Recombinant microorganisms with increased accumulation and / or FLUX of cytidine triphosphate (CTP)

Abstract

Recombinant microorganisms having reduced activity of endogenous nucleoside monophosphate phosphohydrolases (EC 3.1.3.5 or EC 3.1.3.6), endogenous ribonucleotide monophosphatases (EC 3.1.3.5), endogenous nucleotide triphosphate pyrophosphohydrolases (EC 3.6.1.56), and / or endogenous 5'-ribonucleotide phosphohydrolases (EC 3.1.3.5) relative to parental microorganisms; and engineered to increase activity of a pathway that utilizes cytidine triphosphate (CTP). The recombinant microorganisms can be used in methods for producing isoprenoids and / or methods for increasing recycling of cytidine monophosphate (CMP) to cytidine triphosphate (CTP).

Description

RECOMBINANT MICROORGANISMS WITH INCREASED ACCUMULATION AND / OR FLUX OF CYTIDINE TRIPHOSPHATE (CTP) 1. CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of United States provisional application no. 63 / 602,178 filed November 22, 2023, the contents of which are incorporated herein in their entirety 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 November 20, 2024, is named BPC-022WO_SL.xml and is 101,004 bytes in size. 3. BACKGROUND

[0003] Cytidine triphosphate (CTP) is useful in numerous cellular processes across the kingdoms of life, including the synthesis of nucleic acids and as an energy and phosphorus donor in a number of metabolic reactions. As a donor, in some metabolic pathways, CTP loses two phosphate groups to yield cytidine monophosphate (CMP). In metabolic pathways in which CTP is consumed to produce products of bioindustrial interest, CTP consumption can reduce the yield and / or production rate of the products. There is a need in the art for the efficient generation or regeneration of CTP. 4. SUMMARY

[0004] The present disclosure provides recombinant microorganisms engineered to have increased accumulation and / or flux of CTP. This can be effected by improved regeneration of CTP from CMP, reduced direct conversion of CTP to CMP, and / or increased production of CTP via a pathway in which orotidine monophosphate (OMP) is a precursor. Typically, recombinant microorganisms of the disclosure are concurrently engineered to increase activity of a pathway that utilizes (e.g., consumes) CTP.

[0005] Improved regeneration of CTP can be achieved through reduced activity of nucleoside monophosphate phosphohydrolases (EC 3.1.3.5 or EC 3.1.3.6), such as that encoded in E. coli by umpG; ribonucleotide monophosphatases (EC 3.1.3.5), such as that encoded in E. coli by umpH; and / or 5'-ribonucleotide phosphohydrolases (EC 3.1.3.5), such as that encoded in E. coliby ushA. Reduced direct conversion of CTP to CMP can be achieved through reduced activity of nucleoside triphosphate pyrophosphohydrolases (EC 3.6.1.56), such as that encoded in E. coli by nudG. Increased production of CTP via OMP can be achieved by increasing the activity of orotate phosphoribosyltransferases (EC 2.4.2.10), such as that encoded in E. coli by pyrE.

[0006] Examples of pathways in which CTP are utilized are isoprenoid production pathways. Accordingly, in some aspects, recombinant microorganisms of the present disclosure are engineered to have increased isoprenoid production.

[0007] Examples of recombinant microorganisms engineered to have reduced activity of endogenous nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, endogenous nucleoside triphosphate pyrophosphohydrolases, and / or endogenous 5'-ribonucleotide phosphohydrolases are described in Section 6.2.1 and numbered embodiments 1 to 30.

[0008] Examples of recombinant microorganisms engineered to have increased CTP production due to increased activity of cytidylate kinase and / or nucleoside disphosphate kinase are described in Section 6.2.1 and numbered embodiments 126 to 151.

[0009] Examples of recombinant microorganisms engineered to have increased CTP production via OMP are described in Section 6.2.2 and numbered embodiments 152 to 165.

[0010] Examples of recombinant microorganisms engineered to have increased isoprenoid production are described in Section 6.2.3 and numbered embodiments 41to 125.

[0011] Production of the recombinant microorganisms can proceed from parental microorganisms described in Section 6.3.1 and numbered embodiments 161 to 165, by techniques described in Section 6.3.2.

