPE_CLOBH), A5IMJ0 (ISPE_THEP1), A5IQ47 (ISPE_STAA9), A5N3L3 (ISPE_CL0K5), A5U154 (ISPE_MYCTA), A5VDT3 (ISPE_RHIWR), A5VI11 (ISPE_LIMRD), A5VYG1 (ISPE_PSEP1), A5WHA3 (ISPE_PSYWF), A6L5X6 (ISPE_PH0V8), A6L9X1 (ISPE_PARD8), A6LJT9 (ISPE_THEM4), A6LQF2 (ISPE_CL0B8), A6Q782 (ISPE_SULNB), A6QEE8 (ISPE_STAAE), A6T2S0 (ISPE_JANMA), A6TAP2 (ISPE_KLEP7), A6TWV7 (ISPE_ALKMQ), A6TYW9 (ISPE_STAA2), A6U6N3 (ISPE_SINMW), A6VC65 (ISPE_PSEA7), A6VQ56 (ISPE_ACTSZ), A6W1C6 (ISPE_MARMS), A6W6U5 (ISPE_KINRD), A6WSF3 (ISPE_SHEB8), A7FIG4 (ISPE_YERP3), A7FQF0 (ISPE_CL0B1), A7G9M4 (ISPE_CLOBL), A7GJV7 (ISPE_BACCN), A7H2Q3 (ISPE_CAMJD), A7H6J9 (ISPE_ANADF), A7HNL5(ISPE_FERNB), A7HR11 (ISPE_PARL1), A7IF36 (ISPE_XANP2), A7MKB5 (ISPE_CR0S8), A7MY79 (ISPE_VIBC1), A7N9I8 (ISPE_FRATF), A7WYP4 (ISPE_STAA1), A7Z0G9 (ISPE_BACVZ), A7ZKY2 (ISPE_ECO24), A7ZZE4 (ISPE_ECOHS), A8AFZ9 (ISPE_CITK8), A8F7B1 (ISPE_PSELT), A8F913 (ISPE_BACP2), A8FMF7 (ISPE_CAMJ8), A8FYZ3 (ISPE_SHESH), A8G4P2 (ISPE_PR0M2), A8GDA0 (ISPE_SERP5), A8H7A8 (ISPE_SHEPA), A8HU60 (ISPE_AZ0C5), A8LL57 (ISPE_DINSH), A8M223 (ISPE_SALAI), A8ML31 (ISPE_ALKOO), A9AEY9 (ISPE_BURM1), A9BFC2 (ISPE_PETMO), A9BP16(ISPE_DELAS), A9HAC0 (ISPE_GLUDA), A9IQK8 (ISPE_BART1), A9KKE9 (ISPE_LACP7), A9L2D7 (ISPE_SHEB9), A9M421 (ISPE_NEIMO), A9MP98 (ISPE_SALAR), A9MW00 (ISPE_SALPB), A9QZ11 (ISPE_YERPG), A9VN58 (ISPE_BACMK), A9VX42 (ISPE_METEP), B0B931 (ISPE_CHLT2), BOBARO (ISPE_CHLTB), B0BP57 (ISPE_ACTPJ), B0CC88 (ISPE_ACAM1), BOJNBO (ISPE_MICAN), B0K474 (ISPE_THEPX), BOKBHO (ISPE_THEP3), B0KND7 (ISPE_PSEPG), B0RGI6 (ISPE_CLAMS), B0RUA1 (ISPE_XANCB), B0S057 (ISPE_FINM2), B0SA95 (ISPE_LEPBA), B0SKG9 (ISPE_LEPBP), B0T7V9 (ISPE_CAUSK), B0TB90 (ISPE_HELMI), B0TR30 (ISPE_SHEHH), BOTVZO (ISPE_FRAP2), B0UUJ6 (ISPE_HISS2), B1AJP1 (ISPE_UREP2), B1 IE03 (ISPE_CLOBK), B1 IU85 (ISPE_ECOLC), B1JEQO (ISPE_PSEPW), B1JM88 (ISPE_YERPY), B1JYP8 (ISPE_BUROO), B1 KDU6 (ISPE_SHEWM), B1 KSP3 (ISPE_CLOBM), B1 LBU2 (ISPE_THESQ), B1 LH92 (ISPE_ECOSM), B1 LYB4 (ISPEJVIETRJ), B1XAP9 (ISPE_ECODH), B1XK39 (ISPE_SYNP2), B1XS63 (ISPE_POLNS), B1YGP2 (ISPE_EXIS2), B1YN51 (ISPE_BURA4), B1ZIP6 (ISPE_METPB), B2AGT9 (ISPE_CUPTR), B2G5J0 (ISPE_LIMRJ), B2GFD7 (ISPE_LIMF3), B2HDJ9 (ISPE_MYCMM), B2HUM5 (ISPE_ACIBC), B2J0A5 (ISPE_NOSP7), B2JCP0 (ISPE_PARP8), B2K2Y7 (ISPE_YERPB), B2RJI6 (ISPE_PORG3), B2S2W8 (ISPE_TREPS), B2SE44 (ISPE_FRATM), B2SXG6 (ISPE_PARPJ), B2TJM0 (ISPE_CLOBB), B2TZV6 (ISPE_SHIB3), B2UER7 (ISPE_RALPJ), B2UZH3 (ISPE_CLOBA), B2V7G0 (ISPE_SULSY), B2VEI4 (ISPE_ERWT9), B3DP37 (ISPE_BIFLD), B3DVI7 (ISPE_METI4), B3EE40 (ISPE_CHLL2), B3EP19 (ISPE_CHLPB), B3H1B7 (ISPE_ACTP7), B3PJN8 (ISPE_CELJU), B3PRG5 (ISPE_RHIE6), B3QPX7 (ISPE_CHLP8), B3QW77 (ISPE_CHLT3), B4EAR4 (ISPE_BURCJ), B4EVR4 (ISPE_PROMH), B4RKF0 (ISPE_NEIG2), B4S9D9 (ISPE_PROA2), B4SG33 (ISPE_PELPB), B4TKA8 (ISPE_SALHS), B4TXU6 (ISPE_SALSV), B4U041 (ISPE_STREM), B4U5Q9 (ISPE_HYDSO), B4ULC6 (ISPE_ANASK), B5BI72 (ISPE_SALPK), B5EHY0 (ISPE_CITBB), B5F4H2 (ISPE_SALA4), B5FBW7 (ISPE_ALIFM), B5R3J6 (ISPE_SALEP), B5R921 (ISPE_SALG2), B5XW48 (ISPE_KLEP3), B5YG57 (ISPE_THEYD), B5YXM6 (ISPE_ECO5E), B5ZCB5 (ISPE_UREU1), B5ZS25 (ISPE_RHILW), B6EHH2 (ISPE_ALISL), B6I9S3 (ISPE_ECOSE), B6YQC6 (ISPE_AZOPC), B7GPE4 (ISPE_BIFLS), B7GYQ7 (ISPE_ACIB3), B7HIL3 (ISPE_BACC4), B7HPV6 (ISPE_BACC7), B7I7P3 (ISPE_ACIB5), B7IG15 (ISPE_THEAB), B7ISV5 (ISPE_BACC2), B7JK51 (ISPE_BACCO), B7K961 (ISPE_GLOC7), B7KUL9 (ISPE_METC4), B7LGW9(ISPE_ECO55), B7LSI2 (ISPE_ESCF3), B7LXC3 (ISPE_EC08A), B7MKB0 (ISPE_ECO45), B7MTZ2 (ISPE_EC081), B7N419 (ISPE_ECOLU), B7NUX4 (ISPE_ECO7I), B7UQ96 (ISPE_ECO27), B7V0L5 (ISPE_PSEA8), B7VKH0 (ISPE_VIBA3), B8CQV5 (ISPE_SHEPW), B8DGN5 (ISPE_LISMH), B8E818 (ISPE_SHEB2), B8FY51 (ISPE_DESHD), B8H510 (ISPE_CAUVN), B8HF09 (ISPE_PSECP), B8HU41 (ISPE_CYAP4), B8J808 (ISPE_ANAD2), B8ZU60 (ISPE_MYCLB), B9DSY9 (ISPE_STRUO), B9DX96 (ISPE_CL0K1), B9E8V7 (ISPE_MACCJ), B9IZC8 (ISPE_BACCQ), B9KIB9 (ISPE_ANAMF), B9KLF9 (ISPE_CERSK), B9M5U2 (ISPE_GEODF), B9ME49 (ISPE_ACIET), B9MJX8 (ISPE_CALBD), C0MBI2 (ISPE_STRE4), C0MED6 (ISPE_STRS7), C0Q357 (ISPE_SALPC), C0QQW3 (ISPE_PERMH), C0R2S8 (ISPE_WOLWR), C0ZHC9 (ISPE_BREBN), C1AM02 (ISPE_MYCBT), C1 ESX4 (ISPE_BACC3), C1 FQ79 (ISPE_CLOBJ), C1 KYC3 (ISPEJJSMC), C3KDC2 (ISPE_PSEFS), C3KYF7 (ISPE_CL0B6), C3LJ17 (ISPE_BACAC), C3LPI7 (ISPE_VIBCM), C3P9J0 (ISPE_BACAA), C4K7Z0 (ISPE_HAMD5), C4LBL2 (ISPE_TOLAT), C4XHQ9 (ISPE_S0LM1), C4Z205 (ISPE_LACE2), C4ZTP9 (ISPE_ECOBW), C5CYZ4 (ISPE_VARPS), C5D367 (ISPE_GEOSW), C6DHY4 (ISPE_PECCP), C6E4K5 (ISPE_GEOSM), 025984 (ISPE_HELPY), 067060 (ISPE_AQUAE), 081014 (ISPE_ARATH), 083386 (ISPE_TREPA), 084810(ISPE_CHLTR), P30753 (ISPE_SALTY), P37550 (ISPE_BACSU), P42805 (ISPE_PSEAE), P45271 (ISPE_HAEIN), P56848 (ISPE_MENPI), P57267 (ISPE_BUCAI), P57833 (ISPE_PASMU), P62616 (ISPE_ECO57), P65179 (ISPE_MYCBO), P65180 (ISPE_STAAM), P65181 (ISPE_STAAN), P65182 (ISPE_STAAW), P72663 (ISPE_SYNY3), P83700 (ISPE_THET8), P93841 (ISPE_SOLLC), P9WKG6 (ISPE_MYCTO), P9WKG7 (ISPE_MYCTU), Q01QR0 (ISPE_SOLUE), Q02G05 (ISPE_PSEAB), Q03HF4 (ISPE_PEDPA), Q03T54 (ISPE_LEVBA), Q07I91 (ISPE_RHOP5), Q087I7 (ISPE_SHEFN), Q0AC00 (ISPE_ALKEH), Q0AGY8 (ISPE_NITEC), Q0B0T2 (ISPE_SYNWW), Q0BBQ6 (ISPE_BURCM), Q0BP04 (ISPE_FRATO), Q0BR06 (ISPE_GRABC), Q0C4D8 (ISPE_HYPNA), Q0HFC5 (ISPE_SHESM), Q0HYL1 (ISPE_SHESR), Q0I368 (ISPE_HISS1), Q0I9S4 (ISPE_SYNS3), Q0KEP7 (ISPEJDUPNH), Q0S4S3 (ISPE_RHOJR), Q0SQX1 (ISPE_CLOPS), Q0T5I7 (ISPE_SHIF8), Q0TIG2 (ISPE_EC0L5), QOTNAO (ISPE_CL0P1), Q0VS81 (ISPE_ALCBS), Q10VR1 (ISPE_TRIEI), Q11 KG9 (ISPE_CHESB), Q11VT3 (ISPE_CYTH3), Q12DZ8 (ISPE_POLSJ), Q12QS1(ISPE_SHEDO), Q13BT8 (ISPE_RHOPS), Q145X1 (ISPE_PARXL), Q14JH1 (ISPE_FRAT1), Q15SQ6 (ISPE_PSEA6), Q163E9 (ISPE_ROSDO), Q17Z99 (ISPE_HELAH), Q181G8 (ISPE_CL0D6), Q1AXL8 (ISPE_RUBXD), Q1 BTG7 (ISPE_BUR01), Q1C857 (ISPE_YERPA), Q1CJK4 (ISPE_YERPN), Q1CRI6(ISPE_HELPH), Q1GTW9 (ISPE_SPHAL), Q1 H3J0 (ISPE_METFK), Q1IEY5(ISPE_PSEE4), Q1J201 (ISPE_DEIGD), Q1 LRQO (ISPE_CUPMC), Q1 LTH3 (ISPE_BAUCH), Q1MKS2 (ISPE_RHIL3), Q1MQD8 (ISPE_LAWIP), Q1QED3 (ISPE_PSYCK), Q1QIJ6 (ISPE_NITHX), Q1QXD0 (ISPE_CHRSD), Q1 RCNO (ISPE_ECOUT), Q1WV68 (ISPE_LIGS1), Q20YA7 (ISPE_RHOPB), Q21 FL9(ISPE_SACD2), Q21XW4 (ISPE_ALBFT), Q251V5 (ISPE_DESHY), Q255R7(ISPE_CHLFF), Q28V59 (ISPE_JANSC), Q2A5Q2 (ISPE_FRATH), Q2FJE7(ISPE_STAA3), Q2G0S8 (ISPE_STAA8), Q2G7F1 (ISPE_NOVAD), Q2GD50 (ISPE_NEOSM), Q2GG78 (ISPE_EHRCR), Q2GKD6 (ISPE_ANAPZ), Q2IM67 (ISPE_ANADE), Q2J164 (ISPE_RHOP2), Q2JLP6 (ISPE_SYNJB), Q2JQU4 (ISPE_SYNJA), Q2KBV2 (ISPE_RHIEC), Q2LUJ9 (ISPE_SYNAS), Q2NA15(ISPE_ERYLH), Q2NRS1 (ISPE_SODGM), Q2NZW6 (ISPE_XANOM), Q2RMC8(ISPE_MOOTA), Q2RXS7 (ISPE_RHORT), Q2S4Q4 (ISPE_SALRD), Q2SLA0(ISPE_HAHCH), Q2T1 B6 (ISPE_BURTA), Q2VYT6 (ISPE_MAGSA), Q2YBH5 (ISPE_NITMU), Q2YMB5 (ISPE_BRUA2), Q2YW0 (ISPE_STAAB), Q30TG0 (ISPE_SULDN), Q30ZH4 (ISPE_OLEA2), Q31 B18 (ISPE_PROM9), Q31 RH7 (ISPE_SYNE7), Q31ZQ1 (ISPE_SHIBS), Q32GZ9 (ISPE_SHIDS), Q38V25 (ISPE_LATSS), Q39CU1 (ISPE_BURL3), Q39RQ7 (ISPE_GEOMG), Q3A311 (ISPE_SYNC1), Q3AFM4(ISPE_CARHZ), Q3AKD9 (ISPE_SYNSC), Q3AQY2 (ISPE_CHLCH), Q3AXF4 (ISPE_SYNS9), Q3B2S6 (ISPE_CHLL3), Q3BX03 (ISPE_XANC5), Q3IK98 (ISPE_PSET1), Q3J5K7 (ISPE_CERS4), Q3JDR0 (ISPE_NITOC), Q3JW85 (ISPE_BURP1), Q3K3L9 (ISPE_STRA1), Q3K6W5 (ISPE_PSEPF), Q3KKN7 (ISPE_CHLTA), Q3M3F2(ISPE_TRIV2), Q3SLR6 (ISPE_THIDA), Q3SPE5 (ISPE_NITWN), Q3YSE4 (ISPE_EHRCJ), Q3Z0S6 (ISPE_SHISS), Q3Z9E9 (ISPE_DEHM1), Q3ZZE5 (ISPE_DEHMC), Q46L57 (ISPE_PROMT), Q476F8 (ISPE_CUPPJ), Q479M3 (ISPE_DECAR), Q47SX2(ISPE_THEFY), Q47Y90 (ISPE_COLP3), Q48MV8 (ISPE_PSE14), Q492W0 (ISPE_BLOPB), Q49V04 (ISPE_STAS1), Q4FPG0 (ISPE_PELUB), Q4FVB2 (ISPE_PSYA2), Q4JU24 (ISPE_CORJK), Q4K691 (ISPE_PSEF5), Q4L3F2 (ISPE_STAHJ),Q4QL43 (ISPE_HAEI8), Q4URC0 (ISPE_XANC8), Q4ZXX1 (ISPE_PSEU2), Q57EW9 (ISPE_BRUAB), Q57NN2 (ISPE_SALCH), Q5E6T6 (ISPE_ALIF1), Q5F9F6 (ISPE_NEIG1), Q5FHM5 (ISPE_EHRRG), Q5FQP6 (ISPE_GLUOX), Q5GTB0 (ISPE_WOLTR), Q5GWR3 (ISPE_XANOR), Q5HBJ6 (ISPE_EHRRW), Q5HII1 (ISPE_STAAC), Q5HRR0 (ISPE_STAEQ), Q5HU00 (ISPE_CAMJR), Q5L3V4 (ISPE_GEOKA), Q5L568 (ISPE_CHLAB), Q5LC56 (ISPE_BACFN), Q5LX98 (ISPE_RUEPO), Q5N2S7 (ISPE_SYNP6), Q5NI19 (ISPE_FRATT), Q5P725 (ISPE_AROAE), Q5PB05 (ISPE_ANAMM), Q5PCR2 (ISPE_SALPA), Q5QV06 (ISPEJDILO), Q5WLV8 (ISPE_ALKCK), Q5YPY8 (ISPE_NOCFA), Q60A17 (ISPE_METCA), Q62FC4 (ISPE_BURMA), Q63HI8 (ISPE_BACCZ), Q63XL7 (ISPE_BURPS), Q64T40 (ISPE_BACFR), Q65PH5 (ISPE_BACLD), Q65SB8 (ISPE_MANSM), Q66AX8 (ISPE_YERPS), Q67JC2 (ISPE_SYMTH), Q6AAD6 (ISPE_CUTAK), Q6ADP2 (ISPE_LEIXX), Q6AJL6 (ISPE_DESPS), Q6D554 (ISPE_PECAS), Q6F8J0 (ISPE_ACIAD), Q6G0G3 (ISPE_BARQU), Q6G4E4 (ISPE_BARHE), Q6GBZ3 (ISPE_STAAS), Q6GJH6 (ISPE_STAAR), Q6HPX2 (ISPE_BACHK), Q6LNB1 (ISPE_PHOPR), Q6MAT6 (ISPE_PARUW), Q6NAZ1 (ISPE_RHOPA), Q6NIA1 (ISPE_CORDI), Q724M3 (ISPE_LISMF), Q72BQ8 (ISPE_DESVH), Q72GN2 (ISPE_THET2), Q72V75 (ISPE_LEPIC), Q73FG3 (ISPE_BACC1), Q73I23 (ISPE_WOLPM), Q73N18 (ISPE_TREDE), Q741W1 (ISPE_MYCPA), Q74FE9 (ISPE_GEOSL), Q7MMZ0 (ISPE_VIBVY), Q7MVU8 (ISPE_PORGI), Q7N589 (ISPE_PHOLL), Q7NPF3 (ISPE_GLOVI), Q7NQS8 (ISPE_CHRVO), Q7U7D2 (ISPE_PARMW), Q7UEV3 (ISPE_RHOBA), Q7V1 E2 (ISPE_PROMP), Q7V7W1 (ISPE_PROMM), Q7VCH6 (ISPE_PROMA), Q7VL54 (ISPE_HAEDU), Q7VUH0 (ISPE_BORPE), Q7W182 (ISPE_BORPA), Q7WNY5 (ISPE_BORBR), Q81JA2 (ISPE_BACCR), Q81VZ6 (ISPE_BACAN), Q820G3 (ISPE_STRAW), Q821X0 (ISPE_CHLCV), Q82TQ3 (ISPE_NITEU), Q839U9 (ISPE_ENTFA), Q83FU3 (ISPE_TROWT), Q83IA0 (ISPE_TROW8), Q83LD8 (ISPE_SHIFL), Q87A21 (ISPE_XYLFT), Q87RN7 (ISPE_VIBPA), Q888C5 (ISPE_PSESM), Q88PX5 (ISPE_PSEPK), Q88Z91 (ISPE_LACPL), Q899A2 (ISPE_CLOTE), Q89S79 (ISPE_BRADU), Q8AA41 (ISPE_BACTN), Q8CQU6 (ISPE_STAES), Q8D2K6 (ISPE_WIGBR), Q8DFF6 (ISPE_VIBVU), Q8DLJ1 (ISPE_THEVB), Q8DS40 (ISPE_STRMU), Q8E245 (ISPE_STRA5), Q8E7K5 (ISPE_STRA3), Q8EAR0 (ISPE_SHEON), Q8EU37 (ISPE_OCEIH), Q8EZM8 (ISPE_LEPIN), Q8FI04 (ISPE_ECOL6),Q8FQZ4 (ISPE_COREF), Q8G2D0 (ISPE_BRUSU), Q8G6I4 (ISPE_BIFLO), Q8K9X1 (ISPE_BUCAP), Q8KCC7 (ISPE_CHLTE), Q8NRY0 (ISPE_CORGL), Q8PC64 (ISPE_XANCP), Q8PNU1 (ISPE_XANAC), Q8R6C8 (ISPE_FUSNN), Q8R765 (ISPE_CALS4), Q8S2G0 (ISPE_ORYSJ), Q8UHP8 (ISPE_AGRFC), Q8XIA9 (ISPE_CLOPE), Q8Y2E0 (ISPE_RALSO), Q8YAE1 (ISPE_LISMO), Q8YFI3 (ISPE_BRUME), Q8YS61 (ISPE_NOSS1), Q8Z699 (ISPE_SALTI), Q8ZEY1 (ISPE_YERPE), Q92F77 (ISPEJJSIN), Q92RM1 (ISPE_RHIME), Q97F51 (ISPE_CLOAB), Q986C6 (ISPE_RHILO), Q9A8L7 (ISPE_CAUVC), Q9CD51 (ISPE_MYCLE), Q9JUX8 (ISPE_NEIMA), Q9JZW4 (ISPE_NEIMB), Q9K3R6 (ISPE_STRCO), Q9KGK0 (ISPE_HALH5), Q9KQ23 (ISPE_VIBCH), Q9PA75 (ISPE_XYLFA), Q9PLC0 (ISPE_CHLMU), Q9PNJ0 (ISPE_CAMJE), Q9PPN9 (ISPE_UREPA), Q9RR89 (ISPE_DEIRA), Q9X1A3 (ISPE_THEMA), Q9X3W5 (ISPE_ZYMMO), and Q9ZJH3 (ISPE_HELPJ).