[0012] Recombinant microorganisms of the present disclosure can be used in methods for production of isoprenoids as described in Section 6.4.3 and numbered embodiments 166 to 182, and / or in methods for increasing recycling of CMP to CTP as described in Section 6.4.4 and numbered embodiments 183 to 188. 5. BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG.1 schematically depicts three pathways leading to CTP. Abbreviations used: PRPP, phosphoribosyl diphosphate; OMP, orotidine monophosphate; UMP, uridine monophosphate; UDP, uridine diphosphate; UTP, uridine triphosphate; PyrC, dihydroorotase (EC 3.5.2.3); PyrD, dihydroorotate dehydrogenase (EC 1.3.5.2); PyrE, orotate phosphoribosyltransferase (EC2.4.2.10); PyrF, orotidine 5'-phosphate decarboxylase (EC 4.1.1.23); PyrH, uridylate kinase (EC 2.7.4.22); PyrG, CTP synthase (EC 6.3.4.2); RihA, pyrimidine-specific ribonucleoside hydrolase RihA (EC 3.2.2.8) ; RihB, pyrimidine-specific ribonucleoside hydrolase RihB (EC 3.2.2.8); CodA, cytosine deaminase (EC 3.5.4.1); Udp, uridine phosphorylase (EC 2.4.2.3); Udk, uridine kinase (EC 2.7.1.48); CTP, cytidine triphosphate; CDP, cytidine diphosphate; CMP, cytidine monophosphate; UmpG, nucleoside monophosphate phosphohydrolase (EC 3.1.3.5; EC 3.1.3.6); UmpH, ribonucleotide monophosphatase (EC 3.1.3.5) ; Cmk, cytidylate kinase (EC 2.7.4.25); Ndk, nucleoside diphosphate kinase (EC 2.7.4.6); NudG, CTP pyrophosphohydrolase (EC 3.6.1.65); UshA, 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5).

[0014] FIG.2 schematically depicts the DXP pathway, in context with pathways leading from glucose or xylose to DXP, and with pathways leading from DMAPP and IPP to isoprenoids, with certain reactants and products assigned numbers for ease of reference and convenience. Abbreviations used: KDG (3), 2-keto-3-deoxygluconate; KDGP (4), 2-keto-3-deoxy-6- phosphogluconate; GAP (5), glyceraldehyde-3-phosphate; DXP (8), 1-deoxyxylulose-5- phosphate; MEP (9), 2-C-methylerythritol 4-phosphate; CDP-ME (10), 4-diphosphocytidyl-2-C- methylerythritol; CDP-MEP (11), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MEcPP (12), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP (13), (E)-4-Hydroxy-3- methyl-but-2-enyl pyrophosphate; DMAPP (14), dimethylallyl pyrophosphate; IPP (15), isopentenyl pyrophosphate; xylose (16); xylulose (17); xylulose-5-phosphate (18); 1- deoxyxylulose (DX) (19); GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; CTP, cytidine triphosphate; CMP, cytidine monophosphate; Dxs, 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7); Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267); IspD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60); IspE, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148); IspF, 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase (EC 4.6.1.12); IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1); IspH, 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2); Idi, isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2); IspA, farnesyl diphosphate synthase (EC 2.5.1.10); CrtEIB, CrtE, geranylgeranyl diphosphate synthase (EC 2.5.1.29); CrtI, phytoene desaturase (EC 1.3.99.31); and CrtB, 15-cis-phytoene synthase (EC 2.5.1.32); RibB, 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12); YajO, 1-deoxyxylulose-5- phosphate synthase (EC 1.1.-.-); and XylB, xylulose kinase (EC 2.7.1.17). The use of multi- headed arrows (e.g., “→→→”) indicates multiple enzymatic activities are involved in converting the substrate to the reactant in the depicted step. Not all enzymatic activities are shown, and single-headed arrows can be indicative of multi-step processes.

[0015] FIG.3 shows growth (measured as biomass concentration (scattered light)) at various time points for an E. coli base strain and study strains ΔumpG, ΔumpH, ΔumpGΔumpH, ΔushA, and ΔumpGΔushA as described in Example 2. Results are reported as the average of two biological replicates.

[0016] FIG.4 schematically represents the umpG operon of E. coli K-12 substr. MG1655, in which umpG and pcm coding regions are operably linked to the surEp2 promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, Keseler et al., 2021, Front Microbiol 12:711077.

[0017] FIG.5A schematically represents an umpH coding region and promoter of E. coli K-12 substr. MG1655, in which the umpH coding region is operably linked to the umpHp promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.

[0018] FIG.5B schematically represents an operon comprising umpH of E. coli K-12 substr. MG1655, in which nagB, nagA, nagC, and umpH coding regions are operably linked to the nagBp promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.

[0019] FIG.6 schematically represents an ushA coding region and promoter of E. coli K-12 substr. MG1655, in which the ushA coding region is operably linked to the ushAp6 promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.

[0020] FIG.7 schematically represents a nudG coding region and promoter of E. coli K-12 substr. MG1655, in which the nudG coding region is operably linked to the nudGp3 promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.

[0021] FIG.8A schematically represents the pyrE operon of E. coli K-12, in which the rph and pyrE coding regions are operably linked to the rph promoter. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.

[0022] FIG.8B schematically represents the pyrE operon of E. coli K-12 substr. MG1655, in which the rph coding region is truncated by a frameshift mutation. The 5’ to 3’ direction is from left to right. This schematic is not to scale. Prepared with reference to EcoCyc21, ibid.6. DETAILED DESCRIPTION 6.1. Definitions

[0023] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0024] Heterologous: As used herein, the term “heterologous,” when used to describe a first element in reference to a second element indicates that the first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions, or (d) any combination of two or all of (a), (b) and (c). For example, a heterologous regulatory sequence (e.g., promoter, enhancer) can be used to regulate expression of a coding sequence in a way that is different than the coding sequence is normally expressed in nature. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation, wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently or semi-stably for more than one generation (e.g., episomal vector, plasmid or other self-replicating vector).

[0025] Nucleic Acid: The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.

[0026] Operably Linked: The term “operably linked,” when used to describe the relationship between a first nucleic acid or nucleotide sequence and a second nucleic acid or nucleotide sequence, indicates that the first nucleic acid or nucleotide sequence is placed in a functional relationship with the second nucleic acid or nucleotide sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.

[0027] Operon: An “operon” as used herein refers to a nucleic acid sequence encoding multiple coding regions which are transcribed in a single transcript. The coding regions of the operon thus share regulatory sequences that are 5’-ward of the most upstream coding region (which may be termed “operon upstream regulatory sequences”) and 3’-ward of the most downstream coding region (which may be termed “operon downstream regulatory sequences”).

[0028] Parental Microorganism: The terms “parental cell” or “parental microorganism” are used interchangeably to refer to unicellular organisms which can be engineered to increase CTP flux or recycling, e.g., by regeneration of CTP and / or reduction of direct conversion to CMP. In some embodiments, increasing CTP flux can be achieved by reducing the activity of the UmpG, UmpH, NudG, UshA genes, or any combination of two or more of the foregoing. The adjective “parental” indicates that a recombinant cell or recombinant microorganism can be engineered by the introduction into a parental cell or parental microorganism of a heterologous nucleic acid or plurality of heterologous nucleic acids, such as nucleic acid(s) each comprising a coding region or plurality of coding regions each encoding a heterologous polypeptide, and / or by insertion, deletion, substitution, or other modification of coding regions or regulatory sequences in the genome of the parental microorganism.

[0029] A parental microorganism can be a microorganism found in nature or a microorganism that is non-naturally occurring. In other words, a parental microorganism can comprise one or more genetic modifications (e.g., insertion, deletion, or modification of one or more coding regions and / or regulatory sequences) relative to a strain thereof found in nature. In relationship to a recombinant microorganism of the disclosure generated through a series of engineering steps, the terms “parental cell” and “parental microorganism” can refer to an ancestral cell or organism incorporating any of the engineering steps, as well as a cell or microorganism without any of the engineering steps. Sometimes, for ease of reference and comparison, the terms “parental cell” and “parental microorganism” refer to a cell or microorganism which, if havinggenetic modifications, the genetic modification(s) do not relate to one or both aspects of the microorganism engineering described in Section 6.2.1 and Section 6.2.2. Further, the term “parental cell” and “parental microorganism” is intended for use as a reference cell or microorganism and not that the cell or organism was used as a starting point for engineering a microorganism of the disclosure. In some embodiments, a parental cell or parental microorganism has (i) at least wild-type activity of an endogenous nucleoside monophosphate phosphohydrolase (EC 3.1.3.5 or EC 3.1.3.6); (ii) at least wild-type activity of an endogenous ribonucleotide monophosphatase (EC 3.1.3.5); (iii) at least wild-type activity of an endogenous nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.56), (iv) at least wild-type activity of an endogenous 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5); or (v) any combination of two, three or all four of (i), (ii), (iii), and (iv). In further embodiments, a parental cell or parental microorganism comprises an engineered pathway that utilizes CTP (e.g., as described in Section 6.2.3), optionally together with (i) at least wild-type activity of an endogenous nucleoside monophosphate phosphohydrolase (EC 3.1.3.5 or EC 3.1.3.6); (ii) at least wild-type activity of an endogenous ribonucleotide monophosphatase (EC 3.1.3.5); (iii) at least wild-type activity of an endogenous nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.56), (iv) at least wild- type activity of an endogenous 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5); or (v) any combination of two, three or all four of (i), (ii), (iii), and (iv).

[0030] Polypeptide: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide herein may be identified by a name or by a percentage of sequence identity to a reference amino acid sequence. When a polypeptide is identified by a name indicative of an activity performed or enabled by the polypeptide, the name refers to any polypeptide capable of performing or enabling the activity.

[0031] Promoter: A “promoter” as used herein refers to a nucleic acid sequence which is capable of interacting with an RNA polymerase such that transcription of a sequence of interest begins. A typical prokaryotic promoter includes a -35 sequence (a region of about 6 nucleotides, the 5’ end of which is located from 30 to 40 nucleotides, such as 35 nucleotides, upstream (i.e., 5’-ward) of the transcription start site) and a -10 sequence, also known as a Pribnow box (a region of about 6 nucleotides, the 5’ end of which is located from 5 to 15 nucleotides, such as 10 nucleotides, upstream of the transcription initiation site). A prokaryotic promoter typically has from 12 to 22 nucleotides, and in some embodiments 17 ± 3 (e.g., 14, 15, 16, 17, 18, 19, or 20 nucleotides), intervening between the -35 sequence and the -10 sequence. A promoter may include at least a portion of a repressor binding site and / or an activator binding site.