[0130] DXP pathway enzymes also include 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthases (IspF; EC 4.6.1.12), which catalyze the conversion of 4-CDP-2-C-methyl-D- erythritol 2-phosphate to 2-C-methyl-D-erythritol 2,4-cyclic diphosphate. In some embodiments, the IspF is an E. coli IspF, e.g., an IspF identified by UniProt ID number P62617. Other exemplary IspF enzymes include those identified by UniProt ID numbers A0K866 (ISPF_BURCH), A0KU85 (ISPF_SHESA), A0LIS1 (ISPF_SYNFM), A0LQZ7 (ISPF_ACIC1), A0Q3K0 (ISPF_CLONN), A0Q6Y1 (ISPF_FRATN), A0QAB4 (ISPF_MYCA1), A0R8F8 (ISPF_BACAH), A0RN28 (ISPDF_CAMFF), A1AEU0 (ISPF_ECOK1), A1AJY3 (ISPF_PELPD), A1 B890 (ISPDF_PARDP), A1 BE20 (ISPF_CHLPD), A1JJT5 (ISPF_YERE8), A1 K645 (ISPF_AZOSB), A1 KPR7 (ISPF_MYCBP), A1 KUU9 (ISPF_NEIMF), A1RHF6 (ISPF_SHESW), A1S4E0 (ISPF_SHEAM), A1SNY6 (ISPF_NOCSJ), A1TG10 (ISPF_MYCVP), A1TZ51 (ISPF_MARN8), A1 USA2 (ISPDF_BARBK), A1V4Z9 (ISPF_BURMS), A1VDX6 (ISPDF_DESW), A1W1 K9 (ISPDF_CAMJJ), A1WR07 (ISPF_VEREI), A1WWY9 (ISPF_HALHL), A2SBD7 (ISPF_BURM9), A3D792 (ISPF_SHEB5), A3M665 (ISPF_ACIBT), A3MKM3 (ISPF_BURM7), A3N0G3 (ISPF_ACTP2), A3NAL8 (ISPF_BURP6), A3NWD9 (ISPF_BURP0), A3PJQ4 (ISPF_CERS1), A3QC80 (ISPF_SHELP), A4IJG5 (ISPF_GEOTN), A4IYG6 (ISPF_FRATW), A4J0Y4 (ISPF_DESRM), A4JF19 (ISPF_BURVG), A4QH59 (ISPF_CORGB), A4SFZ0 (ISPF_CHLPM), A4TPZ8(ISPF_YERPP), A4VJV0 (ISPF_STUS1), A4WDV1 (ISPF_ENT38), A4WSL4 (ISPF_CERS5), A4XCN1 (ISPF_SALTO), A4XJU4 (ISPF_CALS8), A4XWR4 (ISPF_PSEMY), A4Y940 (ISPF_SHEPC), A4YUQ7 (ISPDF_BRASO), A5CTY4 (ISPDF_CLAM3), A5D5L5 (ISPF_PELTS), A5EIY9 (ISPDF_BRASB), A5F9D7 (ISPF_VIBC3), A5G939 (ISPF_GEOUR), A5HXW3 (ISPF_CLOBH), A5N3R3 (ISPF_CL0K5), A5U8Q6 (ISPFJVIYCTA), A5UE60 (ISPF_HAEIE), A5UHG0 (ISPF_HAEIG), A5V2U9 (ISPDF_RHIWR), A5VQP4 (ISPDF_BRU02), A5W823 (ISPF_PSEP1), A6L0V6 (ISPF_PH0V8), A6LF29 (ISPF_PARD8), A6LQ55 (ISPF_CL0B8), A6Q2K0 (ISPDF_NITSB), A6Q7M3 (ISPDF_SULNB), A6TD38 (ISPF_KLEP7), A6TWK9 (ISPF_ALKMQ), A6U8F8 (ISPDF_SINMW), A6V1G1 (ISPF_PSEA7), A6VQY0 (ISPF_ACTSZ), A6W6E9 (ISPF_KINRD), A6WR25 (ISPF_SHEB8), A6X0N1 (ISPDF_BRUA4), A7FLX7 (ISPF_YERP3), A7FQ95 (ISPF_CL0B1), A7G9H1 (ISPF_CLOBL), A7GJZ8 (ISPF_BACCN), A7GZI1 (ISPDF_CAMC5), A7H5V8 (ISPDF_CAMJD), A7HDA2 (ISPDF_ANADF), A7HXV6 (ISPDF_PARL1), A7I1V2 (ISPDF_CAMHC), A7MJ58 (ISPF_CR0S8), A7MTS5 (ISPF_VIBC1), A7NBK5 (ISPF_FRATF), A7Z0L3 (ISPF_BACVZ), A7ZCQ2 (ISPDF_CAMC1), A7ZQJ0 (ISPF_ECO24), A8A3M5 (ISPF_ECOHS), A8ANW0 (ISPF_CITK8), A8ER37 (ISPDF_ALIB4), A8F959 (ISPF_BACP2), A8FNR8 (ISPDF_CAMJ8), A8FST1 (ISPF_SHESH), A8G687 (ISPF_PR0M2), A8G9Z3 (ISPF_SERP5), A8H1S8 (ISPF_SHEPA), A8IBL6 (ISPDF_AZ0C5), A8LF85 (ISPF_FRASN), A8LKV5 (ISPDF_DINSH), A8M7S7 (ISPF_SALAI), A8MLB2 (ISPF_ALKOO), A9A0H0(ISPF_DESOH), A9AIT6 (ISPF_BURM1), A9BHL8 (ISPF_PETMO), A9II47 (ISPF_BORPD), A9IS87 (ISPDF_BART1), A9KYG7 (ISPF_SHEB9), A9M0U6 (ISPF_NEIMO), A9MF29 (ISPF_SALAR), A9N2D2 (ISPF_SALPB), A9R118 (ISPF_YERPG), A9VN99 (ISPF_BACMK), B0B806 (ISPF_CHLT2), B0BC71 (ISPF_CHLTB), B0BP83 (ISPF_ACTPJ), B0CBC9 (ISPF_ACAM1), B0KSC5 (ISPF_PSEPG), B0RU02 (ISPF_XANCB), B0S2Q7 (ISPF_FINM2), B0SAT2 (ISPF_LEPBA), B0SJL7 (ISPF_LEPBP), B0TK07 (ISPF_SHEHH), B0TYX5 (ISPF_FRAP2), B0URU8 (ISPF_HISS2), B0V6G3 (ISPF_ACIBY), B0VMT6 (ISPF_ACIBS), B1GYT3 (ISPF_ENDTX), B1 HNM6 (ISPF_LYSSC), B1 I0S9 (ISPDF_DESAP), B1 ID41 (ISPF_CLOBK), B1 IUT3 (ISPF_ECOLC), B1JB32 (ISPF_PSEPW), B1JJF7 (ISPF_YERPY), B1JTX0 (ISPF_BUROO), B1 KPT3 (ISPF_SHEWM), B1KRX5 (ISPF_CLOBM), B1 LQ66 (ISPF_ECOSM), B1MGY4(ISPF_MYCA9), B1VIQ0 (ISPF_C0RU7), B1VS88 (ISPF_STRGG), B1XCS2 (ISPF_ECODH), B1YS10 (ISPF_BURA4), B2A4B2 (ISPF_NATTJ), B2HJ22 (ISPF_MYCMM), B2I336 (ISPF_ACIBC), B2JA49 (ISPF_N0SP7), B2JGK3 (ISPF_PARP8), B2K578 (ISPF_YERPB), B2RGP8 (ISPF_P0RG3), B2SDD4 (ISPF_FRATM), B2SUA9 (ISPF_XANOP), B2T3X3 (ISPF_PARPJ), B2TIU0 (ISPF_CLOBB), B2TZI5 (ISPF_SHIB3), B2UFT6 (ISPF_RALPJ), B2URI3 (ISPF_AKKM8), B2USR1 (ISPDF_HELPS), B2UYW8 (ISPF_CLOBA), B2VFZ6 (ISPF_ERWT9), B3E280 (ISPF_TRIL1), B3EMU1 (ISPF_CHLPB), B3H1 E2 (ISPF_ACTP7), B3PJB0 (ISPF_CELJU), B3PYS4 (ISPDF_RHIE6), B3QIL5 (ISPDF_RHOPT), B3QPS3 (ISPF_CHLP8), B3QV82 (ISPF_CHLT3), B3R4U4 (ISPF_CUPTR), B4EC22 (ISPF_BURCJ), B4F226 (ISPF_PROMH), B4S5L1 (ISPF_PR0A2), B4SE23 (ISPF_PELPB), B4T456 (ISPF_SALNS), B4TFW6 (ISPF_SALHS), B4TTW0 (ISPF_SALSV), B4U9N6 (ISPF_HYDSO), B5BEY3 (ISPF_SALPK), B5F410 (ISPF_SALA4), B5FAF6 (ISPF_ALIFM), B5FTS4 (ISPF_SALDC), B5QW17 (ISPF_SALEP), B5RDQ2 (ISPF_SALG2), B5XV35 (ISPF_KLEP3), B5Z3A8 (ISPF_EC05E), B5Z6H4 (ISPDF_HELPG), B5ZNB6 (ISPDF_RHILW), B6EKL5 (ISPF_ALISL), B6I6D6 (ISPF_ECOSE), B6JL03 (ISPDF_HELP2), B6YRV8 (ISPF_AZOPC), B7GT04 (ISPF_BIFLS), B7H1 F3 (ISPF_ACIB3), B7HJ25 (ISPF_BACC4), B7HQS1 (ISPF_BACC7), B7I9W0 (ISPF_ACIB5), B7ISZ6 (ISPF_BACC2), B7JK94 (ISPF_BACCO), B7LEG4 (ISPF_ECO55), B7LWK9 (ISPF_ESCF3), B7LXF8 (ISPF_EC08A), B7MKM0(ISPF_ECO45), B7MZ47 (ISPF_EC081), B7N6X6 (ISPF_ECOLU), B7NT90 (ISPF_ECO7I), B7UHG5 (ISPF_ECO27), B7V8C1 (ISPF_PSEA8), B8CJP9 (ISPF_SHEPW), B8D0A0 (ISPF_HALOH), B8D7U9 (ISPF_BUCAT), B8D9J7 (ISPF_BUCA5), B8DF19 (ISPF_LISMH), B8E8T4 (ISPF_SHEB2), B8F4V8 (ISPF_GLAP5), B8FHQ6 (ISPF_DESAL), B8G1T9 (ISPF_DESHD), B8GQ78 (ISPF_THISH), B8GW85 (ISPDF_CAUVN), B8HCR6 (ISPF_PSECP), B8HVI5 (ISPF_CYAP4), B8ZUA8 (ISPF_MYCLB), B9DXE4 (ISPF_CL0K1), B9IZH1 (ISPF_BACCQ), B9JF01 (ISPDF_AGRRK), B9JW91 (ISPDF_ALLAM), B9KAZ0 (ISPF_THENN), B9KEX3 (ISPDF_CAMLR), B9KSH9 (ISPF_CERSK), B9LZW2 (ISPF_GEODF), B9MRD4 (ISPF_CALBD), C0PXA6 (ISPF_SALPC), C0R0Y4 (ISPF_BRAHW), C0ZPR8 (ISPF_RH0E4), C1A8W3 (ISPF_GEMAT), C1AI40 (ISPF_MYCBT), C1 BAC3 (ISPF_RHOOB), C1 D559 (ISPF_LARHH), C1 DSS0 (ISPF_AZOVD), C1 ET16 (ISPF_BACC3), C1 FQ26 (ISPF_CLOBJ), C1 KYG9 (ISPFJJSMC), C3K703 (ISPF_PSEFS), C3KXW6(ISPF_CL0B6), C3LJ59 (ISPF_BACAC), C3LS19 (ISPF_VIBCM), C3P9N2 (ISPF_BACAA), C4XSW4 (ISPDF_S0LM1), C4Z3N3 (ISPF_LACE2), C4Z8W9 (ISPF_AGARV), C4ZZQ0 (ISPF_ECOBW), C5BGJ0 (ISPF_EDWI9), C5CGG7 (ISPF_KOSOT), C5D3P3 (ISPF_GEOSW), C6DDG1 (ISPF_PECCP), 025664 (ISPDF_HELPY), 067089 (ISPF_AQUAE), 083525 (ISPDF_TREPA), 084441 (ISPF_CHLTR), P44815 (ISPF_HAEIN), P57494 (ISPF_BUCAI), P57708 (ISPF_PSEAE), P57954 (ISPF_PASMU), P62368 (ISPF_PLAF7), P62369 (ISPF_PLAFX), P62618 (ISPF_ECO57), P62619 (ISPF_SHIFL), P65184 (ISPF_MYCB0), P73426 (ISPF_SYNY3), P9WKG4 (ISPF_MYCT0), P9WKG5 (ISPF_MYCTU), Q027G2 (ISPF_S0LUE), Q02R99 (ISPF_PSEAB), Q04VM3 (ISPF_LEPBJ), Q04XV1 (ISPF_LEPBL), Q06756 (ISPF_BACSU), Q07MZ2 (ISPDF_RH0P5), Q086A7 (ISPF_SHEFN), Q0A7K7 (ISPF_ALKEH), Q0AGI1 (ISPF_NITEC), Q0APQ6 (ISPDFJVIARMM), Q0AUF5 (ISPF_SYNWW), Q0BED7 (ISPF_BURCM), Q0BMD2 (ISPF_FRAT0), Q0BTD5 (ISPDF_GRABC), Q0C0N0 (ISPDF_HYPNA), Q0HL69 (ISPF_SHESM), Q0HXG7 (ISPF_SHESR), Q0I5H9 (ISPF_HISS1), Q0IC27 (ISPF_SYNS3), Q0KBM9 (ISPF_CUPNH), Q0RBN8 (ISPF_FRAAA), Q0S889 (ISPF_RHOJR), QOSQNO (ISPF_CLOPS), Q0T1 H7 (ISPF_SHIF8), Q0TEB2 (ISPF_EC0L5), Q0TMY4 (ISPF_CL0P1), Q0VQD3 (ISPF_ALCBS), Q11 HV9 (ISPDF_CHESB), Q11Q93 (ISPF_CYTH3), Q12PZ1 (ISPF_SHEDO), Q137C3 (ISPDF_RHOPS), Q13Z30 (ISPF_PARXL), Q14H93 (ISPF_FRAT1), Q15P30 (ISPF_PSEA6), Q165P5 (ISPF_ROSDO), Q17WU1 (ISPDF_HELAH), Q18CD3 (ISPF_CL0D6), Q1AU09 (ISPF_RUBXD), Q1 BHA4 (ISPF_BUR01), Q1C477 (ISPF_YERPA), Q1CLR5 (ISPF_YERPN), Q1CU78 (ISPDF_HELPH), Q1GGW9 (ISPDF_RUEST), Q1GTN0 (ISPDF_SPHAL), Q1 H2A2 (ISPF_METFK), Q1 I652 (ISPF_PSEE4), Q1 IVA8 (ISPF_KORVE), Q1 LLZ4 (ISPF_CUPMC), Q1MH21 (ISPDF_RHIL3), Q1MR76 (ISPDF_LAWIP), Q1QBM3 (ISPF_PSYCK), Q1QM99 (ISPDF_NITHX), Q1QU74 (ISPF_CHRSD), Q1 R7U5 (ISPF_ECOUT), Q214R1 (ISPDF_RHOPB), Q21 LB9 (ISPF_SACD2), Q21YT7 (ISPDF_ALBFT), Q250Q9 (ISPF_DESHY), Q253F4 (ISPF_CHLFF), Q28Q60 (ISPDF_JANSC), Q2A3Z7 (ISPF_FRATH), Q2G708 (ISPDF_NOVAD), Q2GER2 (ISPF_NEOSM), Q2GHV0 (ISPF_EHRCR), Q2GIK8 (ISPF_ANAPZ), Q2IQG8 (ISPDF_ANADE), Q2IW23 (ISPDF_RH0P2), Q2J543 (ISPF_FRACC), Q2JNA9 (ISPF_SYNJB), Q2JXJ4 (ISPF_SYNJA), Q2K8V5 (ISPDF_RHIEC), Q2KUX6 (ISPF_B0RA1), Q2LUT1(ISPF_SYNAS), Q2NAE1 (ISPDF_ERYLH), Q2NVM3 (ISPF_SODGM), Q2P1L1 (ISPF_XANOM), Q2RFM1 (ISPF_MOOTA), Q2RTS1 (ISPDF_RHORT), Q2S211 (ISPF_SALRD), Q2SKW6 (ISPF_HAHCH), Q2SWT5 (ISPF_BURTA), Q2W4Q8 (ISPDF_MAGSA), Q2Y752 (ISPF_NITMU), Q2YPW1 (ISPDF_BRUA2), Q30QG7 (ISPDF_SULDN), Q310X3 (ISPDF_0LEA2), Q319L1 (ISPF_PR0M9), Q31 P19(ISPF_SYNE7), Q31XA8 (ISPF_SHIBS), Q32CI4 (ISPF_SHIDS), Q39FB9 (ISPF_BURL3), Q39ZL6 (ISPF_GEOMG), Q3A8C7 (ISPF_SYNC1), Q3A9N8 (ISPF_CARHZ), Q3APP2 (ISPF_CHLCH), Q3B2H9 (ISPF_CHLL3), Q3BUS7 (ISPF_XANC5), Q3IDQ5(ISPF_PSET1), Q3J2K8 (ISPF_CERS4), Q3JCS8 (ISPF_NITOC), Q3JRA0 (ISPF_BURP1), Q3KH86 (ISPF_PSEPF), Q3KLR6 (ISPF_CHLTA), Q3MC53 (ISPF_TRIV2), Q3SK37 (ISPF_THIDA), Q3SSN8 (ISPDF_NITWN), Q3YT02 (ISPF_EHRCJ), Q3YYB6 (ISPF_SHISS), Q3ZAD6 (ISPF_DEHM1), Q3ZWG5 (ISPF_DEHMC), Q472F1 (ISPF_CUPPJ), Q47956 (ISPF_HAEDU), Q47EL1 (ISPF_DECAR), Q47KT3(ISPF_THEFY), Q487E8 (ISPF_C0LP3), Q48F85 (ISPF_PSE14), Q493M8 (ISPF_BLOPB), Q4FM31 (ISPDF_PELUB), Q4FSB5 (ISPF_PSYA2), Q4JXJ6 (ISPF_CORJK), Q4KHF0 (ISPF_PSEF5), Q4QMP5 (ISPF_HAEI8), Q4UTP5 (ISPF_XANC8), Q4ZWQ2 (ISPF_PSEU2), Q57D18 (ISPDF_BRUAB), Q57KJ5 (ISPF_SALCH), Q5E329(ISPF_ALIF1), Q5F830 (ISPF_NEIG1), Q5FF92 (ISPF_EHRRG), Q5FQD6 (ISPDF_GLUOX), Q5GSM7 (ISPDF_WOLTR), Q5GYK7 (ISPF_XANOR), Q5HC74 (ISPF_EHRRW), Q5HSI4 (ISPDF_CAMJR), Q5L432 (ISPF_GEOKA), Q5L6S2 (ISPF_CHLAB), Q5L8X2 (ISPF_BACFN), Q5LRN5 (ISPDF_RUEPO), Q5N549 (ISPF_SYNP6), Q5NFU1 (ISPF_FRATT), Q5NYK0 (ISPF_AROAE), Q5P993 (ISPF_ANAMM), Q5PEG2 (ISPF_SALPA), Q5QUC4 (ISPFJDILO), Q5WLT6 (ISPF_ALKCK), Q5Z2R2 (ISPF_NOCFA), Q604M1 (ISPF_METCA), Q62JI6 (ISPF_BURMA), Q63HB3 (ISPF_BACCZ), Q63T71 (ISPF_BURPS), Q64P34 (ISPF_BACFR), Q65PD1 (ISPF_BACLD), Q65Q79 (ISPF_MANSM), Q66EC2 (ISPF_YERPS), Q67JP6 (ISPF_SYMTH), Q6AAV7 (ISPF_CUTAK), Q6ADI0 (ISPDF_LEIXX), Q6ARN9 (ISPDF_DESPS), Q6D1 B4 (ISPF_PECAS), Q6EPN6 (ISPF_ORYSJ), Q6FAU4 (ISPF_ACIAD), Q6G164 (ISPDF_BARQU), Q6G3Z8 (ISPDF_BARHE), Q6HPT1 (ISPF_BACHK), Q6LMT4 (ISPF_PHOPR), Q6N6M5(ISPDF_RHOPA), Q6NFC2 (ISPF_CORDI), Q724H6 (ISPFJJSMF), Q72C30 (ISPDF_DESVH), Q72HP8 (ISPF_THET2), Q72UP7 (ISPF_LEPIC), Q73FC0(ISPF_BACC1), Q73G24 (ISPDF_WOLPM), Q73KC6 (ISPF_TREDE), Q743W4 (ISPF_MYCPA), Q747A0 (ISPF_GEOSL), Q7MHQ5 (ISPF_VIBVY), Q7MQW9 (ISPDF_WOLSU), Q7MXX0 (ISPF_PORGI), Q7N8K6 (ISPF_PHOLL), Q7NC56 (ISPF_MYCGA), Q7NFH8 (ISPF_GLOVI), Q7NYL5 (ISPF_CHRVO), Q7UU80 (ISPF_RHOBA), Q7VFU3 (ISPDF_HELHP), Q7VZN1 (ISPF_BORPE), Q7W5D0 (ISPF_BORPA), Q7WCW4 (ISPF_BORBR), Q81J62 (ISPF_BACCR), Q81VV4 (ISPF_BACAN), Q824F7 (ISPF_CHLCV), Q82GC9 (ISPF_STRAW), Q82US7 (ISPF_NITEU), Q839V8 (ISPF_ENTFA), Q83MX3 (ISPDF_TROWT), Q83NK3 (ISPDF_TR0W8), Q87DY3 (ISPF_XYLFT), Q87LQ3 (ISPF_VIBPA), Q886L7 (ISPF_PSESM), Q88MF3 (ISPF_PSEPK), Q899E9 (ISPF_CLOTE), Q89LQ8 (ISPDF_BRADU), Q8A0Y7 (ISPF_BACTN), Q8D224 (ISPF_WIGBR), Q8DC59 (ISPF_VIBVU), Q8DHC4 (ISPF_THEVB), Q8EBR3 (ISPF_SHEON), Q8F0A5 (ISPF_LEPIN), Q8FEJ6 (ISPF_EC0L6), Q8FMI4 (ISPF_COREF), Q8G0H4 (ISPDF_BRUSU), Q8G5L2 (ISPF_BIFLO), Q8K9D7 (ISPF_BUCAP), Q8KC25 (ISPF_CHLTE), Q8NMB9 (ISPF_CORGL), Q8P9Z0 (ISPF_XANCP), Q8PLR7 (ISPF_XANAC), Q8R6E7 (ISPF_FUSNN), Q8R7S8 (ISPF_CALS4), Q8RQP5 (ISPF_THET8), Q8UFF4 (ISPDF_AGRFC), Q8XI08 (ISPF_CLOPE), Q8XYW2 (ISPF_RALSO), Q8YAB4 (ISPFJJSMO), Q8YHD8 (ISPDF_BRUME), Q8YQF0 (ISPF_NOSS1), Q8Z472 (ISPF_SALTI), Q8ZBP7 (ISPF_YERPE), Q8ZMF7 (ISPF_SALTY), Q92F39 (ISPF_LISIN), Q92Q90 (ISPDF_RHIME), Q97LX0 (ISPF_CLOAB), Q98MX9 (ISPDF_RHILO), Q9A7I5 (ISPDF_CAUVC), Q9CAK8 (ISPF_ARATH), Q9CCW5 (ISPF_MYCLE), Q9JTM4 (ISPF_NEIMA), Q9JYM5 (ISPF_NEIMB), Q9KGF7 (ISPF_HALH5), Q9KUJ1 (ISPF_VIBCH), Q9L0Q7 (ISPF_STRCO), Q9M4W3 (ISPF_CATRO), Q9PDT5 (ISPF_XYLFA), Q9PJV8 (ISPF_CHLMU), Q9PM68 (ISPDF_CAMJE), Q9RNZ1 (ISPDF_ZYMMO), Q9RXS6 (ISPF_DEIRA), Q9WZB5 (ISPF_THEMA), Q9Z805 (ISPF_CHLPN), and Q9ZM19 (ISPDF_HELPJ).

[0131] DXP pathway enzymes also include 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthases (IspG; EC 1.17.7.1 or EC 1.17.7.3), which catalyze the conversion of 2-C-methyl- D-erythritol 2,4-cyclic diphosphate to (2E)-4-hydroxy-3-methylbut-2-enyl diphosphate. In some embodiments, the IspG is an E. coli IspG, e.g., an IspG identified by UniProt ID number P62620. Other exemplary IspG enzymes include those identified by UniProt ID numbers A0JUS8 (ISPG_ARTS2), A0KUJ5 (ISPG_SHESA), A0LDN3 (ISPG_MAGMM),A0LV34 (ISPG_ACIC1 ), A0Q6U8 (ISPG_FRATN), A0RIQ2 (ISPG_BACAH), A0RPL8 (ISPG_CAMFF), A1AE53 (ISPG_EC0K1), A1AW46 (ISPG_RUTMC), A1 B323 (ISPG_PARDP), A1JKS2 (ISPG_YERE8), A1KML3 (ISPGJVIYCBP), A1 KUD8 (ISPG_NEIMF), A1 R501 (ISPG_PAEAT), A1 RHQ3 (ISPG_SHESW), A1S863 (ISPG_SHEAM), A1SU39 (ISPG_PSYIN), A1TZQ0 (ISPG_MARN8), A1 UR54 (ISPG_BARBK), A1V4K1 (ISPG_BURMS), A1VZ41 (ISPG_CAMJJ), A2S2A2 (ISPG_BURM9), A3D6V9 (ISPG_SHEB5), A3M211 (ISPG_ACIBT), A3MK75 (ISPG_BURM7), A3N1 H8 (ISPG_ACTP2), A3NA54 (ISPG_BURP6), A3NVX1 (ISPG_BURPO), A3QCG2 (ISPG_SHELP), A4IQZ4 (ISPG_GEOTN), A4TMT7 (ISPG_YERPP), A4WD93 (ISPG_ENT38), A4X4L9 (ISPG_SALTO), A4XLZ4 (ISPG_CALS8), A4XY32 (ISPG_PSEMY), A4Y8U0 (ISPG_SHEPC), A4YKQ8 (ISPG_BRASO), A5CT01 (ISPG_CLAM3), A5CX35 (ISPG_VESOH), A5ETI5 (ISPG_BRASB), A5FS85 (ISPG_DEHMB), A5FX97 (ISPG_ACICJ), A5IIP2 (ISPG_THEP1), A5U6M2 (ISPG_MYCTA), A5UAB9 (ISPG_HAEIE), A5UGH1 (ISPG_HAEIG), A5VYT5 (ISPG_PSEP1), A6L089 (ISPG_PH0V8), A6LGR5 (ISPG_PARD8), A6Q1 U2 (ISPG_NITSB), A6QC65 (ISPG_SULNB), A6TCD4 (ISPG_KLEP7), A6UE79 (ISPG_SINMW), A6V0W0 (ISPG_PSEA7), A6VQX6 (ISPG_ACTSZ), A6VV06 (ISPG_MARMS), A6WQP7 (ISPG_SHEB8), A6WXY9 (ISPG_BRUA4), A7FFY9 (ISPG_YERP3), A7GSX5 (ISPG_BACCN), A7GZ54 (ISPG_CAMC5), A7H4D8 (ISPG_CAMJD), A7MGV7 (ISPG_CR0S8), A7MU42 (ISPG_VIBC1), A7Z6S2 (ISPG_BACVZ), A7ZCT9 (ISPG_CAMC1), A7ZPV8 (ISPG_ECO24), A8A321 (ISPG_ECOHS), A8AD71 (ISPG_CITK8), A8F4Y8 (ISPG_PSELT), A8FF87 (ISPG_BACP2), A8FLB6 (ISPG_CAMJ8), A8FT70 (ISPG_SHESH), A8GHW5 (ISPG_SERP5), A8H245 (ISPG_SHEPA), A8L6E2 (ISPG_FRASN), A8LI72 (ISPG_DINSH), A8M723 (ISPG_SALAI), A9HJ48 (ISPG_GLUDA), A9IYW1 (ISPG_BART1), A9KXK8 (ISPG_SHEB9), A9LZN8 (ISPG_NEIMO), A9M820 (ISPG_BRUC2), A9MHL5 (ISPG_SALAR), A9N201 (ISPG_SALPB), A9R802 (ISPG_YERPG), A9VH61 (ISPG_BACMK), A9WR14 (ISPG_RENSM), A9WWQ2 (ISPG_BRUSI), B0B9G5 (ISPG_CHLT2), B0BB44 (ISPG_CHLTB), B0BQB9 (ISPG_ACTPJ), B0C6E1 (ISPG_ACAM1), B0JJJ8 (ISPG_MICAN), B0KPI7 (ISPG_PSEPG), B0RDZ5 (ISPG_CLAMS), B0RTS9 (ISPG_XANCB), B0TLI4 (ISPG_SHEHH), B0USG8 (ISPG_HISS2), B0V5G7 (ISPG_ACIBY), B0VKR8 (ISPG_ACIBS), B1 HSS4 (ISPG_LYSSC),B1 IWE6 (ISPG_ECOLC), B1JDV8 (ISPG_PSEPW), B1JS05 (ISPG_YERPY), B1 KKJ2 (ISPG_SHEWM), B1 LC43 (ISPG_THESQ), B1 LNH0 (ISPG_ECOSM), B1VGA4 (ISPG_C0RU7), B1VYP5 (ISPG_STRGG), B1WQF1 (ISPG_CR0S5), B1XAZ0 (ISPG_ECODH), B1XHZ7 (ISPG_SYNP2), B1YLA2 (ISPG_EXIS2), B1YR44 (ISPG_BURA4), B2A390 (ISPG_NATTJ), B2FL74 (ISPG_STRMK), B2GKS3 (ISPG_KOCRD), B2I3E5 (ISPG_ACIBC), B2IE63 (ISPG_BEII9), B2J1G1 (ISPG_N0SP7), B2K9Q0 (ISPG_YERPB), B2S7K6 (ISPG_BRUA1), B2SG03 (ISPG_FRATM), B2TXU0 (ISPG_SHIB3), B2U9U9 (ISPG_RALPJ), B2UKT9 (ISPG_AKKM8), B2UTK0 (ISPGJHELPS), B2V7Y2 (ISPG_SULSY), B2VE89 (ISPG_ERWT9), B3CNN4 (ISPG_WOLPP), B3GY64 (ISPG_ACTP7), B3PDM1 (ISPG_CELJU), B3PR71 (ISPG_RHIE6), B3QBC6 (ISPG_RHOPT), B4EAW9 (ISPG_BURCJ), B4RMG4 (ISPG_NEIG2), B4RV89 (ISPG_ALTMD), B4SRZ0 (ISPG_STRM5), B4T0P9 (ISPG_SALNS), B4TD93 (ISPG_SALHS), B4TR95 (ISPG_SALSV), B4U9V3 (ISPG_HYDSO), B5BAY5 (ISPG_SALPK), B5EJF3 (ISPG_ACIF5), B5F198 (ISPG_SALA4), B5FAX2 (ISPG_ALIFM), B5FR63 (ISPG_SALDC), B5R582 (ISPG_SALEP), B5RCZ2 (ISPG_SALG2), B5XNL5 (ISPG_KLEP3), B5YIQ4 (ISPG_THEYD), B5Z0Y4 (ISPG_EC05E), B5Z6Z7 (ISPG_HELPG), B5ZUA0 (ISPG_RHILW), B6EGY7 (ISPG_ALISL), B6I587 (ISPG_ECOSE), B6JA27 (ISPG_AFIC5), B6JLL3 (ISPG_HELP2), B7GH89 (ISPG_ANOFW), B7H069 (ISPG_ACIB3), B7HCQ3 (ISPG_BACC4), B7HPH8 (ISPG_BACC7), B7I5G7 (ISPG_ACIB5), B7IFY1 (ISPG_THEAB), B7IYD0 (ISPG_BACC2), B7JC30 (ISPG_ACIF2), B7JN03 (ISPG_BACCO), B7K5R5 (ISPG_RIP01), B7KJJ4 (ISPG_GL0C7), B7LDA7 (ISPG_ECO55), B7LKC3 (ISPG_ESCF3), B7M7L9 (ISPG_EC08A), B7MI00 (ISPG_ECO45), B7MYF0 (ISPG_EC081), B7N6A3 (ISPG_ECOLU), B7NRG5 (ISPG_ECO7I), B7UGW1 (ISPG_ECO27), B7UWI8 (ISPG_PSEA8), B7VJT8 (ISPG_VIBA3), B8CKR1 (ISPG_SHEPW), B8E9S7 (ISPG_SHEB2), B8HG44 (ISPG_PSECP), B8HSI6 (ISPG_CYAP4), B8I6E1 (ISPG_RUMCH), B8ZRU5 (ISPG_MYCLB), B9E6U1 (ISPG_MACCJ), B9IY45 (ISPG_BACCQ), B9JE02 (ISPG_AGRRK), B9JV07 (ISPG_ALLAM), B9KD64 (ISPG_CAMLR), B9LA27 (ISPG_NAUPA), C0QRI5 (ISPG_PERMH), C0R5E5 (ISPG_WOLWR), C0RF32 (ISPG_BRUMB), C0ZF58 (ISPG_BREBN), C1AFY4 (ISPG_MYCBT), C1 B2V4 (ISPG_RHOOB), C1DD43 (ISPG_LARHH), C1 DE57 (ISPG_AZOVD), C1ES01 (ISPG_BACC3), C1 L2Z7 (ISPG_LISMC), C3K1 L4(ISPG_PSEFS), C3LKV7 (ISPG_BACAC), C3MAL6 (ISPG_SINFN), C3P8I5 (ISPG_BACAA), C4K4I8 (ISPG_HAMD5), C4L3K5 (ISPG_EXISA), C4ZX90 (ISPG_ECOBW), C5D4Q3 (ISPG_GEOSW), C6BYK9 (ISPG_MARSD), C6DBH4 (ISPG_PECCP), F4K0E8 (ISPG_ARATH), 025342 (ISPG_HELPY), 067496 (ISPG_AQUAE), 083460 (ISPG_TREPA), 084060 (ISPG_CHLTR), P44667 (ISPGJHAEIN), P54482 (ISPG_BACSU), P57374 (ISPG_BUCAI), P57987 (ISPG_PASMU), P58665 (ISPG_AGRFC), P58666 (ISPG_NOSS1), P58667 (ISPG_CLOPE), P58668 (ISPGJJSMO), P58669 (ISPG_RALSO), P58670 (ISPG_SALTI), P58671 (ISPG_SALTY), P58672 (ISPG_YERPE), P62621 (ISPG_EC0L6), P62622 (ISPG_ECO57), P72241 (ISPG_PROST), P73672 (ISPG_SYNY3), P9WKG2 (ISPG_MYCTO), P9WKG3 (ISPG_MYCTU), Q029T9 (ISPG_SOLUE), Q02RV7 (ISPG_PSEAB), Q04UL2 (ISPG_LEPBJ), Q04YW2 (ISPG_LEPBL), Q07V91 (ISPG_RH0P5), Q085U6 (ISPG_SHEFN), Q0AE42 (ISPG_NITEC), Q0BMA6 (ISPG_FRATO), QOBUKO (ISPG_GRABC), Q0C4J3 (ISPG_HYPNA), Q0HKV9 (ISPG_SHESM), Q0HX57 (ISPG_SHESR), Q0I2E9 (ISPG_HISS1), Q0I9J4 (ISPG_SYNS3), Q0RDR2 (ISPG_FRAAA), Q0T204 (ISPG_SHIF8), QOTEXO (ISPG_EC0L5), QOVNEO (ISPG_ALCBS), Q10W70 (ISPG_TRIEI), Q12PT4 (ISPG_SHEDO), Q14IL6 (ISPG_FRAT1), Q17XU2 (ISPG_HELAH), Q1C5I8 (ISPG_YERPA), Q1CK91 (ISPG_YERPN), Q1CTP7 (ISPG_HELPH), Q1GIC1 (ISPG_RUEST), Q1H0U9 (ISPG_METFK), Q1 IEI1 (ISPG_PSEE4), Q1 IRS5 (ISPG_KORVE), Q1LU77 (ISPG_ BAUCH), Q1MAC5 (ISPG_RHIL3), Q1MSE5 (ISPG_LAWIP), Q1QD18 (ISPG_PSYCK), Q1QQI9 (ISPG_NITHX), Q1QTK8 (ISPG_CHRSD), Q1 R8L8 (ISPG_ECOUT), Q21C20 (ISPG_RHOPB), Q21 KT3 (ISPG_SACD2), Q254D2 (ISPG_CHLFF), Q28R10 (ISPG_JANSC), Q2J2S7 (ISPG_RH0P2), Q2J717 (ISPG_FRACC), Q2JPZ9 (ISPG_SYNJB), Q2JS69 (ISPG_SYNJA), Q2K333 (ISPG_RHIEC), Q2NS40 (ISPG_SODGM), Q2P3M7 (ISPG_XANOM), Q2RWE4 (ISPG_RHORT), Q2SDW4 (ISPG_HAHCH), Q2YLG4 (ISPG_BRUA2), Q30TM4 (ISPG_SULDN), Q31QC4 (ISPG_SYNE7), Q31XX5 (ISPG_SHIBS), Q32D47 (ISPG_SHIDS), Q3AK30 (ISPG_SYNSC), Q3AXP3 (ISPG_SYNS9), Q3BUK3 (ISPG_XANC5), Q3ICZ5 (ISPG_PSET1), Q3JRQ3 (ISPG_BURP1), Q3K7B6 (ISPG_PSEPF), Q3KMW4 (ISPG_CHLTA), Q3MG27 (ISPG_TRIV2), Q3SVD0 (ISPG_NITWN), Q3YRZ7 (ISPG_EHRCJ), Q3YZ37 (ISPG_SHISS), Q3ZZC9 (ISPG_DEHMC), Q47BR6 (ISPG_DECAR), Q47RY0(ISPG_THEFY), Q47WC0 (ISPG_C0LP3), Q48LZ4 (ISPG_PSE14), Q492E0 (ISPG_BLOPB), Q4FNA0 (ISPG_PELUB), Q4FTW7 (ISPG_PSYA2), Q4JV28 (ISPG_CORJK), Q4K6U9 (ISPG_PSEF5), Q4QNH4 (ISPG_HAEI8), Q4UTW8 (ISPG_XANC8), Q4ZX23 (ISPG_PSEU2), Q57BA5 (ISPG_BRUAB), Q57LI6 (ISPG_SALCH), Q5E772 (ISPG_ALIF1), Q5F913 (ISPG_NEIG1), Q5FHA6 (ISPG_EHRRG), Q5FUR7 (ISPG_GLUOX), Q5GRK4 (ISPG_WOLTR), Q5H0N8 (ISPG_XANOR), Q5HB57 (ISPG_EHRRW), Q5HV95 (ISPG_CAMJR), Q5KX35 (ISPG_GEOKA), Q5L669 (ISPG_CHLAB), Q5L7W2 (ISPG_BACFN), Q5LQ99 (ISPG_RUEPO), Q5N3W3 (ISPG_SYNP6), Q5NH64 (ISPG_FRATT), Q5NR50 (ISPG_ZYMMO), Q5P7B3 (ISPG_AROAE), Q5PAJ1 (ISPG_ANAMM), Q5PNI2 (ISPG_SALPA), Q5QYA9 (ISPGJDILO), Q5SLI8 (ISPG_THET8), Q5WHB2 (ISPG_ALKCK), Q5YS74 (ISPG_NOCFA), Q604Q5 (ISPG_METCA), Q62JW4 (ISPG_BURMA), Q634Q9 (ISPG_BACCZ), Q63UT3 (ISPG_BURPS), Q64N34 (ISPG_BACFR), Q65HA9 (ISPG_BACLD), Q65R84 (ISPG_MANSM), Q667Z9 (ISPG_YERPS), Q67PA7 (ISPG_SYMTH), Q6A7L2 (ISPG_CUTAK), Q6AEX9 (ISPG_LEIXX), Q6AP32 (ISPG_DESPS), Q6D276 (ISPG_PECAS), Q6FEM3 (ISPG_ACIAD), Q6G104 (ISPG_BARQU), Q6G1X4 (ISPG_BARHE), Q6HDN9 (ISPG_BACHK), Q6K8J4 (ISPG_ORYSJ), Q6LU49 (ISPG_PHOPR), Q6MD85 (ISPG_PARUW), Q6NCF3 (ISPG_RHOPA), Q6NGL3 (ISPG_CORDI), Q71ZM9 (ISPGJJSMF), Q72CD9 (ISPG_DESVH), Q72H18 (ISPG_THET2), Q72TR2 (ISPG_LEPIC), Q730Q8 (ISPG_BACC1), Q73IP1 (ISPG_WOLPM), Q73N90 (ISPG_TREDE), Q73VS3 (ISPG_MYCPA), Q74D60 (ISPG_GEOSL), Q7M8Z2 (ISPG_WOLSU), Q7MNF1 (ISPG_VIBVY), Q7MVT7 (ISPG_PORGI), Q7N706 (ISPG_PHOLL), Q7NBH3 (ISPG_MYCGA), Q7NFA4 (ISPG_GLOVI), Q7NS88 (ISPG_CHRVO), Q7TXN6 (ISPG_MYCBO), Q7U712 (ISPG_PARMW), Q7UWC8 (ISPG_RHOBA), Q7V215 (ISPG_PROMP), Q7V7G9 (ISPG_PROMM), Q7VBS7 (ISPG_PROMA), Q7VI04 (ISPG_HELHP), Q7VME2 (ISPG_HAEDU), Q7VWL0 (ISPG_BORPE), Q7W6P6 (ISPG_BORPA), Q7WHN0 (ISPG_BORBR), Q818H8 (ISPG_BACCR), Q81 LV7 (ISPG_BACAN), Q823I7 (ISPG_CHLCV), Q82K43 (ISPG1_STRAW), Q82ML3 (ISPG2_STRAW), Q82XV0 (ISPG_NITEU), Q83K43 (ISPG_SHIFL), Q83N18 (ISPG_TROWT), Q83NE4 (ISPG_TROW8), Q84GJ3 (ISPG_THETH), Q87A73 (ISPG_XYLFT), Q87S16 (ISPG_VIBPA), Q886Z0(ISPG_PSESM), Q88PJ7 (ISPG_PSEPK), Q895K3 (ISPG_CLOTE), Q89VV9 (ISPG_BRADU), Q8A4T0 (ISPG_BACTN), Q8D1Y3 (ISPG_WIGBR), Q8DEZ8 (ISPG_VIBVU), Q8DK70 (ISPG_THEVB), Q8EUI6 (ISPG_MALP2), Q8F1 H5 (ISPG_LEPIN), Q8FP82 (ISPG_COREF), Q8FYT2 (ISPG_BRUSU), Q8G7Y6 (ISPG_BIFLO), Q8K9P4 (ISPG_BUCAP), Q8KG23 (ISPG_CHLTE), Q8NP12 (ISPG_CORGL), Q8P9R7 (ISPG_XANCP), Q8PLJ8 (ISPG_XANAC), Q8RA30 (ISPG_CALS4), Q8RG40 (ISPG_FUSNN), Q8YJ17 (ISPG_BRUME), Q92L19 (ISPG_RHIME), Q97I56 (ISPG_CLOAB), Q98FG0 (ISPG_RHILO), Q9A9W0 (ISPG_CAUVC), Q9CBU5 (ISPG_MYCLE), Q9HXJ4 (ISPG_PSEAE), Q9JU34 (ISPG_NEIMA), Q9JZ40 (ISPG_NEIMB), Q9KD18 (ISPG_HALH5), Q9KTX1 (ISPG_VIBCH), Q9KYR9 (ISPG2_STRCO), Q9PAE3 (ISPG_XYLFA), Q9PKY3 (ISPG_CHLMU), Q9PPM1 (ISPG_CAMJE), Q9RXC9 (ISPG_DEIRA), Q9WZZ3 (ISPG_THEMA), Q9X7W2 (ISPG1_STRCO), Q9Z8H0 (ISPG_CHLPN), and Q9ZLL0 (ISPG_HELPJ).