[0032] Recombinant microorganism: The terms “recombinant cell” and “recombinant microorganism” are used interchangeably to refer to a cell that has been genetically engineered. It should be understood that this term refers not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, a recombinant counterpart of a parental cell or parental microorganism includes progeny that are not identical to the initial recombinant cell or microorganism engineered from the parent cell or parental microorganism, but are still included within the scope of the terms “recombinant cell” or “recombinant microorganism” as used herein.

[0033] Regulatory Sequence: A “regulatory sequence” as used herein refers to non-coding sequences that influence the expression (e.g., transcription or translation) of a transcribed sequence. Regulatory sequences include different types of regulatory elements such as promoters, operator regions, terminator sequences, intergenic sequences encoding small regulatory RNAs (sRNAs), Shine-Dalgarno (SD) sequences, etc.

[0034] Sequence Identity: “Sequence identity” in relation to nucleotide or amino acid sequence of a nucleic acid or polypeptide molecule, refers to the overall relatedness between two such sequences. Calculation of the percent sequence identity (nucleotide or amino acid sequence identity) of two sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Percent sequence identity can be determined manually once an alignment of nucleotide or amino acid sequences is generated. An alignment of query nucleotide or amino acid sequence and a reference nucleotide or amino acid sequence can be generated using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more reference sequences and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a reference sequence, or both, to maximize sequence alignments. For fast pair wisealignment of nucleotide sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For fast pairwise alignment of amino acid sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. Unless indicated otherwise, the percent sequence identity between a reference nucleotide or amino acid sequence (e.g. a sequence with a defined SEQ ID NO as disclosed herein) and a query nucleotide or amino acid sequence is calculated across the entire length of the reference sequence.

[0035] Transformation: The term “transformation” refers to the introduction of nucleic acid molecules into cells, e.g., into prokaryotic cells. In the context of the present disclosure, the term “transformation” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, e.g., into prokaryotic cells, such as into bacterial cells. Such methods encompass, for example, electroporation, calcium phosphate precipitation, or nanoparticle- based transformation, among other techniques known to the person of ordinary skill in the art having the benefit of the present disclosure.

[0036] Wild-type: The term “wild-type” as used herein to describe a microorganism species or strain refers to a defined species or strain, e.g., as deposited with a depositary such as the American Type Culture Collection (Manassas, Virginia). When describing a nucleic acid or polypeptide, “wild-type” indicates the nucleic acid or polypeptide has a sequence identical to that of the corresponding nucleic acid or polypeptide in a wild-type species or strain. An exemplary microorganism strain that is sometimes referenced herein as a “wild-type” strain is E. coli K12 substrain MG1655. Another “wild-type” E. coli strain is E. coli K12 substrain BW25113. 6.2. Recombinant Microorganisms

[0037] The present disclosure provides recombinant microorganisms engineered to (a) improve CTP regeneration (e.g., recycling of cytidine monophosphate produced though hydrolysis of CTP back to CTP) and / or reduce direct conversion of CTP to CMP, and (b) increase activity of a pathway that utilizes CTP. The improvement in CTP regeneration, reduction in direct conversion of CTP to CMP, and / or increased activity of a pathway that utilizes CTP can be in relation to a parental or wild-type organism. In some embodiments, the improvement in CTP regeneration, reduction in direct conversion of CTP to CMP, and / or increased activity of a pathway that utilizes CTP is in relation to a wild-type organism. In some embodiments, the improvement in CTP regeneration, reduction in direct conversion of CTP to CMP, and / or increased activity of a pathway that utilizes CTP is in relation to a parental organism, e.g., a parental microorganismthat is not a wild-type organism. In some embodiments, a parental microorganism is an organism that is engineered to increase activity of a pathway that utilizes CTP in relation to a wild-type organism.

[0038] Improving CTP regeneration can be achieved through reducing activity of endogenous nucleoside monophosphate phosphohydrolases (EC 3.1.3.5 or EC 3.1.3.6); endogenous ribonucleotide monophosphatases (EC 3.1.3.5), and / or endogenous 5’-ribonucleotide phosphohydrolases (EC 3.1.3.5).. Exemplary modifications to improve CTP regeneration are described in Section 6.2.1.

[0039] Reducing direct conversion of CTP to CMP can be achieved through reducing activity of endogenous nucleoside triphosphate pyrophosphohydrolases (EC 3.6.1.56). Exemplary modifications to reduce direct conversion of CTP to CMP are also described in Section 6.2.1.

[0040] A pathway that yields commercially valuable products and which utilizes CTP is the isoprenoid pathway. The present disclosure provides microorganisms engineered to increase flux or production of an isoprenoid pathway component or precursor, e.g., any of components (1) through (19) of FIG.2, in addition to increasing CTP regeneration. Exemplary modifications to increase flux or production of an isoprenoid pathway component or precursor are described in Section 6.2.2. 6.2.1. Increasing CTP Flux By Regeneration of CTP and / or Reduction of Direct Conversion to CMP

[0041] The recombinant microorganisms of the disclosure are engineered to increase CTP regeneration and / or reduce direct conversion of CTP to CMP, while at the same time are engineered to increase activity of a pathway which utilizes (e.g., consumes) CTP.

[0042] Increasing CTP regeneration can be achieved by reducing activity of endogenous nucleoside monophosphate phosphohydrolases (EC 3.1.3.5 or EC 3.1.3.6); endogenous ribonucleotide monophosphatases (EC 3.1.3.5), and / or endogenous 5’-ribonucleotide phosphohydrolases (EC 3.1.3.5). Reducing the activity of endogenous nucleoside triphosphate pyrophosphohydrolases (EC 3.6.1.56) can reduce direct conversion of CTP to CMP.

[0043] Reduction of a cell’s endogenous nucleoside monophosphate phosphohydrolase activity, endogenous ribonucleotide monophosphatase activity, endogenous nucleoside triphosphate pyrophosphohydrolase activity, and / or endogenous 5’-ribonucleotide phosphohydrolase activity can be achieved by modifications to coding sequences encoding nucleoside monophosphate phosphohydrolases, ribonucleotide monophosphatases, nucleosidetriphosphate pyrophosphohydrolases, and / or 5’-ribonucleotide phosphohydrolases and / or modifications to regulatory sequences (e.g., promoters) operably linked to such coding sequences. In either situation, modification can include deletion of all or a portion of a coding sequence and / or a regulatory sequence; replacement of all or a portion of a coding sequence and / or a regulatory sequence; and / or introduction of one or more mutations in a coding sequence and / or a regulatory sequence.

[0044] In coding regions, introduced mutations can be missense mutations (mutations that change amino acids encoded at particular residues in a polypeptide), insertion mutations (mutations that add amino acids into the primary sequence of a polypeptide), nonsense mutations (mutations that replace codons coding amino acids with stop codons), or frameshift mutations (mutations that insert or delete a number of nucleotides not divisible by three, which thereby are highly likely to change amino acids encoded by downstream codons). Missense, insertion, nonsense, and / or frameshift mutations can disrupt enzymatic activities of a polypeptide and / or introduce degradation tags that lower the intracellular half-life of a polypeptide. Modified coding sequences can comprise multiple mutations.

[0045] Modifications of regulatory sequences can include elimination of one or more regulatory sequences such that a coding sequence is not transcribed, a transcript is not translated, a constitutive or strong promoter is replaced with an inducible (in which case, activity can be reduced by withholding the inducer from the recombinant microorganisms) or weak promoter, or the like.

[0046] RNA interference (RNAi) techniques can alternatively or additionally be used to reduce a cell’s endogenous nucleoside monophosphate phosphohydrolase activity endogenous ribonucleotide monophosphatase activity, endogenous nucleoside triphosphate pyrophosphohydrolase activity, and / or endogenous 5’-ribonucleotide phosphohydrolase activity.

[0047] It should be borne in mind, depending on the cell, that an endogenous nucleoside monophosphate phosphohydrolase activity, an endogenous ribonucleotide monophosphatase activity, an endogenous nucleoside triphosphate pyrophosphohydrolase activity, and / or an endogenous 5’-ribonucleotide phosphohydrolase activity may be provided by an enzyme encoded by a coding region that is a part of an operon. Accordingly, the techniques referred to herein for reducing these activities may be tailored to maintain the activities of other enzymes encoded by the same operon. For example, mutations of coding regions encoding endogenous nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, endogenous nucleoside triphosphate pyrophosphohydrolases, and / orendogenous 5’-ribonucleotide phosphohydrolases would be expected to reduce these activities without modifying the activities of other enzymes encoded by the same operon. For another example, if coding regions encoding endogenous nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, endogenous nucleoside triphosphate pyrophosphohydrolases, and / or endogenous 5’-ribonucleotide phosphohydrolases are the most 3’-ward of the coding regions in the operons, transcription termination sites can be engineered into the nucleic acid sequences upstream of the 5’ end of the coding regions encoding endogenous nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, and / or endogenous 5’-ribonucleotide phosphohydrolases and 3’-ward of next nearest upstream coding region.

[0048] More specific description of particular modification techniques will follow with reference to particular nucleoside monophosphate phosphohydrolases, ribonucleotide monophosphatases, nucleoside triphosphate pyrophosphohydrolases, and 5’-ribonucleotide phosphohydrolases.