[0132] DXP pathway enzymes also include 4-hydroxy-3-methylbut-2-enyl diphosphate reductases (IspH; EC 1 .17.7.4), which catalyze the conversion of (2E)-4-hydroxy-3- methylbut-2-enyl diphosphate to isopentenyl diphosphate and dimethylallyl diphosphate. In some embodiments, the IspH is an E. coli IspH, e.g., an IspH identified by UniProt ID number P62623. Other exemplary IspH enzymes include those identified by UniProt ID numbers A0A2Z5WA18 (ISPH_BOTBR), A0KG42 (ISPH_AERHH), A0LD71 (ISPH_MAGMM), A0LW20 (ISPH_ACIC1), A0PKQ3 (ISPH_MYCUA), A0Q4T9 (ISPH_FRATN), A0RIR1 (ISPH_BACAH), A0RQC4 (ISPH_CAMFF), A1A777 (ISPH_ECOK1), A1ANP9 (ISPH_PELPD), A1AXS1 (ISPH_RUTMC), A1 B842 (ISPH_PARDP), A1 BDJ5 (ISPH_CHLPD), A1JJE4 (ISPH_YERE8), A1 K4R4 (ISPH_AZOSB), A1 KS64 (ISPH_NEIMF), A1SZP0 (ISPH_PSYIN), A1TE06 (ISPH_MYCVP), A1TYZ2 (ISPH_MARN8), A1 UKL5 (ISPH_MYCSK), A1 URX2 (ISPH_BARBK), A1VHK1 (ISPH.DESVV), A1VZM8 (ISPH.CAMJJ), A2SJA8 (ISPH_METPP), A3M9H2 (ISPH_ACIBT), A3N2G8 (ISPH_ACTP2), A3PGE9 (ISPH_CERS1), A3Q4N0 (ISPH_MYCSJ), A4IR05 (ISPH_GEOTN), A4IYW2 (ISPH_FRATW), A4QCY9 (ISPH_CORGB), A4SGC0 (ISPH_CHLPM), A4SIX8 (ISPH_AERS4), A4T986 (ISPH_MYCGI), A4TQF0 (ISPH_YERPP), A4W6E4 (ISPH_ENT38), A4WVQ4 (ISPH_CERS5), A5CVK1 (ISPH_VESOH), A5FPY7 (ISPH_DEHMB), A5GE10 (ISPH_GEOUR), A5IME1 (ISPH_THEP1), A5UD69(ISPH_HAEIE), A5UID9 (ISPH_HAEIG), A5WCZ2 (ISPH_PSYWF), A6L1 P5 (ISPH_PH0V8), A6LIB3 (ISPH_PARD8), A6Q2R7 (ISPH_NITSB), A6Q7P9 (ISPH_SULNB), A6T4G3 (ISPH_KLEP7), A6TRF9 (ISPH_ALKMQ), A6VQH8 (ISPH_ACTSZ), A6WWG6 (ISPH_BRUA4), A7FMD4 (ISPH_YERP3), A7GSY4 (ISPH_BACCN), A7GXB5 (ISPH_CAMC5), A7H3F3 (ISPH_CAMJD), A7HCQ8 (ISPH_ANADF), A7I0N0 (ISPH_CAMHC), A7MIM0 (ISPH_CR0S8), A7NA22 (ISPH_FRATF), A7Z6T1 (ISPH_BACVZ), A7ZCD0 (ISPH_CAMC1), A7ZHB8 (ISPH_ECO24), A7ZVX7 (ISPH_ECOHS), A8ALT3 (ISPH_CITK8), A8FFA1 (ISPH_BACP2), A8FLU3 (ISPH_CAMJ8), A8G9L7 (ISPH_SERP5), A8H1 H6 (ISPH_SHEPA), A8LLC7 (ISPH_DINSH), A8MFE2 (ISPH_ALKOO), A9BGS3 (ISPH_PETMO), A9IQQ9 (ISPH_BART1), A9M1J9 (ISPH_NEIMO), A9MR43 (ISPH_SALAR), A9MYG8 (ISPH_SALPB), A9R005 (ISPH_YERPG), A9VHR7 (ISPH_BACMK), B0B991 (ISPH_CHLT2), BOBAXO (ISPH_CHLTB), B0BRB3 (ISPH_ACTPJ), B0C4N8 (ISPH_ACAM1), B0JVA7 (ISPH_MICAN), B0K197 (ISPH_THEPX), B0K9L0 (ISPH_THEP3), B0RY26 (ISPH_XANCB), B0TJB2 (ISPH_SHEHH), B0U064 (ISPH_FRAP2), B0U3Q7 (ISPH_XYLFM), B0UV08 (ISPH_HISS2), B0V8K4 (ISPH_ACIBY), B0VQ53 (ISPH_ACIBS), B1 HTG5 (ISPH_LYSSC), B1 IRE6 (ISPH_ECOLC), B1JKZ5 (ISPH_YERPY), B1 LBI2 (ISPH_THESQ), B1 LFV9 (ISPH_ECOSM), B1W2D7 (ISPH_STRGG), B1WTZ2 (ISPH_CROS5), B1XBF4 (ISPH_ECODH), B1YL84 (ISPH_EXIS2), B1ZXD4 (ISPH_OPITP), B2FU83 (ISPH_STRMK), B2HZW3 (ISPH_ACIBC), B2I6V0 (ISPH_XYLF2), B2IED0 (ISPH_BEII9), B2IZV5 (ISPH_NOSP7), B2K3M9 (ISPH_YERPB), B2S3D9 (ISPH_TREPS), B2S9W9 (ISPH_BRUA1), B2SES8 (ISPH_FRATM), B2STC6 (ISPH_XANOP), B2U248 (ISPH_SHIB3), B2UUG0 (ISPH_HELPS), B2VH04 (ISPH_ERWT9), B3CM01 (ISPH_WOLPP), B3E441 (ISPH_TRIL1), B3EFF5 (ISPH_CHLL2), B3EM42 (ISPH_CHLPB), B3GYK1 (ISPH_ACTP7), B3PSB9 (ISPH_RHIE6), B3QQE0 (ISPH_CHLP8), B3QSU2 (ISPH_CHLT3), B4R8T7 (ISPH_PHEZH), B4RPZ2 (ISPH_NEIG2), B4S4X8 (ISPH_PROA2), B4SCU0 (ISPH_PELPB), B4SP20 (ISPH_STRM5), B4T6H5 (ISPH_SALNS), B4TIE9 (ISPH_SALHS), B4TWP0 (ISPH_SALSV), B4UEK4 (ISPH_ANASK), B5BLL3 (ISPH_SALPK), B5E9S9 (ISPH_CITBB), B5ERJ7 (ISPH_ACIF5), B5F728 (ISPH_SALA4), B5FA63 (ISPH_ALIFM), B5FHE3 (ISPH_SALDC), B5R1 N9 (ISPH_SALEP), B5RG84 (ISPH_SALG2), B5Y232 (ISPH_KLEP3), B5YEC2 (ISPH_DICT6), B5YYC1 (ISPH_ECO5E), B5ZSV9(ISPH_RHILW), B6ENC7 (ISPH_ALISL), B6HYX7 (ISPH_ECOSE), B6JMP4 (ISPH_HELP2), B6YQB8 (ISPH_AZOPC), B7GH78 (ISPH_ANOFW), B7GVR1 (ISPH_ACIB3), B7HCR2 (ISPH_BACC4), B7HPI8 (ISPH_BACC7), B7IAU0 (ISPH_ACIB5), B7IYD9 (ISPH_BACC2), B7JBD6 (ISPH_ACIF2), B7JN13 (ISPH_BACCO), B7K4V8 (ISPH_RIP01), B7KEG3 (ISPH_GL0C7), B7L4F1 (ISPH_ECO55), B7LVN5 (ISPH_ESCF3), B7M0C5 (ISPH_EC08A), B7MAE9 (ISPH_ECO45), B7MNN4 (ISPH_EC081), B7N7Q3 (ISPH_ECOLU), B7NHD3 (ISPH_ECO7I), B7UI74 (ISPH_ECO27), B8D752 (ISPH_BUCAT), B8D8U8 (ISPH_BUCA5), B8DMK7 (ISPH_DESVM), B8E2T7 (ISPH_DICTD), B8EQB9 (ISPH_METSB), B8F6E6 (ISPH_GLAP5), B8GQR4 (ISPH_THISH), B8HWD3 (ISPH_CYAP4), B8J3G9 (ISPH_DESDA), B9E6U8 (ISPH_MACCJ), B9IY56 (ISPH_BACCQ), B9JAQ2 (ISPH_AGRRK), B9K8E2 (ISPH_THENN), B9KD74 (ISPH_CAMLR), B9KIX9 (ISPH_ANAMF), B9L9K5 (ISPH_NAUPA), B9LZG4 (ISPH_GEODF), C0Q4I9 (ISPH_SALPC), C0R4T5 (ISPH_WOLWR), C0ZL73 (ISPH_BREBN), C1A2V7 (ISPH_RH0E4), C1AYI4 (ISPH_RHOOB), C1 DPH8 (ISPH_AZOVD), C1 ESI2 (ISPH_BACC3), C1 KV98 (ISPH_LISMC), C3LKW7 (ISPH_BACAC), C3P8J4 (ISPH_BACAA), C4K4R2 (ISPH_HAMD5), C4L3Z5 (ISPH_EXISA), C4LD10(ISPH_TOLAT), C4XUD2 (ISPH_S0LM1), C4ZPV5 (ISPH_ECOBW), C5B7M7 (ISPH_EDWI9), C5CIU9 (ISPH_KOSOT), C5D4R3 (ISPH_GEOSW), C6DF01 (ISPH_PECCP), C6E2B3 (ISPH_GEOSM), I1WW99 (ISPH_BURP2), 067625 (ISPH_AQUAE), 083558 (ISPH_TREPA), 084867 (ISPH_CHLTR), P0A5I1 (ISPH1_MYCB0), P0A5I3 (ISPH2_MYCB0), P0C632 (ISPH_CAMJE), P0DMK8 (ISPH2_BURPS), P44976 (ISPH_HAEIN), P54473 (ISPH_BACSU), P57247 (ISPH_BUCAI), P57960 (ISPH_PASMU), P58673 (ISPH_AGRFC), P58674 (ISPH_N0SS1), P58675 (ISPH_CL0PE), P58676 (ISPHJJSMO), P58677 (ISPH_RALSO), P58678 (ISPH_SALTI), P58679 (ISPH_SALTY), P58680 (ISPH_YERPE), P62624 (ISPH_EC0L6), P62625 (ISPH_ECO57), P65185 (ISPH_HELPY), P65186 (ISPH_HELPJ), P65191 (ISPH_NEIMA), P65192 (ISPH_NEIMB), P65193 (ISPH_XYLFA), P65194 (ISPH_XYLFT), P9WKF8 (ISPH1_MYCT0), P9WKF9 (ISPH1_MYCTU), P9WKG0 (ISPH2_MYCT0), P9WKG1 (ISPH2_MYCTU), Q0BNK1 (ISPH_FRATO), QOBYQO (ISPH_HYPNA), Q0S489 (ISPH_RHOJR), Q0STT3 (ISPH_CLOPS), Q0T8G5 (ISPH_SHIF8), Q0TLW2(ISPH_EC0L5), Q0TRF3 (ISPH_CL0P1), Q0VSD8 (ISPH_ALCBS), Q10WA8(ISPH_TRIEI), Q11 KC7 (ISPH_CHESB), Q11YY5 (ISPH_CYTH3), Q12EY5 (ISPH_POLSJ), Q14I06 (ISPH_FRAT1), Q16AJ6 (ISPH_ROSDO), Q187C6 (ISPH_CL0D6), Q1B4H4 (ISPH_MYCSS), Q1C0J0 (ISPH_YERPA), Q1CMU8 (ISPH_YERPN), Q1CSL3 (ISPH_HELPH), Q1GDG5 (ISPH_RUEST), Q1 LSS7 (ISPH_BAUCH), Q1MKH8 (ISPH_RHIL3), Q1Q974 (ISPH_PSYCK), Q1 0B2 (ISPH_CHRSD), Q1 RGH3 (ISPH_ECOUT), Q21 HK6 (ISPH_SACD2), Q255J4 (ISPH_CHLFF), Q2A587 (ISPH_FRATH), Q2GDX3 (ISPH_NEOSM), Q2NVY3 (ISPH_SODGM), Q2P5A8 (ISPH_XANOM), Q2S9U2 (ISPH_HAHCH), Q30S81 (ISPH_SULDN), Q316F5 (ISPH_0LEA2), Q326J4 (ISPH_SHIBS), Q32K68 (ISPH_SHIDS), Q39XB6 (ISPH_GEOMG), Q3AT23 (ISPH_CHLCH), Q3B241 (ISPH_CHLL3), Q3BW40 (ISPH_XANC5), Q3J5X0 (ISPH_CERS4), Q3KKH8 (ISPH_CHLTA), Q3M8X6 (ISPH_TRIV2), Q3Z5Y1 (ISPH_SHISS), Q3Z6U4 (ISPH_DEHM1), Q3ZYL6 (ISPH_DEHMC), Q47BK8 (ISPH_DECAR), Q486R1 (ISPH_C0LP3), Q493S1 (ISPH_BLOPB), Q4FQY8 (ISPH_PSYA2), Q4QLQ5 (ISPH_HAEI8), Q4US42 (ISPH_XANC8), Q57EP6 (ISPH_BRUAB), Q57TL2 (ISPH_SALCH), Q5E7N1 (ISPH_ALIF1), Q5FAF2 (ISPH_NEIG1), Q5FFL5 (ISPH_EHRRG), Q5FUH7 (ISPH_GLUOX), Q5GTN6 (ISPH_WOLTR), Q5H2D9 (ISPH_XANOR), Q5HB13 (ISPH_EHRRW), Q5HUR4 (ISPH_CAMJR), Q5KX24 (ISPH_GEOKA), Q5L5D3 (ISPH_CHLAB), Q5L9K3 (ISPH_BACFN), Q5LNJ7 (ISPH_RUEPO), Q5NGK4 (ISPH_FRATT), Q5NP61 (ISPH_ZYMMO), Q5P224 (ISPH_AROAE), Q5PAE7 (ISPH_ANAMM), Q5PKI4 (ISPH_SALPA), Q5QZR7 (ISPHJDILO), Q5SMC8 (ISPH_THET8), Q5WHC6 (ISPH_ALKCK), Q5YQ94 (ISPH_NOCFA), Q607E5 (ISPH_METCA), Q629Z7 (ISPH1_BURMA), Q62HM8 (ISPH2_BURMA), Q634Q0 (ISPH_BACCZ), Q63WH0 (ISPH1_BURPS), Q64PU1 (ISPH_BACFR), Q65H97 (ISPH_BACLD), Q65RQ4 (ISPH_MANSM), Q66ES1 (ISPH_YERPS), Q67QZ8 (ISPH_SYMTH), Q6AA89 (ISPH_CUTAK), Q6ADV0 (ISPH_LEIXX), Q6AL80 (ISPH_DESPS), Q6AVG6 (ISPH_ORYSJ), Q6D0C6 (ISPH_PECAS), Q6G0C9 (ISPH_BARQU), Q6G4C5 (ISPH_BARHE), Q6HDN0 (ISPH_BACHK), Q6LUK8 (ISPH_PHOPR), Q6MC97 (ISPH_PARUW), Q6N1Y1 (ISPH2_RHOPA), Q6N3G0 (ISPH1_RHOPA), Q6NI36 (ISPH_CORDI), Q71ZL9 (ISPH_LISMF), Q72G08 (ISPH_DESVH), Q72G65 (ISPH_THET2), Q72S57 (ISPH_LEPIC), Q730P8 (ISPH_BACC1), Q73FQ1 (ISPH_WOLPM), Q73NQ6 (ISPH_TREDE), Q73WH6(ISPH_MYCPA), Q749Y8 (ISPH_GEOSL), Q7M8Y6 (ISPH_WOLSU), Q7MNM5 (ISPH_VIBVY), Q7MWK6 (ISPH_PORGI), Q7N8W9 (ISPH_PHOLL), Q7NG74 (ISPH_GLOVI), Q7NS59 (ISPH_CHRVO), Q7U9K4 (ISPH_PARMW), Q7UDT8 (ISPH_SHIFL), Q7ULU1 (ISPH_RHOBA), Q7VJV5 (ISPH_HELHP), Q7VPK4 (ISPH_HAEDU), Q7VYS2 (ISPH_BORPE), Q7W9B4 (ISPH_BORPA), Q7WHF2 (ISPH_BORBR), Q81 LU9 (ISPH_BACAN), Q822D9 (ISPH_CHLCV), Q82IE8 (ISPH_STRAW), Q82WM1 (ISPH_NITEU), Q83MR9 (ISPH_TROWT), Q83NB2 (ISPH_TR0W8), Q87S87 (ISPH_VIBPA), Q895G2 (ISPH_CLOTE), Q89QW7 (ISPH2_BRADU), Q89UU5 (ISPH1_BRADU), Q8A625 (ISPH_BACTN), Q8D2R2 (ISPH_WIGBR), Q8DET0 (ISPH_VIBVU), Q8DK29 (ISPH_THEVB), Q8EWR9 (ISPH_MALP2), Q8F3I3 (ISPH_LEPIN), Q8FQP0 (ISPH_COREF), Q8G257 (ISPH_BRUSU), Q8G4L8 (ISPH_BIFLO), Q8I295 (ISPH_PLAF7), Q8K9Z4 (ISPH_BUCAP), Q8KFN9 (ISPH_CHLTE), Q8NRM2 (ISPH_CORGL), Q8PBG4 (ISPH_XANCP), Q8PN17 (ISPH_XANAC), Q8RA76 (ISPH_CALS4), Q8RI52 (ISPH_FUSNN), Q8YFR1 (ISPH_BRUME), Q92RG2 (ISPH_RHIME), Q94B35 (ISPH_ARATH), Q97I09 (ISPH_CLOAB), Q985W3 (ISPH_RHILO), Q9FBM1 (ISPH_STRCO), Q9KD37 (ISPH_HALH5), Q9KU44 (ISPH_VIBCH), Q9PL59 (ISPH_CHLMU), Q9RSG0 (ISPH_DEIRA), Q9X1F7 (ISPH_THEMA), Q9X781 (ISPH_MYCLE), and Q9Z6P2 (ISPH_CHLPN).