[0049] Which endogenous nucleotide sequences encode nucleoside monophosphate phosphohydrolases, ribonucleotide monophosphatases, nucleoside triphosphate pyrophosphohydrolases, and / or 5’-ribonucleotide phosphohydrolases will depend on the species of recombinant microorganisms of interest. In E. coli, an endogenous nucleoside monophosphate phosphohydrolase is umpG (UniProt Accession No. A0A4C7A4I9; SEQ ID NO:41) and an endogenous ribonucleotide monophosphatase is umpH (also known as nagD; UniProt Accession No. P0AF24; SEQ ID NO:42). An endogenous nucleoside triphosphate pyrophosphohydrolase is nudG (UniProt Accession No. P77788; SEQ ID NO:43). An endogenous 5’-ribonucleotide phosphohydrolase is ushA (UniProt Accession No. P07024; SEQ ID NO: 44).

[0050] E. coli umpG requires a divalent metal cation cofactor which binds at amino acid residues 8D, 9D, 39S, and 92N of SEQ ID NO:41. In some embodiments, mutations introduced to the umpG coding region change one or more of the endogenous amino acids 8D, 9D, 39S, and 92N of SEQ ID NO:41 to reduce nucleoside monophosphate phosphohydrolase activity.

[0051] As shown in FIG.4, E. coli K12 substr. MG1655 umpG is encoded by an operon that also encodes pcm. The sequence of the operon is provided as SEQ ID NO:1. Mutations of the umpG coding region (SEQ ID NO:2) would be expected to reduce nucleoside monophosphate phosphohydrolase activity or polypeptide half-life with minimal interference with pcm expression. Alternatively or additionally, disruption of the surEp2 promoter and / or other operon upstreamregulatory sequences (SEQ ID NO:3), combined with the engineering of a promoter and any other appropriate regulatory sequences into a suitable position upstream of pcm, would also be expected to reduce nucleoside monophosphate phosphohydrolase activity in E. coli with minimal interference with pcm expression. Another option would be disruption of the surEp2 promoter and / or other operon upstream regulatory sequences, combined with the introduction of a heterologous pcm into another location of the E. Coli genome or into a non-genomic vector (e.g., a plasmid or a bacterial artificial chromosome (BAC)), would also be expected to reduce nucleoside monophosphate phosphohydrolase activity in a manner that need not interfere with pcm expression. Other approaches will be apparent to persons of ordinary skill in the arts having the benefit of the present disclosure.

[0052] E. coli umpH requires Mg2+as cofactor which binds at amino acid residues D9, D11, and D201. E. coli umpH binds substrate at amino acid residues D11, T42, N43, K176, N202, L203, R204, and T205 of SEQ ID NO:42. Amino acid residue R55 plays a role in the orientation of D11 for proton transfer, and amino acid residue D146 is involved in substrate specificity. In some embodiments, mutations introduced to the umpG coding region change one or more of the endogenous amino acids 8D, 9D, 39S, and 92N to reduce cofactor binding. In some embodiments, mutations introduced to the umpH coding region change one or more of the endogenous amino acids D9, D11, T42, N43, R55, D146, K176, D201, N202, L203, R204, and T205 of SEQ ID NO:42 to reduce ribonucleotide monophosphatase activity.

[0053] As shown in FIG.5A and FIG.5B, E. coli K12 substr. MG1655 umpH is expressed in two ways. One is from a monocistronic transcript promoted by umpHp (FIG.5A; SEQ ID NO:4); the other is as part of the nagBAC-umpH operon operably linked to the nagBp promoter (FIG.5B; SEQ ID NO:7). Routes that can be considered for reduction of ribonucleotide monophosphatase activity in E. coli with minimal interference with nagB, nagA, and / or nagC expression include mutations of the umpH coding region (SEQ ID NO:5), disruption of the umpHp promoter (SEQ ID NO:6), and insertion of a transcription terminator between nagC and umpH in SEQ ID NO:7, among others that will be apparent to persons of ordinary skill in the arts having the benefit of the present disclosure.

[0054] E. coli ushA requires Zn2+as cofactor which binds at amino acid residues D41, H43, D84, N116, H217, H252, and Q254 of SEQ ID NO:44. Amino acid residues H117 and D120 are involved in stabilization of the transition state. Substrate is bound at amino acid residues R375, D376, K377, V378, R379, F498, N499, A500, T501, G502, G503, and D504. In some embodiments, mutations introduced to the ushA coding region change one or more of theendogenous amino acids D41, H43, D84, N116, H117, D120, H217, H252, Q254, R375, D376, K377, V378, R379, F498, N499, A500, T501, G502, G503, and D504 of SEQ ID NO:44 to reduce 5'-ribonucleotide phosphohydrolase activity.

[0055] As shown in FIG.6, E. coli K12 substr. MG1655 ushA is expressed from a monocistronic transcript promoted by promoter ushAp6 (FIG.6; SEQ ID NO:45; -35 region and -10 region in bold). Reduction of ushA activity can be effected by any technique described herein.