[0133] DXP pathway enzymes also include isopentenyl-diphosphate Delta-isomerases (Idi; EC 5.3.3.2), which catalyze the isomerization of isopentenyl diphosphate and dimethylallyl diphosphate. In some embodiments, the Idi is an E. coli Idi, e.g., an Idi identified by UniProt ID number Q46822. Other exemplary Idi enzymes include those identified by UniProt ID numbers A0A1 D8PLI2 (IDI1_CANAL), A0AII5 (IDI2_LISW6), A0B6E1 (IDI2_METTP), A0PL81 (IDI2_MYCUA), A0RBV4 (IDI2_BACAH), A1AF79 (IDI_ECOK1), A1 BDG7 (IDI2_CHLPD), A1 KJG2 (IDI_MYCBP), A1 R1M6 (IDI_PAEAT), A1 RTI3 (I DI2_PYRIL), A1T5G2 (IDI_MYCVP), A1T741 (IDI2_MYCVP), A1WXH5 (IDI_HALHL), A3PL07 (IDI_CERS1), A4FK76 (IDI_SACEN), A4QG13 (IDI_CORGB), A4SGE6 (IDI2_CHLPM), A4TE63 (IDI2_MYCGI), A4VR98 (IDI2_STUS1), A4WE41 (IDI_ENT38), A4WH01 (IDI2_PYRAR), A4WRA6 (IDI_CERS5), A4YIM3 (IDI2_METS5), A5CV36 (IDI_CLAM3), A5IDN6 (IDI2_LEGPC), A5IVC7 (IDI2_STAA9), A5U3A5 (IDI_MYCTA), A5VK00 (IDI2JJMRD), A6QJI7 (IDI2_STAAE), A6TDP3 (IDI_KLEP7), A6U473 (IDI2_STAA2),A6UW89 (IDI2_META3), A7GN36 (IDI2_BACCN), A7IAG4 (IDI2_METB6), A7IMW3 (IDI2_XANP2), A7MPA0 (IDI2_CROS8), A7N787 (IDI2_VIBC1), A7X5W0 (IDI2_STAA1), A7Z638 (IDI2_BACVZ), A7ZQZ8 (IDI_ECO24), A8A430 (IDI_ECOHS), A8AAN4 (IDI2JGNH4), A8AP95 (IDI_CITK8), A8AUV1 (IDI2_STRGC), A8EYM2 (IDI2_RICCK), A8FEM3 (IDI2_BACP2), A8GDW2 (IDI_SERP5), A8GNY6 (IDI2_RICAH), A8GSH4 (IDI2_RICRS), A8GWR2 (I DI2_RICB8), A8LQ20 (I DI_DINSH), A8Z536 (IDI2_STAAT), A9AST0 (IDI_BURM1), A9MRI5 (IDI_SALAR), A9N3L5 (IDI_SALPB), A9VMA7 (IDI2_BACMK), B0BXY6 (IDI2_RICR0), B0RIB7 (IDI_CLAMS), B1 ITB3 (IDI_ECOLC), B1 LR71 (IDI_ECOSM), B1VTW2 (IDI_STRGG), B1XEH6 (IDI_ECODH), B1YA32 (IDI2_PYRNV), B2G7E3 (IDI2_LIMRJ), B2GFH1 (IDI_KOCRD), B2HGA4 (IDI2_MYCMM), B2ILS5 (IDI2_STRPS), B2U0Q6 (IDI_SHIB3), B3EFC7 (IDI2_CHLL2), B3QQG6 (IDI2_CHLP8), B3WEJ5 (IDI2_LACCB), B4SCG2 (IDI2_PELPB), B4T534 (IDI_SALNS), B4TGV9 (IDI_SALHS), B4TUQ7 (IDI_SALSV), B5BFK4 (IDI_SALPK), B5F5G3 (IDI_SALA4), B5F9P6 (IDI_ALIFM), B5FUF2 (IDI_SALDC), B5QXG6 (IDI_SALEP), B5RE00 (IDI_SALG2), B5XUF5 (IDI_KLEP3), B5YQ81 (IDI_EC05E), B6I722 (IDI_ECOSE), B6YST3 (IDI2_THE0N), B7HHQ2 (IDI2_BACC4), B7HL09 (IDI2_BACC7), B7IP77 (IDI2_BACC2), B7JGY4 (IDI2_BACC0), B7LYF3 (IDI_EC08A), B7MM76 (IDI_ECO45), B7MZ42 (IDI_EC081), B7N7D3 (IDI_ECOLU), B7NW30 (IDI_ECO7I), B7UHT8 (IDI_ECO27), B8DFU4 (IDI2_LISMH), B8ZLF5 (IDI2_STRPJ), B9IVM2 (IDI2_BACCQ), B9KK15 (IDI_CERSK), C0PY11 (IDI_SALPC), C1AP18 (IDI_MYCBT), C1C5C3 (IDI2_STRP7), C1 EMZ6 (IDI2_BACC3), C1 L2T9 (IDI2JJSMC), C3L9F9 (IDI2_BACAC), C3MJQ6 (IDI2_SULIL), C3MZ14 (IDI2_SULIM), C3N063 (IDI2_SULIA), C3N8S7 (IDI2_SULIY), C3NMP1 (IDI2_SULIN), C3P586 (IDI2_BACAA), C3PNP9 (IDI2_RICAE), C4K1D6 (IDI2_RICPU), C4KJA2 (IDI2_SULIK), C5A0G1 (IDI_ECOBW), C5D3G3 (IDI2_GE0SW), C6A3U0 (IDI2_THESM), I1 RZ92 (IDI 1_GIBZE), 026154 (IDI2_METTH), 027997 (IDI2_ARCFU), 035586 (IDI1_MESAU), 035760 (IDI1_RAT), 042641 (IDI1_PHARH), 048964 (IDI1_CAMAC), 048965 (IDI2_CAMAC), 051627 (IDI2_B0RBU), 058893 (IDI2_PYRH0), P0DB80 (IDI2_STRP3), P0DB81 (IDI2_STRPQ), P15496 (IDI1_YEAST), P50740 (IDI2_BACSU), P58044 (IDI1_M0USE), P58052 (IDI2_STAAU), P60923 (IDI_C0RDI), P61615 (IDI2_SACSH), P65102 (IDI2_STAAM), P65103 (IDI2_STRP1), P65105 (IDI2_STRP8), P74287 (IDI2_SYNY3), P95997 (IDI2_SACS2), P99172 (IDI2_STAAN), P9WKK4 (IDI_MYCTO), P9WKK5 (IDI_MYCTU), Q01335 (IDI2_PSEVU),Q038V3 (IDI2_LACP3), Q03S19 (IDI2_LEVBA), Q04M86 (IDI2_STRP2), Q0RBQ7 (IDI_FRAAA), Q0SMG9 (IDI2_B0RAP), Q0T107 (IDI_SHIF8), Q0TDW3 (IDI_EC0L5), Q10132 (IDI1_SCHP0), Q12TH8 (IDI2_METBU), Q13907 (IDI1 JHUMAN), Q16DR2 (IDI_ROSDO), Q1 LZ95 (IDI 1_B0VIN), Q1 R7E2 (IDI_ECOUT), Q1 RIK2 (IDI2_RICBR), Q28W27 (IDI_JANSC), Q2FEF1 (IDI2_STAA3), Q2FVR9 (IDI2_STAA8), Q2RIU8 (IDI2_M00TA), Q2YYY9 (IDI2_STAAB), Q31 EW3 (IDI2_HYDCU), Q31WF1 (IDI_SHIBS), Q32BV2 (IDI_SHIDS), Q38929 (IDI1_ARATH), Q38X74 (IDI2_LATSS), Q39471 (IDI2_CLABR), Q39472 (IDI1_CLABR), Q39664 (IDI2_CLAXA), Q3AQM4 (IDI2_CHLCH), Q3B213 (IDI2_CHLL3), Q3IUB0 (IDI2_NATPD), Q3MAB0 (IDI2_TRIV2), Q3YXY0 (IDI_SHISS), Q42553 (IDI2_ARATH), Q46CL4 (IDI2_METBF), Q48U28 (IDI2_STRPM), Q49ZS3 (IDI2_STAS1), Q4L8K4 (IDI2_STAHJ), Q4R4W5 (IDI1_MACFA), Q4ULD7 (I DI2_RICFE), Q4WM52 (IDI1_ASPFU), Q57K77 (IDI_SALCH), Q58272 (IDI2_METJA), Q5E7U8 (IDI_ALIF1), Q5HDL0 (IDI2_STAAC), Q5HLP8 (IDI2_STAEQ), Q5LWT6 (IDI_RUEPO), Q5N019 (IDI2_SYNP6), Q5NWG5 (IDI2_AR0AE), Q5P011 (IDI1_AR0AE), Q5PL31 (IDI_SALPA), Q5R8R6 (IDI1_P0NAB), Q5UX45 (IDI_HALMA), Q5WUY8 (IDI2_LEGPL), Q5X3K0 (IDI2_LEGPA), Q5XCM6 (IDI2_STRP6), Q5YXN4 (IDI2_N0CFA), Q5YYB6 (IDI_NOCFA), Q5ZTV7 (IDI2_LEGPH), Q63DN3 (IDI2_BACCZ), Q65I10 (IDI2_BACLD), Q660I6 (IDI2_B0RGP), Q67NT4 (IDI2_SYMTH), Q68WS6 (IDI2_RICTY), Q6A5Z1 (IDI_CUTAK), Q6AC73 (I DI_LEIXX), Q6D3F5 (IDI_PECAS), Q6G6X4 (IDI2_STAAS), Q6GE88 (IDI2_STAAR), Q6HL56 (IDI2_BACHK), Q6L1S1 (IDI2_PICT0), Q6LUX5 (IDI_PHOPR), Q6M174 (IDI2_METMP), Q6MMK2 (IDI2_BDEBA), Q71ZT7 (IDI2_LISMF), Q73AZ6 (IDI2_BACC1), Q746I8 (IDI2_THET2), Q76CZ1 (IDI2_THEK0), Q7N0A6 (IDI2_PH0LL), Q7N1V4 (IDI1_PH0LL), Q7VEU0 (IDI_MYCBO), Q7X5H2 (IDI_CITFR), Q81 FS0 (IDI2_BACCR), Q81SX4 (IDI2_BACAN), Q82MJ7 (IDI_STRAW), Q837E2 (IDI2_ENTFA), Q83MJ9 (IDI_SHIFL), Q87JH5 (IDI2_VIBPA), Q88WB6 (IDI2_LACPL), Q8BFZ6 (IDI2_M0USE), Q8CRB6 (IDI2_STAES), Q8DJ26 (IDI2_THEVB), Q8DR48 (IDI2_STRR6), Q8DUI9 (IDI2_STRMU), Q8EST0 (IDI2_0CEIH), Q8FE75 (IDI_EC0L6), Q8FND7 (IDI_COREF), Q8KFR5 (IDI2_CHLTE), Q8KP37 (IDI_AGRME), Q8L1 I4 (IDI2_PARZE), Q8NN99 (IDI_CORGL), Q8NV55 (IDI2_STAAW), Q8PW37 (IDI2_METMA), Q8TT35 (IDI2_METAC), Q8TX99 (IDI2_METKA), Q8U2H9 (IDI2_PYRFU), Q8XD58 (IDI_ECO57), Q8Y7A5 (IDI2_LISM0), Q8YNH4 (IDI2_N0SS1), Q8Z3X9 (IDI_SALTI), Q8ZM82 (IDI_SALTY), Q8ZYF6 (IDI2_PYRAE), Q92BX2 (I DI2_LISI N),Q92HM7 (IDI2_RICCN), Q96YW9 (IDI2_SULT0), Q97CC2 (IDI2_THEV0), Q97SH8 (IDI2_STRPN), Q989L5 (IDI2_RHIL0), Q9BXS1 (IDI2_HUMAN), Q9CIF5 (IDI2_LACLA), Q9HHE4 (IDI2_HALSA), Q9HLX2 (IDI2_THEAC), Q9HP40 (IDI_HALSA), Q9KK75 (IDI_BRELN), Q9KWD1 (IDI_AGRRH), Q9KWF6 (IDI2_KITGR), Q9KWG2 (IDI2_STRC1), Q9NH02 (IDI_DICDI), Q9RVE2 (IDI2_DEIRA), Q9UZS9 (IDI2_PYRAB), Q9X7Q6 (IDI_STRCO), Q9YB30 (IDI2_AERPE), Q9Z5D3 (IDI_CERS4), and Q9ZD90 (IDI2_RICPR).

[0134] In some embodiments, two or more DXP pathway enzymes are proteins of interest, under the control of one or more gluconate-regulated promoters as described herein. In some embodiments, DXP pathway enzymes Dxs and Dxr are under the control of a gluconate-regulated promoter or promoters. In other embodiments, DXP pathway enzymes Dxs and Idi are under the control of a gluconate-regulated promoter or promoters. In further embodiments, DXP pathway enzymes Dxr and Idi are under the control of a gluconate- regulated promoter or promoters. In yet further embodiments, DXP pathway enzymes Dxs, Dxr, and Idi are under the control of a gluconate-regulated promoter or promoters.

[0135] One gluconate-regulated promoter can be operably linked to one, two, or three DXP pathway enzymes, and hence, one, two, or three gluconate-regulated promoters can be present. In embodiments wherein two or three gluconate-regulated promoters are present, the gluconate-regulated promoters, operator regions, and GntR binding site(s) can have identical sequences or different sequences.

[0136] In some embodiments, proteins of interest are terpene synthases, such as myrcene synthases, which catalyze the conversion of geranyl diphosphate to myrcene, a linear monoterpene. In some embodiments, terpene synthases comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:50.

[0137] In some embodiments, proteins of interest are transcriptional regulators controlling the expression of regulatory proteins. For example, by controlling the expression of crp*, a variant of the crp gene that codes for a mutated CRP protein which does not require cAMP for activity, the expression of more than 300 genes in E. coli can be modified. The use of CRP* in E. coli to enhance sugars utilization for xylitol production from corncob hydrolysates has been reported (Yuan, et al., 2020, Appl. Microbiol. Biotechnol. 104:2039-2050).Furthermore, to create specific phenotypes with different applications, regulatory circuits can be designed based on the use of the E. coli repressors cl (UniProt identifier P03034, SEQ ID NO:53), Lacl (UniProt identifier P03023, SEQ ID NO:54), AraC (UniProt identifier P0A9E0, SEQ ID NO:55), and TetR (UniProt identifier P0ACT4, SEQ ID NO:56), and Aliivibrio fischeri LuxR (UniProt identifier P12746, SEQ ID NO:57).

[0138] For another example, catalytically inactive Cas9 (dCas9) enzymes ( / .e., Cas9s incapable of cleaving DNA) can be used to create gene knockdowns. If dCas9s are proteins of interest with expression induced as described herein, and specific guide RNAs are present, dCas9s can bind to DNA and repress gene transcription. The use of different transcriptional regulators for applications in synthetic biology has been reviewed by Engstrom et al., 2017, Synt. Systems. Biotechnol. 2:176-191. Exemplary dCas9s include Streptococcus thermophilus Cas9 (UniProt identifier G3ECR1 , SEQ ID NO:58) comprising one or both mutations D31A and H868A (Sapranauskas et al., 2011 , Nucleic Acids Res. 39:9275-9282; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. U.S.A. 109:E2579-E2586) and Streptococcus pyogenes serotype M1 Cas9 (UniProt identifier Q99ZW2, SEQ ID NO:59) comprising one, some, or all mutations D10A, R70A, A97-150, A312-409, H840A, A1099- 1368, R1333A / R1135A (Jinek ef a / ., 2012, Science 337:816-821 ; Fonfara et al., 2014, Nucleic Acids Res. 42:2577-2590; Nishimasu et al., 2014, Cell 156:935-949; Anders et al., 2014, Nature 513:569-573).6.2.5.2. Non-Coding Sequences of Interest

[0139] Expression systems of the present disclosure can be used in the production of any sequences of interest that do not encode a protein or have a reduced potential to be translated to a protein. Putting non-coding or non-translated sequences of interest under regulation by gluconate can allow such sequences of interest to be transcribed only at desired times or under desired conditions.

[0140] For example, expression systems can be used to generate RNA molecules that play functional roles in cells (e.g., ribosomal RNAs; tRNAs; ribozymes) or that can disrupt one or more activities of cells (e.g., RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs) for RNA interference (RNAi) applications; or antisense RNAs (asRNAs)). RNAs that disrupt one or more activities of cells may be referred to herein as “regulatory RNAs”.

[0141] Expression of regulatory RNAs can be used to silence genes by interfering with the translation or stability of their messenger RNAs. By operably linking expression of sequences of interest from which regulatory RNAs can be transcribed to regulatory regions as disclosed herein, the silencing effect of regulatory RNAs can be made conditional upon induction of expression, making it possible to apply to genes essential for growth. Furthermore, creating expression vectors for regulatory RNAs can be less laborious than gene knockout methods. RNA-mediated regulatory mechanisms and their potential applications in synthetic biology and metabolic engineering have been described (e.g., Na et al., 2013, Nat. Biotechnol. 31 :170-174).

[0142] In some embodiments, sequences of interest are transcribed to microRNAs (miRNAs).

[0143] In some embodiments, sequences of interest are transcribed to small interfering RNAs (siRNAs).

[0144] In some embodiments, sequences of interest are transcribed to antisense RNAs (asRNAs)).

[0145] In some embodiments, sequences of interest are transcribed to single guide RNAs (sgRNAs) for use in CRISPR-based techniques (e.g., CRISPR interference (CRISPRi); CRISPR gene editing.6.3. Engineered Prokaryotic Cells6.3.1. Parental Microorganisms

[0146] Parental microorganisms that can be engineered to comprise expression systems of the disclosure can be any prokaryote. In some embodiments, the prokaryote has the ability to import gluconate from a medium or produce gluconate when provided with one or more of sucrose, glucose, or fructose.

[0147] In some embodiments, the microorganism is E. coll. In particular aspects, the E. coll is E. coli-K 2 or a strain derived therefrom, such as E. coli MG1655. In other aspects, the E. coli is E. coli W.

[0148] In some embodiments, the microorganism is Bacillus subtilis.

[0149] In some embodiments, the microorganism is Pseudomonas putida.

[0150] In some embodiments, the microorganism is Klebsiella oxytoca.

[0151] In some embodiments, the microorganism is Pantoea ananatis.

[0152] In some embodiments, the microorganism is Tatumella citrea.

[0153] In some embodiments, the microorganism is Zymomonas mobilis.

[0154] In some embodiments, the microorganism is Corynebacterium glutamicum.

[0155] In some embodiments, the microorganism is E. coli SuA6 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS HIcrr, Amak, AgntK, AidnK).

[0156] In some embodiments, the microorganism is E. coli SuA7.1 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS HIcrr, Amak, AidnK, Aglk).

[0157] In some embodiments, the microorganism is E. coli SuA5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR).

[0158] In some embodiments, the microorganism is E. coli K-12 strain further comprising the genotype Apts HIcrr; AgntK; AidnK; Aedd::Km.eda. The E. coli K-12 strain with this genotype has the following phenotype: reduced ability to use glucose, reduced ability to phosphorylate gluconate, reduced ability to dehydrate gluconate-6P, and increased expression of KDGP aldolase relative to parental E. coli K-12. As will be apparent to a person of ordinary skill in the art, other genotypes can impart the same phenotype.

[0159] In some embodiments, the microorganism is E. coli K-12 strain further comprises the genotype Apts HIcrr; AgntK; AidnK; Aedd::Km.eda + pLac-Achro-TK 81.6.3.2. Engineering Methods

[0160] Parental microorganisms can be engineered using techniques known in the art. For example, expression systems described herein can be introduced or engineered into parental microorganism via techniques known in the art.

[0161] In some embodiments, expression systems are engineered into parental microorganisms by introducing nucleic acids comprising all of promoters, operator regions, gntR binding sites, and sequences of interest into cells of the parental microorganism.

[0162] In some embodiments, expression systems are formed in situ by replacing one, two, three, or all four of endogenous promoters, operator regions, gntR binding sites, and / or endogenous sequences with promoters, operator regions, gntR binding sites, and / or sequences of interest described herein. For example, in some embodiments, expression systems are engineered into parental microorganisms by replacing endogenous regulatory elements with promoters, operator regions, and / or gntR binding sites as described herein, such that endogenous sequences previously regulated by the endogenous regulatory elements are operably linked to the promoters, operator regions, and / or gntR binding sites described herein, thereby forming expression systems of the present disclosure in situ. Replacement of endogenous regulatory elements with promoters, operator regions, and / or gntR binding sites described herein can render gluconate-inducible the expression of endogenous sequences that are constitutively expressed or are inducibly expressed by inducers other than gluconate in parental microorganisms, or increase expression if the endogenous sequences are under control of a relatively weak gluconate-inducible promoter / operator region (such as that of the GntKU promoter / operator region of E. coli) in parental microorganisms.

[0163] For another example, in some embodiments, sequences of interest (optionally with operator regions and / or GntR binding sites) described herein replace endogenous sequences (e.g., GntK and / or GntU coding regions of parental E. coli) with sequences of interest, thereby forming expression systems of the present disclosure in situ. These expression systems allow expression of sequences of interest by endogenous gluconate- inducible regulatory elements (e.g., GntKU regulatory elements). Co-replacement of endogenous operator regions and / or GntR binding sites can modify expression levels relative to those of GntK and / or GntU coding regions of parental E. coli.

[0164] Alternatively or additionally, enhancers described herein can replace endogenous enhancers and / or can be added to increase expression of endogenous promoters and / or promoters described herein. Enhancers can be included in expression systems along with any one or more of promoters, operator regions, and / or GntR binding sites described herein.

[0165] In some embodiments, expression systems are introduced into the microorganism by any appropriate transformation technique. Nucleic acids comprising expression systems can be extrachromosomal, on a vector (such as a plasmid or a phage), such as a low copynumber vector, an intermediate copy number vector, or a high copy number vector. Nucleic acids comprising expression systems can be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, nucleic acids comprising expression systems or portions thereof (e.g., one, two, three, or all four of promoters, operator regions, gntR binding sites, and / or sequences of interest described herein) 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, as is well known in the art.

[0166] 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 region to a regulatory region 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 region from an inducible promoter), to form expression systems.

[0167] Additionally or alternatively, other techniques can be used in engineering parental microorganisms to include the expression systems. Non-specific mutagens can be used to delete or disrupt genes in a parental microorganism, and cells can be screened for a phenotype indicative of deletion or disruption of a gene of interest. Cells found to have the desired phenotype can then receive nucleic acids comprising expression systems described herein.

[0168] In some embodiments, in addition to nucleic acids comprising gluconate-regulated expression systems described herein, parental microorganisms can be engineered to further comprise nucleic acids comprising expression systems that are not regulated by gluconate. Such further nucleic acids can comprise constitutive promoters or inducible promoters that are not induced by gluconate.

[0169] In some embodiments, parental microorganisms can convert or can be engineered to convert gluconate to 2-keto-3-deoxy-D-gluconate (KDG). Examples of such parental microorganisms and enzymes that can be engineered into such parental microorganisms include those disclosed in PCT / US2020 / 041801 , which is hereby incorporated herein byreference. In some further embodiments, parental microorganisms can convert or can be engineered to convert KDG to 2-keto-3-deoxy-D-gluconate-phosphate (KDGP). In some embodiments, one or more proteins of interest (subject to gluconate-regulated expression as described herein) are DXP pathway enzymes, e.g., as described in Section 6.2.5.1.6.4. Methods of Use

[0170] The present disclosure also relates to methods for producing sequences of interest, such as proteins of interest or RNAs of interest, by use of expression systems. The present disclosure also relates to methods for producing products, such as products the formation of which can be catalyzed by enzymes that can be expressed by expression systems of the present disclosure.6.4.1. Culture media

[0171] Generally, methods disclosed herein comprise growing cells of a recombinant microorganism ( / .e., cells of a prokaryotic species into which nucleic acids disclosed herein have been introduced) in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration, and culturing the cells in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of the sequence of interest. Culturing can be in a batch mode or a continuous mode.

[0172] Examples of media that can be used in batch mode culturing include M9 medium and Hi-Def medium. In some embodiments, M9 medium comprises the following: sodium phosphate dibasic heptahydrate, 1.28 w / v%; potassium phosphate monobasic, 0.3 w / v%; sodium chloride, 0.05 w / v%; ammonium chloride, 0.1 w / v%; glucose, 0.4 w / v%; MgSO4, 0.024 w / v%; and CaCI2, 0.001 w / v%. In some embodiments, Hi-Def medium comprises ingredients known to the person of ordinary skill in the art, and it is commercially available (Teknova Inc. Hollister, CA).

[0173] In some embodiments, a culture medium comprises sucrose, fructose, glucose, and / or gluconate. In some embodiments, culture media comprise at least 0.1 w / v% sucrose, fructose, glucose, and / or gluconate. 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. Aculture 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).

[0174] 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).

[0175] In some embodiments, culture media comprise at least 0.1 w / v% fructose, at least 0.2 w / v% fructose, at least 0.3 w / v% fructose, at least 0.4 w / v% fructose, at least 0.5 w / v% fructose, at least 0.6 w / v% fructose, at least 0.7 w / v% fructose, at least 0.8 w / v% fructose, at least 0.9 w / v% fructose, or at least 1 w / v% fructose. A culture medium typically comprises less than 5 w / v% fructose, more typically less than 2 w / v% fructose (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% fructose; from 0.1 w / v% to 2 w / v% fructose; from 0.1 w / v% to 1 w / v% fructose; or from 1 w / v% to 2 w / v% fructose, among other possible ranges).

[0176] 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 induce expression of sequences of interest and provide a carbon source for recombinant microorganisms.

[0177] In some embodiments, culture medium are M9 medium or Hi-Def medium supplemented with sucrose, glucose, fructose, and / or gluconate. Such a medium may be referred to herein as a “production medium.”

[0178] In some embodiments, a production medium comprises sucrose. In some embodiments, a production medium comprises fructose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises gluconate. In some embodiments, a production medium comprises any two, any three, or all four of sucrose, fructose, glucose, or gluconate. The inclusion of gluconate or precursors thereof in production media can induce expression of sequences of interest in recombinant microorganisms comprising nucleic acids comprising expression systems described herein.

[0179] In some embodiments, a production medium comprises an inducer other than gluconate, i.e., a molecule other than gluconate which binds to a repressor other than GntR and thereby induces translation of a coding region regulated by an inducible promoter.

[0180] In some embodiments, wherein culture media comprise two or more carbon sources, culture media comprise at least 0.5 w / v% total carbon sources, at least 0.6 w / v% total carbon sources, at least 0.7 w / v% total carbon sources, at least 0.8 w / v% total carbon sources, at least 0.9 w / v% total carbon sources, or at least 1 w / v% total carbon sources. A culture medium typically comprises less than 5 w / v% total carbon sources, more typically less than 2 w / v% total carbon sources (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% total carbon sources; from 0.1 w / v% to 2 w / v% total carbon sources; from 0.1 w / v% to 1 w / v% total carbon sources; or from 1 w / v% to 2 w / v% total carbon sources, among other possible ranges).

[0181] In some embodiments of some methods described herein, it may be desirable to allow growth of a recombinant microorganism without expression of sequences of interest by the gluconate-regulated expression systems until a desired biomass of the recombinant microorganism has been reached. This can be effected by use of a growth medium comprising a carbon source other than glucose, fructose, and / or sucrose, and not comprising gluconate. In some embodiments, growth media comprise 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).

[0182] 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.

[0183] In particular embodiments, a growth medium lacks added glucose, fructose, and / or sucrose, i.e., one or more 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, fructose, and / or sucrose.

[0184] The selection of particular concentrations of sucrose, glucose, fructose, 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.

[0185] For fed-batch and / or continuous mode culturing, the ranges of sucrose, glucose, fructose, gluconate, glycerol, or combinations thereof given above can be initially provided to the medium. The consumption of the carbon source(s) during culturing can be repeatedly or continuously monitored and additional carbon source(s) can be provided as needed to sustain a desired respiratory coefficient, growth rate, rate of expression of sequences of interest, and / or a rate of production of desired compound(s). The feed rate may be adjusted to avoid accumulation of carbon source(s), which may maximize output of desired compound(s) and minimize waste of carbon source(s). The person of ordinary skill in the art having the benefit of the present disclosure can select the medium composition and the amount of carbon source added thereto during the process to enable the expression of sequences of interest to a desired level and / or production of desired product(s) to a desired concentration, such as at least 20 g / L, at least 50 g / L, or at least 100 g / L.6.4.2. Culture conditions

[0186] Recombinant cells comprising expression systems of the disclosure may be cultured under suitable conditions in a medium, such as a medium described in Section 6.4.1. 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.

[0187] The fermentation process is typically run at a temperature that is optimal for growth of a recombinant microorganism. Fermentation for a mesophilic microorganism is typically carried out at a temperature within the range of from 20°C to 45°C, from 25°C to 40°C, from 35°C to 40°C, or from 30°C to 37°C. In some embodiments wherein a recombinant microorganism is derived from one of the exemplary microorganisms described herein, culturing comprises maintaining the recombinant microorganism at a mesophilic temperature. In some embodiments, the mesophilic temperature is selected from any of the foregoing ranges.

[0188] 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. In certain embodiments, fermentation is 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 46 to 48 hours.6.4.3. Methods of Expressing Sequences of Interest

[0189] The present disclosure also relates to methods for producing sequences of interest in prokaryotic species, i.e., in cells of the prokaryotic species comprising nucleic acidscomprising expression systems described herein. Cells can be grown to a desired cell concentration in media lacking sucrose, fructose, glucose, and gluconate. After growth to the desired cell concentration, cells can be cultured in media containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of sequences of interest.

[0190] In some embodiments, sequences of interest are transcribed to yield RNAs of interest. RNAs of interest include coding RNAs and non-coding RNAs.

[0191] In some embodiments, RNAs of interest are microRNAs (miRNAs) and small interfering RNAs (siRNAs). Both miRNAs and siRNAs can be extracted and purified from recombinant microorganisms by known techniques. Purified miRNAs and / or siRNAs can be used in RNAi techniques in eukaryotic cells and / or in in vitro studies.

[0192] In some embodiments, RNAs of interest are antisense RNAs (asRNAs), which can be extracted, purified, and used in antisense RNA techniques in cells other than the recombinant cells expressing them. Also, asRNAs can be chosen that pair with and neutralize transcripts of interest in the recombinant cells expressing them, and / or can be used in in vitro studies.

[0193] In some embodiments, RNAs of interest are single guide RNAs (sgRNAs), which can be produced for use in CRISPR-based techniques (e.g., CRISPR interference (CRISPRi); CRISPR gene editing) either in cells of the recombinant microorganism expressing them, in other prokaryotic cells, and / or in in vitro studies, after any extraction and purification that may be appropriate.

[0194] In some embodiments, sequences of interest are translated to yield proteins of interest. In some embodiments, proteins of interest are extracted from cells of recombinant microorganisms and purified. In some embodiments, proteins of interest are retained in cells of recombinant microorganisms.

[0195] Proteins of interest can include both enzymes and non-catalytic proteins. Non- catalytic proteins include structural proteins, proteins configured for transporting other molecules (e.g., serum albumin), signaling polypeptides, antibodies and antigen-binding fragments thereof, and transcriptional regulators, among others. Enzymes includepolypeptides catalyzing any reaction by which a precursor can be converted to a product or a second precursor which can lead to the product by the action of another enzyme.

[0196] Proteins of interest that are enzymes can be parts of existing pathways in cells, such as by increasing the amount of a protein endogenous to parental microorganisms and / or recombinant microorganisms at a desired point in the growth curve of the cells, or having an activity level different than a corresponding endogenous protein of the cells. Proteins of interest that are enzymes can be parts of pathways in cells of recombinant microorganisms which are not present in parental microorganisms.

[0197] In some embodiments, proteins of interest are KDG aldolases or KDGP aldolases. In some embodiments, proteins of interest comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:48 or SEQ ID NO:60.

[0198] In some embodiments, proteins of interest are DXP pathway enzymes, such as DXP synthases. In some embodiments, proteins of interest comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO:49.

[0199] In some embodiments, proteins of interest are terpene synthases, such as myrcene synthases. In some embodiments, proteins of interest comprise an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NQ:50.

[0200] In some embodiments, proteins of interest are transcriptional regulators. In some embodiments, transcriptional regulators are Cas9 proteins incapable of cleaving DNA. Such Cas9 proteins can be paired with a guide RNA specific for a promoter or an operator region for which regulation of transcription is desired, to bind to that promoter or operator region and repress transcription of sequences operably linked thereto.6.4.4. Methods of Producing a Product

[0201] The present disclosure also relates to methods for producing products. The methods comprise introducing nucleic acids comprising expression systems described herein, wherein ORFs of the expression systems encode proteins of interest that catalyze reactions in pathways between precursors and the products, into cells of prokaryotic species. Similarly to methods of expressing sequences of interest to yield proteins of interest, cells can be grown in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration. After growth to the desired cell concentration, cells can be cultured in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of proteins of interest.

[0202] After expression, proteins of interest can catalyze the reactions. In some embodiments, catalysis are in vitro. Proteins of interest can be extracted and purified from cells using known techniques. The purified proteins of interest can be used in solution, after immobilization on a substrate, or under other conditions to catalyze the reactions upon contact with substrates for the proteins of interest.

[0203] In some embodiments, catalysis is in vivo, e.g., in cells of the recombinant microorganisms. Substrates can be added to cells by incorporating the substrates in media in which the cells are cultured and / or cells can produce substrates by metabolic pathways which can be present in parental microorganisms or can be engineered into recombinant microorganisms.

[0204] Regardless whether catalyzing is performed in vitro or in vivo, proteins of interest can catalyze reactions by which substrates can be converted directly to products, i.e., the substrates are direct precursors of the products, and / or reactions by which substrates can be converted into intermediates which can be further converted by actions of one or more other enzymes to the products.6.5. Numbered Embodiments