[0056] E. coli nudG requires Mn2+as a cofactor, although the amino acid residues where cofactor binds have not been elucidated at this time. Amino acid residues F34, A35, G36, G37, K38, V39, R72, and D118 are believed to be involved in substrate binding. In some embodiments, mutations introduced to the nudG coding region change one or more of the endogenous amino acids F34, A35, G36, G37,K38, V39, R72, and D118 to reduce nucleoside triphosphate pyrophosphohydrolase activity.

[0057] As shown in FIG.7, E. coli K12 substr. MG1655 nudG is expressed from a monocistronic transcript promoted by promoter nudGp3 (FIG.7; SEQ ID NO:46; -35 region and -10 region in bold). Reduction of nudG activity can be effected by any technique described herein.

[0058] The discussion above has focused on E. coli K12 substr. MG1655 by way of example. Other parental microorganisms, e.g., other substrains of E. coli K12, other strains of E. coli, other bacteria, or other microorganisms can be engineered to have reduced endogenous nucleoside monophosphate phosphohydrolase activity, endogenous ribonucleotide monophosphatase activity, endogenous nucleoside triphosphate pyrophosphohydrolase activity, and / or endogenous 5’-ribonucleotide phosphohydrolase activity. Particular parental microorganisms which can be engineered to produce recombinant microorganisms of the present disclosure are described in Section 6.3.1. 6.2.2. Particular techniques by which coding regions and / or regulatory sequences can be modified are described in Section 6.3.2.Improved CTP Production from de Novo Pathway

[0059] In addition to being engineered for improved CTP regeneration and / or reduced direct conversion of CTP to CMP, recombinant microorganisms of the present disclosure can be engineered to have improved CTP production via the de novo pathway relative to a wild-type parental microorganism.

[0060] To improve CTP production via the de novo pathway in a microorganism, pyrE activity can be increased.

[0061] In some embodiments, increasing pyrE activity can be achieved by engineering a strain to include a heterologous pyrE expression cassette. In some embodiments, the pyrE coding region is operably linked to a heterologous promoter, for example by replacement of a coding region in the microorganism genome with a pyrE coding region, by insertion of an expression cassette comprising a pyrE coding region operably linked to a promoter into the microorganism genome, or by inclusion of such an expression cassette in a plasmid or bacterial artificial chromosome, among other techniques. For yet another example, pyrE activity can be increased by codon optimization. These techniques and others that will be apparent to persons of ordinary skill in the art having the benefit of the present disclosure can be used alone or in any desired combination.

[0062] As shown in FIG.8A and FIG.8B, in E. coli, the pyrE (orotate phosphoribosyltransferase, EC 2.4.2.10) coding region is the 3’-most coding region in a bicistronic operon, downstream of rph, which encodes a phosphorolytic exoribonuclease.

[0063] Thus in E. coli, increasing pyrE activity can be achieved by incorporating a promoter between the rph and pyrE coding regions in the microorganism genome,

[0064] In certain E. coli strains, pyrE activity is reduced by virtue of a mutation in an upstream gene. In particular, in certain E. coli K12 substrains, a frameshift deletion in rph (relative to rph in E. coli K12) gives the rph coding region the sequence of SEQ ID NO:62. This frameshift deletion is schematically depicted in FIG.8B. This leads to reduced expression of pyrE compared to E. coli K12. As shown in FIG.1, pyrE catalyzes the formation of OMP and increases flux through the de novo pathway to generate CTP. Reduced expression of pyrE is currently understood to explain the reduced ability of certain E. coli K12 substrains (including E. coli MG1655) to reach high cell density in pyrimidine-restricted media, compared to their common ancestor W1485 strain.

[0065] Thus, in E. coli strains having this mutation, pyrE activity can be increased by correcting the frameshift deletion in rph, e.g., by CRISPR gene editing among other techniques, to yield a coding region such as that having the sequence of SEQ ID NO:63. 6.2.3. CTP Utilizing Pathways

[0066] The recombinant microorganisms of the disclosure are engineered to increase activity of a pathway which utilizes CTP, while at the same time engineered to increase CTP regeneration or reduce direct conversion of CTP to CMP.

[0067] One pathway that yields commercially valuable products and which utilizes CTP is the isoprenoid pathway. The present disclosure provides microorganisms engineered to increase flux or production of an isoprenoid pathway component or precursor, e.g., any of components (1) through (19) of FIG.2, in addition to increasing CTP regeneration.

[0068] In some embodiments, the microorganism is engineered to increase the activity of the 1- deoxyxylulose-5-phosphate (DXP) pathway, such as by recombinantly expressing one or more DXP pathway enzymes. FIG.2 schematically depicts the DXP pathway, in context with pathways leading from glucose (1) or xylose (14) to DXP (8), and with pathways leading from dimethylallyl pyrophosphate (DMAPP) (14) and isopentenyl pyrophosphate (IPP) (15) to isoprenoids. In the DXP pathway, CTP is incorporated into 4-diphosphocytidyl-2-C- methylerythritol (CDP-ME) (10) by a 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD; EC 2.7.7.60), and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF; EC 4.6.1.12) releases CMP from 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP- MEP) (11).