[0205] 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 nucleic acid comprising a system for expression of a sequence of interest in a prokaryotic species, comprising the sequence of interest operably linked to a regulatory element, wherein the regulatory element comprises:(a) a promoter, comprising from 5’ to 3’:(i) a -35 sequence, optionally comprising TTGACA or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof; and(ii) a -10 sequence having a 5’ end and a 3’ end, optionally comprising TATAAT or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof;(b) an operator region, comprising from 5’ to 3’ when the sequence of interest is an open reading frame (ORF) encoding a protein of interest:(i) a ribosome binding site (RBS), optionally comprising TAAGGAGG or a sequence having at least 4, at least 5, at least 6, or at least 7 nucleotides thereof; and(ii) a spacer, optionally having 3 or fewer (e.g., 3, 2, 1, or O) cytosine nucleotides;(c) a first GntR binding site and optionally a second GntR binding site each having a 5’ end and a 3’ end, optionally wherein:(i) the 5’ end of the first GntR binding site overlaps the 3’ end of the -10 sequence,(ii) the 3’ end of the first GntR binding site overlaps the 5’ end of the second GntR binding site, if present, or(iii) both (i) and (ii); and wherein:A. the regulatory element nucleic acid comprises one or more of the optional elements of (a)(i), (a)(ii), (b)(i), and (b)(ii), and / orB. the sequence of interest is an ORF encoding a protein of interest and comprising an initiation codon, wherein the initiation codon is the most prevalent initiation codon in the prokaryotic species and / or the ORF does not encode gntK.2. The nucleic acid of embodiment 1 , wherein the promoter comprises a sequence having at least 90% identity to SEQ ID NO: 12.3. The nucleic acid of embodiment 1 or embodiment 2, wherein the 5’ end of the first GntR binding site overlaps the 3’ end of the -10 sequence, the 3’ end of the first GntR binding site overlaps the 5’ end of the second GntR binding site, if present, or both.4. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTGACA.5. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTGATA.6. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTGATG.7. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTGATC.8. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTTATA.9. The nucleic acid of any one of embodiments 1 to 3, wherein the -35 sequence comprises TTGAGA.10. The nucleic acid of any one of embodiments 1 to 9, wherein the -10 sequence comprises TACAAT.11 . The nucleic acid of any one of embodiments 1 to 9, wherein the -10 sequence comprises TATAAT.12. The nucleic acid of any one of embodiments 1 to 11 , wherein the first GntR binding site and the second GntR binding site, if present, each independently comprise a nucleotide sequence having at least 70% identity to TAATGTTACCSGTAACATTT (SEQ ID NO:1) or TTTACAATGSCCATTGTAAT (SEQ ID NO:52), wherein S is C or G.13. The nucleic acid of embodiment 12, wherein the first GntR binding site comprises a nucleotide sequence having at least 70% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.14. The nucleic acid of embodiment 12 or embodiment 13, wherein the first GntR binding site comprises a nucleotide sequence having at least 75% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.15. The nucleic acid of any one of embodiments 12 to 14, wherein the first GntR binding site comprises a nucleotide sequence having at least 80% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.16. The nucleic acid of any one of embodiments 12 to 15, wherein the first GntR binding site comprises a nucleotide sequence having at least 85% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.17. The nucleic acid of any one of embodiments 12 to 16, wherein the first GntR binding site comprises a nucleotide sequence having at least 90% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.18. The nucleic acid of any one of embodiments 12 to 17, wherein the first GntR binding site comprises a nucleotide sequence having at least 95% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.19. The nucleic acid of any one of embodiments 12 to 18, wherein the first GntR binding site comprises a nucleotide sequence having 100% identity to AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2.20. The nucleic acid of embodiment 12, wherein the first GntR binding site comprises a nucleotide sequence having at least 70% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.21 . The nucleic acid of embodiment 12 or embodiment 20, wherein the first GntR binding site comprises a nucleotide sequence having at least 75% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.22. The nucleic acid of any one of embodiments 12 or 20 to 21 , wherein the first GntR binding site comprises a nucleotide sequence having at least 80% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.23. The nucleic acid of any one of embodiments 12 or 20 to 22, wherein the first GntR binding site comprises a nucleotide sequence having at least 85% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.24. The nucleic acid of any one of embodiments 12 or 20 to 23, wherein the first GntR binding site comprises a nucleotide sequence having at least 90% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.25. The nucleic acid of any one of embodiments 12 or 20 to 24, wherein the first GntR binding site comprises a nucleotide sequence having at least 95% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.26. The nucleic acid of any one of embodiments 12 or 20 to 25, wherein the first GntR binding site comprises a nucleotide sequence having 100% identity to CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3.27. The nucleic acid of embodiment 12, wherein the first GntR binding site comprises a nucleotide sequence having at least 70% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.28. The nucleic acid of embodiment 12 or embodiment 27, wherein the first GntR binding site comprises a nucleotide sequence having at least 75% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.29. The nucleic acid of any one of embodiments 12 or 27 to 28, wherein the first GntR binding site comprises a nucleotide sequence having at least 80% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.30. The nucleic acid of any one of embodiments 12 or 27 to 29, wherein the first GntR binding site comprises a nucleotide sequence having at least 85% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.31 . The nucleic acid of any one of embodiments 12 or 27 to 30, wherein the first GntR binding site comprises a nucleotide sequence having at least 90% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.32. The nucleic acid of any one of embodiments 12 or 27 to 31 , wherein the first GntR binding site comprises a nucleotide sequence having at least 95% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.33. The nucleic acid of any one of embodiments 12 or 27 to 32, wherein the first GntR binding site comprises a nucleotide sequence having 100% identity to TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4.34. The nucleic acid of embodiment 12, wherein the first GntR binding site comprises a nucleotide sequence having at least 70% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.35. The nucleic acid of embodiment 12 or embodiment 34, wherein the first GntR binding site comprises a nucleotide sequence having at least 75% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.36. The nucleic acid of any one of embodiments 12 or 34 to 35, wherein the first GntR binding site comprises a nucleotide sequence having at least 80% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.37. The nucleic acid of any one of embodiments 12 or 34 to 36, wherein the first GntR binding site comprises a nucleotide sequence having at least 85% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.38. The nucleic acid of any one of embodiments 12 or 34 to 37, wherein the first GntR binding site comprises a nucleotide sequence having at least 90% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.39. The nucleic acid of any one of embodiments 12 or 34 to 38, wherein the first GntR binding site comprises a nucleotide sequence having at least 95% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.40. The nucleic acid of any one of embodiments 12 or 34 to 39, wherein the first GntR binding site comprises a nucleotide sequence having 100% identity to TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5.41 . The nucleic acid of embodiment 12, wherein the first GntR binding site comprises a nucleotide sequence having at least 70% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.42. The nucleic acid of embodiment 12 or embodiment 41 , wherein the first GntR binding site comprises a nucleotide sequence having at least 75% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.43. The nucleic acid of any one of embodiments 12 or 41 to 42, wherein the first GntR binding site comprises a nucleotide sequence having at least 80% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.44. The nucleic acid of any one of embodiments 12 or 41 to 43, wherein the first GntR binding site comprises a nucleotide sequence having at least 85% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.45. The nucleic acid of any one of embodiments 12 or 41 to 44, wherein the first GntR binding site comprises a nucleotide sequence having at least 90% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.46. The nucleic acid of any one of embodiments 12 or 41 to 45, wherein the first GntR binding site comprises a nucleotide sequence having at least 95% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.47. The nucleic acid of any one of embodiments 12 or 41 to 46, wherein the first GntR binding site comprises a nucleotide sequence having 100% identity to TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6.48. The nucleic acid of any one of embodiments 12 to 47, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 70% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.49. The nucleic acid of any one of embodiments 12 to 48, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 75% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.50. The nucleic acid of any one of embodiments 12 to 49, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 80% identity to TTATTTTACCGGTAACATGA (SEQ ID NO:7) or a sequence reverse to SEQ ID NO:7.51 . The nucleic acid of any one of embodiments 12 to 50, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 85% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.52. The nucleic acid of any one of embodiments 12 to 51 , wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 90% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.53. The nucleic acid of any one of embodiments 12 to 52, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 95% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.54. The nucleic acid of any one of embodiments 12 to 53, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 100% identity to TTATTTTACCGGTAACATGA (SEQ ID NOT) or a sequence reverse to SEQ ID NOT.55. The nucleic acid of any one of embodiments 12 to 47, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 70% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.56. The nucleic acid of any one of embodiments 12 to 47 and 55, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 75% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.57. The nucleic acid of any one of embodiments 12 to 47 and 55 to 56, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 80% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.58. The nucleic acid of any one of embodiments 12 to 47 and 55 to 57, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 85% identity to AAATGTTACGGGTAACTGTT (SEQ ID NO:8) or a sequence reverse to SEQ ID NO:8.59. The nucleic acid of any one of embodiments 12 to 47 and 55 to 58, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 90% identity to AAATGTTACGGGTAACTGTT (SEQ ID NO:8) or a sequence reverse to SEQ ID NO:8.60. The nucleic acid of any one of embodiments 12 to 47 and 55 to 59, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 95% identity to AAATGTTACGGGTAACTGTT (SEQ ID NO:8) or a sequence reverse to SEQ ID NO:8.61 . The nucleic acid of any one of embodiments 12 to 47 and 55 to 60, wherein the second GntR binding site, if present, comprises a nucleotide sequence having 100% identity to AAATGTTACGGGTAACTGTT (SEQ ID NO:8) or a sequence reverse to SEQ ID NO:8.62. The nucleic acid of any one of embodiments 12 to 47, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 70% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.63. The nucleic acid of any one of embodiments 12 to 47 and 62, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 75% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.64. The nucleic acid of any one of embodiments 12 to 47 and 62 to 63, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 80% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.65. The nucleic acid of any one of embodiments 12 to 47 and 62 to 64, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 85% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.66. The nucleic acid of any one of embodiments 12 to 47 and 62 to 65, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least90% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.67. The nucleic acid of any one of embodiments 12 to 47 and 62 to 66, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 95% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.68. The nucleic acid of any one of embodiments 12 to 47 and 62 to 67, wherein the second GntR binding site, if present, comprises a nucleotide sequence having 100% identity to GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.69. The nucleic acid of any one of embodiments 12 to 47, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 70% identity to TCACGTTATGCGTAACATAG (SEQ ID NQ:10) or a sequence reverse to SEQ ID NQ:10.70. The nucleic acid of any one of embodiments 12 to 47 and 69, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 75% identity to TCACGTTATGCGTAACATAG (SEQ ID NQ:10) or a sequence reverse to SEQ ID NQ:10.71 . The nucleic acid of any one of embodiments 12 to 47 and 69 to 70, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 80% identity to TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NQ:10.72. The nucleic acid of any one of embodiments 12 to 47 and 69 to 71 , wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 85% identity to TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NQ:10.73. The nucleic acid of any one of embodiments 12 to 47 and 69 to 72, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 90% identity to TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NQ:10.74. The nucleic acid of any one of embodiments 12 to 47 and 69 to 73, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 95% identity to TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NQ:10.75. The nucleic acid of any one of embodiments 12 to 47 and 69 to 74, wherein the second GntR binding site, if present, comprises a nucleotide sequence having 100% identity to TCACGTTATGCGTAACATAG (SEQ ID NO:10) or a sequence reverse to SEQ ID NQ:10.76. The nucleic acid of any one of embodiments 12 to 47, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 70% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.77. The nucleic acid of any one of embodiments 12 to 47 and 76, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 75% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.78. The nucleic acid of any one of embodiments 12 to 47 and 76 to 77, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 80% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.79. The nucleic acid of any one of embodiments 12 to 47 and 76 to 78, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 85% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.80. The nucleic acid of any one of embodiments 12 to 47 and 76 to 79, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least 90% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.81 . The nucleic acid of any one of embodiments 12 to 47 and 76 to 80, wherein the second GntR binding site, if present, comprises a nucleotide sequence having at least95% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.82. The nucleic acid of any one of embodiments 12 to 47 and 76 to 81 , wherein the second GntR binding site, if present, comprises a nucleotide sequence having 100% identity to AACAGTTACCCGTAACATTT (SEQ ID NO:11) or a sequence reverse to SEQ ID NO:11.83. The nucleic acid of any one of embodiments 1 to 82, wherein the -10 sequence and the 5’ end of the first GntR binding site overlap.84. The nucleic acid of any one of embodiments 1 to 83, wherein the 3’ end of the first GntR binding site and the 5’ end of the second GntR binding site, if present, overlap.85. The nucleic acid of any one of embodiments 1 to 84, wherein the RBS comprises GGAGG, GGAG, or AAGGA.86. The nucleic acid of any one of embodiments 1 to 85, wherein the spacer comprises 2 or fewer cytosine nucleotides.87. The nucleic acid of any one of embodiments 1 to 86, wherein the ORF encodes a 2-keto-3-deoxy-gluconate (KDG) aldolase.88. The nucleic acid of embodiment 87, wherein the KDG aldolase comprises an amino acid sequence having at least 70% identity to SEQ ID NO:48.89. The nucleic acid of embodiment 87 or embodiment 88, wherein the KDG aldolase comprises an amino acid sequence having at least 75% identity to SEQ ID NO:48.90. The nucleic acid of any one of embodiments 87 to 89, wherein the KDG aldolase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:48.91 . The nucleic acid of any one of embodiments 87 to 90, wherein the KDG aldolase comprises an amino acid sequence having at least 85% identity to SEQ ID NO:48.92. The nucleic acid of any one of embodiments 87 to 91 , wherein the KDG aldolase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:48.93. The nucleic acid of any one of embodiments 87 to 92, wherein the KDG aldolase comprises an amino acid sequence having at least 92.5% identity to SEQ ID NO:48.94. The nucleic acid of any one of embodiments 87 to 93, wherein the KDG aldolase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:48.95. The nucleic acid of any one of embodiments 87 to 94, wherein the KDG aldolase comprises an amino acid sequence having at least 96% identity to SEQ ID NO:48.96. The nucleic acid of any one of embodiments 87 to 95, wherein the KDG aldolase comprises an amino acid sequence having at least 97% identity to SEQ ID NO:48.97. The nucleic acid of any one of embodiments 87 to 96, wherein the KDG aldolase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:48.98. The nucleic acid of any one of embodiments 87 to 97, wherein the KDG aldolase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:48.99. The nucleic acid of any one of embodiments 87 to 98, wherein the KDG aldolase comprises an amino acid sequence having 100% identity to SEQ ID NO:48.100. The nucleic acid of any one of embodiments 1 to 86, wherein the ORF encodes a 2-dehydro-3-deoxy-6-phospho-D-gluconate (KDGP) aldolase.101. The nucleic acid of embodiment 100, wherein the KDGP aldolase comprises an amino acid sequence having at least 70% identity to SEQ ID NQ:60.102. The nucleic acid of embodiment 100 or embodiment 101 , wherein the KDGP aldolase comprises an amino acid sequence having at least 75% identity to SEQ ID NQ:60.103. The nucleic acid of any one of embodiments 100 to 102, wherein the KDGP aldolase comprises an amino acid sequence having at least 80% identity to SEQ ID NQ:60.104. The nucleic acid of any one of embodiments 100 to 103, wherein the KDGP aldolase comprises an amino acid sequence having at least 85% identity to SEQ ID NQ:60.105. The nucleic acid of any one of embodiments 100 to 104, wherein the KDGP aldolase comprises an amino acid sequence having at least 90% identity to SEQ ID NQ:60.106. The nucleic acid of any one of embodiments 100 to 105, wherein the KDGP aldolase comprises an amino acid sequence having at least 92.5% identity to SEQ ID NO:60.107. The nucleic acid of any one of embodiments 100 to 106, wherein the KDGP aldolase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:60.108. The nucleic acid of any one of embodiments 100 to 107, wherein the KDGP aldolase comprises an amino acid sequence having at least 96% identity to SEQ ID NQ:60.109. The nucleic acid of any one of embodiments 100 to 108, wherein the KDGP aldolase comprises an amino acid sequence having at least 97% identity to SEQ ID NQ:60.110. The nucleic acid of any one of embodiments 100 to 109, wherein the KDGP aldolase comprises an amino acid sequence having at least 98% identity to SEQ ID NQ:60.111. The nucleic acid of any one of embodiments 100 to 110, wherein the KDGP aldolase comprises an amino acid sequence having at least 99% identity to SEQ ID NQ:60.112. The nucleic acid of any one of embodiments 100 to 111 , wherein the KDGP aldolase comprises an amino acid sequence having 100% identity to SEQ ID NQ:60.113. The nucleic acid of any one of embodiments 1 to 86, wherein the ORF encodes a DXP pathway enzyme.114. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a 1-deoxy-D-xylulose-5-phosphate synthase (Dxs; EC 2.2.1.7).115. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr; EC 1.1.1.267).116. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD; EC 2.7.7.60).117. The nucleic acid embodiment 113, wherein the DXP pathway enzyme is a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148).118. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; EC 4.6.1.12).119. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG; EC 1.17.7.1 or EC 1.17.7.3).120. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH; EC 1.17.7.4).121. The nucleic acid of embodiment 113, wherein the DXP pathway enzyme is an isopentenyl-diphosphate Delta-isomerase (Idi; EC 5.3.3.2).122. The nucleic acid of any one of embodiments 1 to 86, wherein the ORF encodes a terpene synthase.123. The nucleic acid of embodiment 122, wherein the terpene synthase comprises an amino acid sequence having at least 70% identity to SEQ ID NO:50.124. The nucleic acid of embodiment 122 or embodiment 123, wherein the terpene synthase comprises an amino acid sequence having at least 75% identity to SEQ ID NO:50.125. The nucleic acid of any one of embodiments 122 to 124, wherein the terpene synthase comprises an amino acid sequence having at least 80% identity to SEQ ID NQ:50.126. The nucleic acid of any one of embodiments 122 to 125, wherein the terpene synthase comprises an amino acid sequence having at least 85% identity to SEQ ID NQ:50.127. The nucleic acid of any one of embodiments 122 to 126, wherein the terpene synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NQ:50.128. The nucleic acid of any one of embodiments 122 to 127, wherein the terpene synthase comprises an amino acid sequence having at least 92.5% identity to SEQ ID NQ:50.129. The nucleic acid of any one of embodiments 122 to 128, wherein the terpene synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NQ:50.130. The nucleic acid of any one of embodiments 122 to 129, wherein the terpene synthase comprises an amino acid sequence having at least 96% identity to SEQ ID NQ:50.131. The nucleic acid of any one of embodiments 122 to 130, wherein the terpene synthase comprises an amino acid sequence having at least 97% identity to SEQ ID NQ:50.132. The nucleic acid of any one of embodiments 122 to 131 , wherein the terpene synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:50.133. The nucleic acid of any one of embodiments 122 to 132, wherein the terpene synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:50.134. The nucleic acid of any one of embodiments 122 to 133, wherein the terpene synthase comprises an amino acid sequence having 100% identity to SEQ ID NQ:50.135. The nucleic acid of any one of embodiments 1 to 86, wherein the ORF encodes a transcriptional regulator.136. The nucleic acid of embodiment 135, wherein the transcriptional regulator is a catalytically inactive Cas9 (dCas9).137. The nucleic acid of any one of embodiments 1 to 86, wherein the sequence of interest is not a protein (e.g., the sequence of interest is transcribed to a microRNA (miRNA), a small interfering RNA (siRNA), an antisense RNA (asRNA), or a guide RNA).138. The nucleic acid of any one of embodiments 1 to 137, wherein the regulatory element has at least 90% sequence identity to nucleotides 1-85 of SEQ ID NO:51 or comprises a nucleotide sequence having at least 90% sequence identity to nucleotides 1-85 of SEQ ID NO:51.139. The nucleic acid of embodiment 138, wherein the regulatory element has 100% sequence identity to nucleotides 1-85 of SEQ ID NO:51 or comprises a nucleotide sequence having 100% sequence identity to nucleotides 1-85 of SEQ ID NO:51.140. The nucleic acid of any one of embodiments 1 to 137, wherein the regulatory element has at least 90% sequence identity to nucleotides 1-86 of SEQ ID NO:27 or comprises a nucleotide sequence having at least 90% sequence identity to nucleotides 1-86 of SEQ ID NO:27.141. The nucleic acid of embodiment 140, wherein the regulatory element has 100% sequence identity to nucleotides 1-86 of SEQ ID NO:27 or comprises a nucleotide sequence having 100% sequence identity to nucleotides 1-86 of SEQ ID NO:27.142. The nucleic acid of any one of embodiments 1 to 141 , wherein the nucleic acid is incorporated into a plasmid.143. The nucleic acid of any one of embodiments 1 to 141 , wherein the nucleic acid is integrated into the genome of the prokaryotic species.144. The nucleic acid of any one of embodiments 1 to 143, further comprising one or more additional sequences of interest operably linked to a promoter, an operator region, a first GntR binding site, and optionally a second GntR binding site.145. The nucleic acid of embodiment 144, wherein the one or more additional sequences of interest are operably linked to the same promoter, operator region, first GntR binding site, and optionally second GntR binding site as the sequence of interest.146. The nucleic acid of embodiment 144 or embodiment 145, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Dxr.147. The nucleic acid of embodiment 144 or embodiment 145, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Idi.148. The nucleic acid of embodiment 144 or embodiment 145, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxr and Idi.149. The nucleic acid of embodiment 144 or embodiment 145, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs, Dxr, and Idi.150. The nucleic acid of any one of embodiments 1 to 149, wherein the regulatory element further comprises an enhancer.151. The nucleic acid of embodiment 150, wherein the enhancer is 5’-ward of the promoter.152. The nucleic acid of embodiment 151 , wherein the enhancer and the -35 sequence are separated by a number of nucleotides in the range of 5 nt to 35 nt (e.g., in therange of 5 nt to 10 nt, 10 nt to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, or 30 nt to 35 nt, and in some embodiments 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt or 35 nt).153. The nucleic acid of embodiment 150 or embodiment 151 , wherein the enhancer and the -35 sequence are separated by from 26 nt to 32 nt.154. The nucleic acid of any one of embodiments 150 to 153, wherein the enhancer is derived from the lambda bacteriophage PLupstream (UP) element.155. The nucleic acid of any one of embodiments 150 to 154, wherein the enhancer comprises SEQ ID NO:62, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77.156. A prokaryotic cell comprising the nucleic acid of any one of embodiments 1 to 149.157. A prokaryotic cell comprising the nucleic acid of any one of embodiments 150 to 155.158. The prokaryotic cell of embodiment 156 or embodiment 157, wherein the nucleic acid is incorporated into a plasmid.159. The prokaryotic cell of embodiment 156 or embodiment 157, wherein the nucleic acid is integrated into the genome of the prokaryotic species.160. The prokaryotic cell of any one of embodiments 156 to 159, which is capable of producing (e.g., is engineered to produce) 2-keto-3-deoxy-D-gluconate (KDG).161. The prokaryotic cell of any one of embodiments 156 to 160, which is capable of converting (e.g., is engineered to convert) gluconate to 2-keto-3-deoxy-D-gluconate (KDG).162. The prokaryotic cell of any one of embodiments 156 to 161 , wherein the prokaryotic species is E. coli.163. The prokaryotic cell of embodiment 162, wherein the E. coli is a K-12 strain.164. The prokaryotic cell of embodiment 162 or embodiment 163, which has a reduced ability to use glucose as compared to a parental cell.165. The prokaryotic cell of embodiment 164, which has a loss of function mutation (e.g., deletion) in pts, err, or both, optionally wherein the loss of function mutation (e.g., deletion) in pts is a loss of function mutation (e.g., deletion) in ptsH, ptsl, or both.166. The prokaryotic cell of any one of embodiments 162 to 165, which has a reduced ability to phosphorylate gluconate as compared to a parental cell.167. The prokaryotic cell of embodiment 166, which has a loss of function mutation (e.g., deletion) in gntK, idnK, or both.168. The prokaryotic cell of any one of embodiments 162 to 167, which has a reduced ability to dehydrate gluconate-6P as compared to a parental cell.169. The prokaryotic cell of embodiment 168, which has a loss of function mutation (e.g., deletion) in edd.170. The prokaryotic cell of any one of embodiments 162 to 169, which has increased expression of KDGP aldolase as compared to a parental cell.171. The prokaryotic cell of embodiment 170, which is engineered to recombinantly express or overexpress eda.172. The prokaryotic cell of any one of embodiments 162 to 171 , wherein the E. coli further comprises plasmid pLac-Achro-TK 81.173. A method of producing a sequence of interest in a prokaryotic species, comprising:(a) growing prokaryotic cells of any one of embodiments 156 to 172 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration; and(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of the sequence of interest.174. The method of embodiment 173, wherein the medium for growing comprises glycerol.175. The method of embodiment 173 or embodiment 174, wherein the sequence of interest encodes a KDG aldolase.176. The method of embodiment 173 or embodiment 174, wherein the sequence of interest encodes a KDGP aldolase.177. The method of embodiment 173 or embodiment 174, wherein the sequence of interest encodes a DXP pathway enzyme.178. The method of embodiment 177, wherein the DXP pathway enzyme is a 1- deoxy-d-xylulose-5-phosphate synthase (Dxs; EC 2.2.1.7).179. The method of embodiment 177, wherein the DXP pathway enzyme is a 1- deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr; EC 1.1.1.267).180. The method of embodiment 177, wherein the DXP pathway enzyme is a 2-C- methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD; EC 2.7.7.60).181. The method of embodiment 177, wherein the DXP pathway enzyme is a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148).182. The method of embodiment 177, wherein the DXP pathway enzyme is a 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; EC 4.6.1.12).183. The method of embodiment 177, wherein the DXP pathway enzyme is a 4- hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG; EC 1.17.7.1 or EC 1.17.7.3).184. The method of embodiment 177, wherein the DXP pathway enzyme is a 4- hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH; EC 1.17.7.4).185. The method of embodiment 177, wherein the DXP pathway enzyme is an isopentenyl-diphosphate Delta-isomerase (Idi; EC 5.3.3.2).186. The method of embodiment 173 or embodiment 174, wherein the sequence of interest encodes a terpene synthase.187. The method of embodiment 173 or embodiment 174, wherein the sequence of interest encodes a transcriptional regulator.188. The method of any one of embodiments 173 to 187, wherein the nucleic acid or the prokaryotic cell further comprises one or more additional sequences of interest operably linked to a promoter, an operator region, a first GntR binding site, and optionally a second GntR binding site.189. The method of embodiment 188, wherein the one or more additional sequences of interest are operably linked to the same promoter, operator region, first GntR binding site, and optionally second GntR binding site as the sequence of interest.190. The method of embodiment 188 or embodiment 189, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Dxr.191. The method of embodiment 188 or embodiment 189, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Idi.192. The method of embodiment 188 or embodiment 189, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxr and Idi.193. The method of embodiment 188 or embodiment 189, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs, Dxr, and Idi.194. A method of producing a product, comprising:(a) growing prokaryotic cells of any one of embodiments 156 to 172 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration;(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of the protein of interest; and(c) catalyzing a reaction by the protein of interest, thereby producing or leading to production of the product.195. The method of embodiment 194, wherein the medium for culturing comprises a substrate for the reaction.196. The method of embodiment 194 or embodiment 195, wherein the medium for growing comprises glycerol.197. The method of any one of embodiments 194 to 196, wherein the protein of interest is KDG aldolase.198. The method of any one of embodiments 194 to 196, wherein the protein of interest is KDGP aldolase.199. The method of any one of embodiments 194 to 196, wherein the protein of interest is a DXP pathway enzyme.200. The method of embodiment 199, wherein the DXP pathway enzyme is a 1- deoxy-d-xylulose-5-phosphate synthase (Dxs; EC 2.2.1.7).201. The method of embodiment 199, wherein the DXP pathway enzyme is a 1- deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr; EC 1.1.1.267).202. The method of embodiment 199, wherein the DXP pathway enzyme is a 2-C- methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD; EC 2.7.7.60).203. The method of embodiment 199, wherein the DXP pathway enzyme is a 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148).204. The method of embodiment 199, wherein the DXP pathway enzyme is a 2-C- methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; EC 4.6.1.12).205. The method of embodiment 199, wherein the DXP pathway enzyme is a 4- hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG; EC 1.17.7.1 or EC 1.17.7.3).206. The method of embodiment 199, wherein the DXP pathway enzyme is a 4- hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH; EC 1.17.7.4).207. The method of embodiment 199, wherein the DXP pathway enzyme is an isopentenyl-diphosphate Delta-isomerase (Idi; EC 5.3.3.2).208. The method of any one of embodiments 194 to 196, wherein the protein of interest is a terpene synthase.209. The method of any one of embodiments 194 to 208, wherein the nucleic acid or the prokaryotic cell further comprises one or more additional sequences of interest operably linked to a promoter, an operator region, a first GntR binding site, and optionally a second GntR binding site.210. The method of embodiment 209, wherein the one or more additional sequences of interest are operably linked to the same promoter, operator region, first GntR binding site, and optionally second GntR binding site as the sequence of interest.211. The method of embodiment 209 or embodiment 210, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Dxr.212. The method of embodiment 209 or embodiment 210, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs and Idi.213. The method of embodiment 209 or embodiment 210, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxr and Idi.214. The method of embodiment 209 or embodiment 210, wherein the sequence of interest and the one or more additional sequences of interest comprise ORFs encoding Dxs, Dxr, and Idi.215. The method of any one of embodiments 194 to 214, wherein the product is an isoprenoid.216. The method of any one of embodiments 194 to 215, further comprising converting gluconate to KDG, wherein the prokaryotic cell is capable of converting (e.g., is engineered to convert) gluconate to KDG.217. The method of any one of embodiments 173 to 216, wherein in the culturing the medium contains gluconate.218. The method of any one of embodiments 173 to 217, wherein in the culturing the medium contains glucose.219. The method of any one of embodiments 173 to 218, wherein in the culturing the medium contains sucrose.220. The method of any one of embodiments 194 to 219, wherein the catalyzing is in the cells of the prokaryotic species.221. The method of any one of embodiments 194 to 220, further comprising purifying the protein of interest, wherein the catalyzing is in vitro.222. A nucleic acid comprising a system for expression of a target gene in a prokaryotic species, comprising:(a) a promoter;(b) an operator region;(c) a first means for binding GntR and a second means for binding GntR; and(d) an open reading frame (ORF); provided at least one of elements (a)-(d) is not naturally occurring or the combination of elements (a)-(d) is not naturally occurring.223. A prokaryotic cell, comprising means inducible by gluconate for expressing a target gene, wherein the means comprise the nucleic acid of embodiment 222.224. A method of producing a protein of interest in a prokaryotic species, comprising:(a) growing cells of the prokaryotic species of embodiment 223 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration; and(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, or gluconate, thereby inducing expression of the protein of interest.225. A method of producing a product, comprising:(a) growing cells of the prokaryotic species of embodiment 223 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration;(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, or gluconate, thereby inducing expression of the protein of interest; and(c) catalyzing the reaction by the protein of interest, thereby producing or leading to production of the product.7. EXAMPLES7.1. Example 1 : Engineering of E. coli gntK promoter for increased expression

[0206] The naturally occurring GntK promoter / operator region / initiation codon region of E. coli MG1655 (depicted in context in FIG. 2; SEQ ID NO:12) contains a number of features that were believed to lead to relatively low expression of gntK. These features include (1) a - 35 sequence (TTGCTT) with 3 mismatches from the consensus sequence (TTGACA); (2) a -10 sequence (GACAAT) with 2 mismatches from the consensus sequence (TATAAT); (3) a ribosome binding site (RBS) (TGTGGGGG) with relatively low identity to the consensus sequence (TAAGGAGG); (4) a relatively short spacer sequence (CACCACT) with four cytosines out of seven nucleotides; and (5) an open reading frame (ORF) initiation codon (TTG) present in only about 3% of E. coli genes. These characteristics suggested that expression of sequences of interest operably linked to gntK regulatory elements could be increased by one or more modifications relative to gntK regulatory elements of E. coli MG1655.

[0207] To develop gntK promoter variants that enabled higher levels of gene expression, but still retained its capacity to be induced by gluconate, a series of plasmids were constructed. To do so, first a 1304 bp DNA fragment containing the E. coli MG1655 gntR gene, including its regulatory region, as well as the gntK promoter region, was amplified by PCR, utilizing primers gntR-F5 and gntR-R5 (SEQ ID NO:13-14).To clone this regulatory region in front of a reporter gene, primers gntR-F6 and gntR-R6 (SEQ ID NO: 15-16) were used to add 15 bp tails for cloning using the NEBuilder HiFi DNA- Assembly kit (New England Biolabs). Primer gntR-R6 also added an optimized RBS with the sequence GGAGG. In addition, the spacer was modified by lengthening to 8 nucleotides (AATAAACC). All these changes are shown in FIG. 5 and set forth in SEQ ID NO:17. Furthermore, primer gntR-R6, also added the coding region for the first 5 amino acids of the mScarlet red fluorescent protein (Bindels, et al., 2017. Nat. Methods. 14: 53-56). The resulting 1345 bp DNA fragment was cloned into plasmid mScarlet-pUC57 digested with the restriction enzymes Xbal and Ncol (SEQ ID NO: 18).

[0208] After transformation into E. coli Stellar competent cells (TaKaRa Bio. San Jose CA), it was noted that most of the colonies were red, but in a few of them, the red color was more intense. After sequencing some of these colonies, it was found that the DNA from coloniesthat had a more intense red color, contained a 2 bp deletion overlapping the 3’ end of the ribosome binding site. These differences are shown below, with nucleotides of ribosome binding sites in bold:Original design GGAGGAATAAACCATGG (SEQ ID NO:46) Intense Red color GGAG ATAAACCATGG (SEQ ID NO:47)

[0209] Based on these results, it was concluded that the 2bp deletion enhanced translation of the mScarlet mRNA, producing more mScarlet protein. This mutated RBS region was used for further plasmid constructions.

[0210] For a more accurate evaluation of the gntK promoter region, a pBR322 derivative named pBR-GntRK-lacZ 710, containing the gntRK region (described above) fused to the lacZ gene was constructed. A scheme of the most relevant characteristics of this plasmid are shown in Fig. 4A. The sequence of the plasmid is set for in SEQ ID NO: 19.

[0211] To investigate whether expression from a modified gluconate-induced promoter could be increased over its level in the GntK promoter region of E. coli MG1655, three gntK promoter variants were prepared. This was accomplished by amplifying the gntK regulatory region from plasmid pBR-GntRK-lacZ 710 with three different forward mutagenic primers, GntRK-M1 (SEQ ID NQ:20), GntRK-M2 (SEQ ID NO:21), and GntRK-M12 (SEQ ID NO:22). The reverse primer used for these 3 PCR reactions was primer gntRK-R71 (SEQ ID NO:23). In Mut1 , the -35 sequence TGCTT was mutated to the consensus sequence (TTGACA); in Mut2, the -10 sequence GACAAT was mutated to the sequence TACAAT; and in Mut12, both the -35 sequence and the -10 sequence were mutated, to TTGACA and TACAAT, respectively (SEQ ID NO:24). The resulting PCR products were cloned into pBR-GntR-lacZ 710 previously digested with restriction enzymes Aflll and Ncol to remove the wild type gntK regulatory region (see Fig. 4A). A comparison of these promoters is depicted in context in FIG. 4B.

[0212] To test the strength of the promoter variants, as well as to find out if the mutated promoters remained inducible by gluconate, the three GntK promoter variant plasmids were introduced using conventional techniques into an E. coli strain having a genotype comprising ptsHIcrr, gntK dnK Aecfc Km. This strain may be referred to herein as A4EL-Km. The genotype of strain A4EL-Km yielded two relevant phenotypes: (1) the strain was unable to produce cAMP, and thereby was expected to cause CRP-dependentpromoters like gntK to have very low levels of expression, and (2) the strain was unable to use gluconate via the Entner-Doudoroff or the pentose pathways. To enable the strain containing the GntK promoter variant plasmids to grow on gluconate, a second plasmid, pLac-Achro-TK 81 (SEQ ID NO:25), was introduced into the strain. pLac-Achro-TK 81 is a low copy plasmid in which the LacllV5 promoter is operably linked to the Achromobacter sp. gluconate dehydratase, E. coli gluconate permease (gntT), and E. coli KDG kinase (kdgK) genes. This plasmid allows the strain to use gluconate as a carbon source, via the 2-Keto-3 deoxy-gluconate (KDG) pathway. Strains containing both a GntK promoter variant plasmid, and pLac-Achro-TK 81 were identified by selecting for growth on LB plates containing the antibiotics carbenicill in and spectinomycin. It should be mentioned that parental plasmid pBR-GntRK-lacZ 710 expressed lacZ at very low levels in strain A4EL-Km, presumably due to the lack of activation by CRP (data not shown), and was not further studied.

[0213] After strains containing both plasmids were identified, they were grown in Hi-Def medium supplemented with 2% gluconate or 2% glycerol. The Hi-Def medium contains ingredients known to the person of ordinary skill in the art, and it is commercially available (Teknova Inc. Hollister, CA). Liquid cultures were incubated in a shaker for 24 hr at 37°C, at which time, samples were taken and the p-Galactosidase activity was measured using a modified Miller assay, utilizing ONPG as a substrate. From these assays, the relative p- galactosidase activity (calculated as OD42o / OD6oo) was determined. The results are shown in FIG. 6. All three promoters showed negligible lacZ activity when grown on glycerol and roughly 50-fold to 160-fold higher activity when lacZ expression was induced by gluconate. Surprisingly, the Mut12 promoter had lower relative lacZ activity than either Mut1 or Mut2.

[0214] In conclusion, the GntK promoter variants were well regulated by gluconate and, after induction, expressed lacZ at relatively high levels.7.2. Example 2: Engineering of bacteriophage T5 PH207 promoter for induction by gluconate

[0215] Some viral promoters are among the strongest promoters in cells which their parental virus can infect. For example, promoter PH207 from bacteriophage T5 (SEQ ID NO:26) and promoter PM from bacteriophage T7 have relative strengths in E. coli expression systems that are roughly 10-fold to 13-fold greater than E. coli Piacpromoter even after induction of the latter (Deuschle, et al., 1996, EMBO J. 5:2987-2994).

[0216] To study whether PH207 could be engineered to be induced by gluconate, a hybrid T5- GntR promoter, Pys-GntR-wr, was synthesized (Azenta Life Sciences, La Jolla, CA). The hybrid T5-GntR promoter comprised a portion of the promoter from the parental promoter and operator regions (shown in context in FIG. 7, upper; SEQ ID NO:27). Weaker variants of the hybrid promoter Pys-GntR-wr were prepared (see below) by replacing the naturally occurring - 35 sequence from PH207 ((TTGCTA) with TTGATA (to yield promoter Pys-GntR-wt),TTGATA) with TTGATG (to yield promoter Pys-GntR-so), TTGATC (to yield promoter Pys-GntR-4o), TTTATA (to yield promoter Pys-GntR-is), or TTGAGA (to yield promoter Pys-GntR-s), as shown in FIG. 8.

[0217] The percentage values shown in FIG. 8 represent expected reductions in expression relative to the unmodified PH2O? -35 sequence. The predicted reductions in promoter strength were based on the effect of similar mutations in P22 ant promoter (Moyle, et al., 1991. J.Bact. 173:1944-1950).

[0218] The original hybrid PH207-WT promoter (SEQ ID NO:27) and its variants, as well as optimized RBS and spacer sequences, were operably linked to the red fluorescent protein mScarlet gene. This was accomplished by amplifying the mentioned regions by PCR using five different forward primers (SEQ ID NOS:28-32) and one reverse primer (SEQ ID NO:33). Using the NEBuilder Hi Fi DNAS Assembly kit (New England Biolabs), these PCR products were cloned into plasmid mScarlet-pUC57 digested with restriction enzymes EcoRI and Ncol (SEQ ID NO:34). After transforming the assemblies into Stellar competent cells (TaKaRa Bio, San Jose, CA), in the resulting colonies some level of mScarlet expression was observed from all the variant promoters, although observed expression did not decrease linearly with the expected reductions. DNA from some colonies for each promoter variants was purified and the correct sequence for the promoter variants was confirmed. The resulting plasmids were named: pT5-GntR-WT-Scarlet original Pys-GntR- T Promoter sequence pT5-GntR-50-Scarlet TTGATA mutated to TTGATG pT5-GntR-40-Scarlet TTGATA mutated to TTGATC pT5-GntR-15-Scarlet TTGATA mutated to TTTATA pT5-GntR-5-Scarlet TTGATA mutated to TTGAGADuring this process, it was also observed that after subsequent cultivations of the original strains in liquid cultures, and re-plating them on Lb-Carbenicillin plates, numerous white colonies were observed, suggesting plasmid instability. This was particularly evident with plasmids containing the PTS-GHIR-WT and PTS-GAIR-SO promoters. We reasoned that this plasmid instability may be caused by an excessive mScarlet expression, due to the high-copy number of the cloning vector, and that the repression of the hybrid promoter by GntR, was incomplete because only one chromosomal copy of the GntR repressor gene. Because of this, a new plasmid construction was designed, using a lower copy plasmid that also encoded the GntR repressor and the lacZ gene as a reporter.

[0219] Plasmid pT5-GntR-LacZ-50 (SEQ ID NO:35) was constructed. A map of the most relevant regions present on this plasmid are shown in FIG. 9. The sequence of the hybrid T5-GntR promoter is shown in FIG. 10. To evaluate the regulation of the PTS-GHIR-WT by gluconate, plasmid pT5-GntR-LacZ-50 was co-introduced with pLac-Achro-TK 81 into E. coli A4EL-Km as described in Example 1. After strains containing both plasmids were identified, lacZ expression was analyzed as described in Example 1.

[0220] The results are shown in FIG. 11. In the absence of gluconate in both Luria broth and Hi-Def media, lacZ activity was minimal, even after 48 hr. When gluconate was added to Hi- Def, lacZ activity increased roughly 5-fold after 24 hr compared to negative control, and roughly 28-fold after 48 hr compared to negative control.

[0221] In conclusion, the engineered PH2o? variant promoter PTS-GHIR-SW was well regulated by gluconate and, after induction, expressed lacZ at relatively high levels.

[0222] Plasmid pT5-GntR-LacZ-50 was used to clone the four promoter variants. This plasmid was digested with the restriction enzymes Swal and Ncol. As can be seen in FIG. 9, this removed the PT5-GntR-wr promoter. The four promoter variants described above were amplified by PCR by using four forward primers, one specific for each variant (SEQ ID NO:36-39), and a common reverse primer (SEQ ID NQ:40). The purified PCR products were cloned into the Swal-Ncol-linearized pT5-GntR-LacZ-50 vector using the N EBuilder HiFi DNAS Assembly kit. After transforming the assembling reactions into Stellar competent cells, plasmid DNAs from colonies that were carbenicillin-resistant and produced a blue color with X-Gal were purified and sequenced. Plasmids containing the correct promoter variant were named:pT5 GntR-v5-lacZ pT5 GntR-v15-lacZ pT5 GntR-v40-lacZ pT5 GntR-v50-lacZ

[0223] To characterize the strength of the promoter variants, these plasmids are transformed into strain A4EL-Km containing plasmid pLac-Achro-TK 81 , and evaluated as described in Example 1. As shown in Table 1 below these studies showed that in Luria broth and Hi-Def media with glycerol, lacZ was produced at very low levels. However, in Hi-Def media with gluconate, and compared with Pys-GntR- r, the T5 GntR variants produced different levels of lacZ expression, but retained their regulation by gluconate. Furthermore, the promoter variants, pT5 GntR-v40-lacZ and pT5 GntR-v50-lacZ, produced lacZ at the same level than the wild-type promoter, indicating that the introduced mutations had no impact on promoter strength, while the mutation in variant 5, decreased promoter strength by 42%, and the mutation in variant 15 was very detrimental to promoter strength.7.3. Example 3: Integration of the Mut2 GntK and the Hybrid PH207-WT promoters into the E. coli chromosome

[0224] In additional studies, utilizing CRISPR and homology-directed repair methods known to those skilled in the art, the GntK-Mut2 and Pys-GntR-wr promoters were integrated into the E. coli chromosome at the GntRKU region. For such a purpose, and to isolate the Mut2 GntK and PT5-GntR-wT promoters completely from possible transcription coming from the GntR gene located upstream of the GntK gene, the CRP binding site was replaced by a first heterologous transcriptional terminator (SEQ ID NO:41). Also, to study regulation bygluconate of these new constructions, three more modifications were performed: (1) the GntK gene was replaced by the gene of the red fluorescent protein mScarlet; (2) a second heterologous transcriptional terminator (SEQ ID NO:42), was inserted downstream of the red fluorescent protein gene; and (3) the GntU gene was inactivated by deleting its amino terminal region. A scheme of these genomic modifications is shown in FIG. 12. SEQ ID NO:43-44 are relevant portions of the sequences of the integrated promoter regions. SEQ ID NO:45 depicts the end of mScarlet, transcriptional terminator 2, and the junction with the deleted gntU gene. To study the regulation by gluconate of the chromosomal integrants, strains were grown in modified MOPS media (Neidhardt et al., 1974, J. Bact. 119:736-747), where the concentration of the MOPS buffer was increased to 200mM. The media contained 0.5% Glycerol or 0.5% gluconate. After 24 hr at 37 °C, samples were taken, and mScarlet was quantified by measuring its fluorescence, using excitation at 570 nm, and emission at 591 nm, with a 70% gain. Relative activity of each promoter was calculated by the ratio of the 591 nm emission value to the OD 600 of the sample.

[0225] The results are shown in Table 2. As can be seen, expression in glycerol media was negligible for both promoters, indicating essentially full repression by GntR. Gluconate clearly induced high levels of expression from both promoters. Furthermore, compared with GntK-Mut2, the PTS -GntR- r promoter was 3 times stronger. These results indicate that by engineering particular promoter and operator region sequences, gluconate-induced expression levels can be tuned as desired for a given sequence of interest.7.4. Example 4: Increasing the strength of the pT5-GntR-WT promoter by using an enhancer element upstream promoter core in E. coli.7.4.1. Materials and Methods

[0226] To study the impact of an enhancer on the strength of the pT5-GntR-WT promoter ofExample 2, the upstream region (UP) (SEQ ID NO:62) of the lambda bacteriophage P promoter region (SEQ ID: 61) was integrated upstream of the pT5-GntR-WT promoter onthe E. coli chromosome using the CRISPR / MAD7-associated Lambda-RED recombineering tool to yield UP+pT5-GntR-WT (FIG. 15B, FIG. 16). Gene expression was measured by operably linking UP+pT5-GntR-WT to mScarlet as a reporter (FIG. 15B). To study the effect of the UP element on gene expression, strains without (FIG. 15A) and with (FIG. 15B) the UP element upstream of pT5-GntR-WT promoter were grown in modified MOPS media (Neidhardt, et al., 1974, J. Bad. 119:736-747), where concentration of MOPS was increased to 200 mM to maintain pH levels around 7.0. The media initially contained either 0.5% (wt / vol) glycerol or 1% (wt / vol) sodium gluconate. Cultures grown on glycerol were treated with sodium gluconate (final concentration of 0.5% (wt / vol)) after 7 h of growth. All cultures were incubated at 37°C for a total of 37 h in a 96-well plate using a plate reader. Respective cell densities and relative mScarlet levels were quantified by measuring OD 600nm and reporter fluorescence using excitation at 570 nm and emission at 591 nm with a 75% gain every 30 min over the course of cultures.7.4.2. Results

[0227] Time-course data for cell densities and relative mScarlet levels were obtained from mean of three biological replicates and are shown in FIG. 17A (promoter induction by gluconate at time = 0 h) and FIG. 17B (promoter induction by gluconate at time = 7 h). Fold Change (FC) values for cell densities and specific relative mScarlet levels (relative mScarlet level normalized to cell density at a given time) were calculated as the ratio of values obtained from strains containing UP element and strains without UP element. FC values at the end of cultures are summarized in 0 3.

[0228] These data demonstrate that the presence of the P promoter’s UP element upstream of pT5-GntR-WT promoter 1) did not adversely affect cell growth and 2) enhanced specific mScarlet level by up to 50%-60%, depending on time of exposure to gluconate (inducer) and sampling time.7.5. Example 5: Development of a leaky gluconate-regulated promoter for controlled gene expression during growth and production phases.7.5.1. Materials and Methods

[0229] As discussed in Example 2 and shown in FIG. 18A, the pT5-GntR-WT promoter region has two tandem GntR-binding sites, which enables strong repression of the promoter by the GntR repressor in the absence of gluconate (inducer). To develop a leaky promoter, GntR-binding site 2 was eliminated using the CRISPR / MAD7-associated Lambda-RED recombineering tool on the chromosome to yield pT5-GntR-leaky promoter (SEQ ID: 63) (FIG. 18B and FIG. 19). mScarlet reporter was used to study the effect of gene expression driven by the pT5-GntR-leaky promoter.

[0230] Briefly, strains carrying pT5-GntR-WT or pT5-GntR-leaky promoter were grown in modified MOPS media (Neidhardt, et al., 1974, J. Bact. 119:736-747), where concentration of MOPS was increased to 200 mM to maintain pH levels around 7.0. The media initially contained either 0.5% (wt / vol) glycerol or 1% (wt / vol) sodium gluconate to measure leaky or fully induced gene expression, respectively. To compare accumulated relative mScarlet levels driven by both promoters at the end of cultures, strains grown on glycerol were treated with sodium gluconate (final concentration of 0.5% (wt / vol)) after 7 h of growth. All cultures were incubated at 37°C for total of 27 h in a 96-well plate using a plate reader. Respective cell densities and relative mScarlet levels were quantified by measuring OD 600nm and reporter fluorescence using excitation at 570 nm and emission at 591 nm with a 75% gain every 30 min over the course of cultures. Leakiness for mScarlet expression was defined as ratio of specific mScarlet levels in the absence of gluconate and specific mScarlet level in the presence of gluconate during growth phase (7 h post-inoculation), where specific mScarlet values were obtained by normalizing relative mScarlet levels to respective cell densities.7.5.2. Results

[0231] Table 4 shows leakiness levels during growth (time = 7h) and relative accumulated mScarlet levels at the end of culture (time = 27 h) for the WT and leaky promoters obtained from the mean of three biological replicates. These data demonstrate that leaky gene expression from pT5-GntR-leaky promoter was about 5.4-fold greater than that from pT5-GntR-WT promoter. Additionally, the accumulated mScarlet level yielded from the pT5- GntR-leaky promoter was almost 75% higher than that from pT5-GntR-WT.8. SEQUENCES

[0232] Exemplary sequences referred to herein are provided in Table 5 below (where “SEQ” refers to the SEQ ID NO).9. INCORPORATION BY REFERENCE

[0233] 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.

[0234] 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 nucleic acid comprising a system for expression of a protein of interest prokaryotic species, comprising(a) a regulatory element comprising:(i) a promoter, comprising from 5’ to 3’:A. a -35 sequence, optionally comprising TTGACA or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof; andB. a -10 sequence having a 5’ end and a 3’ end, optionally comprising TATAAT or a sequence having at least 3, at least 4, or at least 5 nucleotides thereof;(ii) an operator region comprising:A. a ribosome binding site (RBS), optionally comprising TAAGGAGG or a sequence having at least 4, at least 5, at least 6, or at least 7 nucleotides thereof; andB. a spacer, optionally having 3 or fewer (e.g., 3, 2, 1, or 0) cytosine nucleotides; and(iii) a first GntR binding site and optionally a second GntR binding site, each having a 5’ end and a 3’ end; and(b) an open reading frame (ORF) encoding a protein of interest other than gntK, optionally wherein the ORF comprises the most prevalent initiation codon in the prokaryotic species.

2. The nucleic acid of claim 1 , wherein the promoter comprises a sequence having at least 90% identity to SEQ ID NO:12.

3. The nucleic acid of claim 1 or claim 2, wherein the 5’ end of the first GntR binding site overlaps the 3’ end of the -10 sequence, the 3’ end of the first GntR binding site overlaps the 5’ end of the second GntR binding site, if present, or both.

4. The nucleic acid of any one of claims 1 to 3, wherein the -35 sequence comprises TTGACA, TTGATA, TTGATG, TTGATC, TTATTA, or TTGAGA and / or the -10 sequence comprises TACAAT or TATAAT.

5. The nucleic acid of any one of claims 1 to 4, wherein the first GntR binding site and the second GntR binding site, if present, each independently comprise a nucleotide sequence having at least 70% identity to:(a) TAATGTTACCSGTAACATTT (SEQ ID NO:1), wherein S is C or G;(b) TTTACAATGSCCATTGTAAT (SEQ ID NO:52), wherein S is C or G;(c) AATTGTTACGCGTAACAAAT (SEQ ID NO:2) or a sequence reverse to SEQ ID NO:2;(d) CAATGTTACCGATAACAGTT (SEQ ID NO:3) or a sequence reverse to SEQ ID NO:3;(e) TGACGTTACCCATAACAAAT (SEQ ID NO:4) or a sequence reverse to SEQ ID NO:4;(f) TATTTCTACTGATAAGAATT (SEQ ID NO:5) or a sequence reverse to SEQ ID NO:5;(g) TAATGTTACCGATAACAGTT (SEQ ID NO:6) or a sequence reverse to SEQ ID NO:6;(h) TTATTTTACCGGTAACATGA (SEQ ID NO:7) or a sequence reverse to SEQ ID NO:7;(i) AAATGTTACGGGTAACTGTT (SEQ ID NO:8) or a sequence reverse to SEQ ID NO:8;(j) GAATGATACGGGTAACATCT (SEQ ID NO:9) or a sequence reverse to SEQ ID NO:9.(k) TCACGTTATGCGTAACATAG (SEQ ID NO: 10) or a sequence reverse to SEQ ID NO: 10; or(l) AACAGTTACCCGTAACATTT (SEQ ID NO: 11) or a sequence reverse to SEQ ID NO:11.

6. The nucleic acid of any one of claims 1 to 5, wherein the RBS comprises GGAGG, GGAG, or AAGGA.

7. The nucleic acid of any one of claims 1 to 6, wherein the spacer comprises 2 or fewer cytosine nucleotides.

8. The nucleic acid of any one of claims 1 to 7, wherein the ORF encodes a 2- keto-3-deoxy-gluconate (KDG) aldolase.

9. The nucleic acid of any one of claims 1 to 7, wherein the ORF encodes a 2- dehydro-3-deoxy-6-phospho-D-gluconate (KDGP) aldolase.

10. The nucleic acid of any one of claims 1 to 7, wherein the ORF encodes a DXP pathway enzyme, optionally wherein the wherein the DXP pathway enzyme is a 1- deoxy-d-xylulose-5-phosphate synthase (Dxs; EC 2.2.1.7), a 1-deoxy-D-xylulose-5- phosphate reductoisomerase (Dxr; EC 1.1.1.267), a 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD; EC 2.7.7.60), a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148), a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; EC 4.6.1.12), a 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG; EC 1.17.7.1 or EC 1.17.7.3), a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH; EC 1.17.7.4), or an isopentenyl-diphosphate Delta-isomerase (Idi; EC 5.3.3.2).11 . The nucleic acid of any one of claims 1 to 7, wherein the ORF encodes a terpene synthase.

12. The nucleic acid of any one of claims 1 to 11 , wherein the regulatory element comprises a nucleotide sequence having at least 90% sequence identity to nucleotides 1-85 of SEQ ID NO:51 or nucleotides 1-86 of SEQ ID NO:27.

13. The nucleic acid of any one of claims 1 to 12, which is incorporated into a plasmid.

14. The nucleic acid of any one of claims 1 to 12, which is integrated into the genome of the prokaryotic species.

15. The nucleic acid of any one of claims 1 to 14, wherein the regulatory element further comprises an enhancer.

16. The nucleic acid of claim 15, wherein the enhancer is derived from the lambda bacteriophage PL upstream (UP) element, optionally wherein the enhancer comprises the nucleotide sequence of any one of SEQ ID NOS:62 and 75-77.

17. A prokaryotic cell comprising the nucleic acid of any one of claims 1 to 16.

18. The prokaryotic cell of claim 17, wherein the prokaryotic species is E. coli, optionally wherein the E. coli is a K-12 strain.

19. The prokaryotic cell of claim 18, which has:(a) a reduced ability to use glucose as compared to a parental cell, optionally wherein the prokaryotic cell has a loss of function mutation (e.g., deletion) in pts, err, or both, optionally wherein the loss of function mutation (e.g., deletion) in pts is a loss of function mutation (e.g., deletion) in ptsH, ptsl, or both.(b) a reduced ability to phosphorylate gluconate as compared to a parental cell, optionally wherein the prokaryotic cell has a loss of function mutation (e.g., deletion) in gntK, idnK, or both;(c) a reduced ability to dehydrate gluconate-6P as compared to a parental cell, optionally wherein the prokaryotic cell has a loss of function mutation (e.g., deletion) in edd;(d) increased expression of KDGP aldolase as compared to a parental cell, optionally wherein the prokaryotic cell is engineered to recombinantly express or overexpress eda.

20. A method of producing a protein of interest in a prokaryotic species, comprising:(a) growing prokaryotic cells according to any one of claims 17 to 19 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration; and(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of the protein of interest.21 . The method of claim 20, wherein the medium for growing comprises glycerol.

22. A method of producing a product, comprising:(a) growing prokaryotic cells according to any one of claims 17 to 19 in a medium lacking sucrose, fructose, glucose, and gluconate to a desired cell concentration;(b) culturing the cells in a medium containing at least one of sucrose, fructose, glucose, and / or gluconate, thereby inducing expression of the protein of interest; and(c) catalyzing a reaction by the protein of interest, thereby producing or leading to production of the product.

23. The method of claim 22, wherein the medium for culturing comprises a substrate for the reaction.

24. The method of claim 22 or claim 23, wherein the medium for growing comprises glycerol.

Images