[0069] Isoprenoid production can be increased by increasing the activity of one or more enzymes of the DXP pathway as shown in FIG.2. Alternatively or additionally, isoprenoid production can be increased by increasing the activity of one or more enzymes that interconvert DMAPP and IPP and / or convert DMAPP and / or IPP to isoprenoids, such as GPP; FPP; isoprene; limonene; ^-myrcene; nerol; geraniol; linalool; ^-ocimene;1,8-cineole; farnesene; or farnesol, among others.

[0070] Further additionally or alternatively, isoprenoid production can be increased by increasing the activity of one or more enzymes that convert glucose or xylose to DXP. For example, recombinant microorganisms can be engineered to increase isoprenoid production in part by being engineered to produce 1-deoxyxylulose-5-phosphate (DXP) from 2-keto-3- deoxygluconate (KDG). Such organisms can be further engineered to produce KDG from glucose. One approach by which microorganisms can be further engineered to produce KDG from glucose is described in PCT / US2020 / 041801, the contents of which are hereby incorporated herein by reference in their entirety.

[0071] For another example, recombinant microorganisms can be engineered to increase isoprenoid production in part by being engineered to produce DXP from ribulose-5-P. For another example, recombinant microorganisms can be engineered to increase isoprenoid production in part by being engineered to produce DXP from 1-deoxyxylulose (DX). The threepathways for DXP production shown in FIG.2 are not mutually exclusive; any one, any two, or all three can be active in a single recombinant microorganism.

[0072] The activity of one or more enzymes can be increased throughout the cell cycle of the recombinant microorganism and / or during a production phase of the cell cycle.

[0073] The activity of one or more enzymes can be increased by replacing endogenous regulatory sequences of genes encoding enzymes with regulatory sequences that drive higher expression. Such regulatory sequences include, but are not limited to, constitutive promoters, inducible promoters (and optionally operator regions operably linked thereto), RBSs, spacers, and / or 3’ UTRs.

[0074] Alternatively or additionally, the activity of one or more enzymes can be increased by modifying polypeptide sequences (e.g., by modifying nucleotide sequences of coding regions) to have improved translation (e.g., codon-optimized coding regions), higher activity, higher stability, higher resistance to inhibitory protein-protein interactions, and / or lower resistance to excitatory protein-protein interactions, among other properties, relative to corresponding polypeptide sequences in parental microorganisms. Comparable effects can be achieved by replacing all or part of coding regions in parental microorganisms with heterologous coding regions. Heterologous coding regions can be operably linked to regulatory sequences that are not native to parental microorganisms and / or the coding regions. For example, heterologous coding regions can be operably linked to inducible promoters, e.g., gluconate-inducible promoters.

[0075] As yet another addition or alternative, the activity of one or more enzymes can be increased by increasing the copy number in recombinant microorganisms of nucleotide sequences comprising genes for the enzymes. This encompasses the addition into cells of heterologous nucleotide sequences comprising genes for enzymes not present in parental microorganisms, thereby increasing the copy number from zero to one (or a higher number).

[0076] Any nucleotide sequences comprising coding regions for the enzymes can be present in extrachromosomal nucleic acids and / or incorporated into the chromosome of the recombinant microorganism. Incorporations into the chromosome can be insertions into non-coding regions, insertions into and disruptions of coding regions, and / or replacements of coding regions and / or non-coding regions.

[0077] Turning to particular enzymatic activities, isoprenoid production can be increased by increasing the activity of Dxs, 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7) relative toa wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase Dxs activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous Dxs polypeptides, such as endogenous Dxs polypeptides comprising sequences having 100% identity to SEQ ID NO:14. This can also be effected by engineering recombinant microorganisms to express heterologous Dxs polypeptides, such as heterologous Dxs polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:15.

[0078] Isoprenoid production can also or additionally be increased by increasing the activity of Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase Dxr activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous Dxr polypeptides, such as endogenous Dxr polypeptides comprising sequences having 100% identity to SEQ ID NO:16. This can also be effected by engineering recombinant microorganisms to express heterologous Dxr polypeptides, such as heterologous Dxr polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:17.

[0079] Isoprenoid production can also or additionally be increased by increasing the activity of IspD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60) relative to a wild- type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspD activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspD polypeptides, such as endogenous IspD polypeptides comprising sequences having 100% identity to SEQ ID NO:18. This can also be effected by engineering recombinant microorganisms to express heterologous IspD polypeptides, such as heterologous IspD polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:19.

[0080] Additionally or alternatively, isoprenoid production can be increased by increasing the activity of IspE, 4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspE activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspE polypeptides, such as endogenous IspE polypeptides comprising sequence having 100% identity to SEQ ID NO:20. This can also be effected by engineeringrecombinant microorganisms to express heterologous IspE polypeptides, such as heterologous IspE polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:21.

[0081] Alternatively or additionally, isoprenoid production can be increased by increasing the activity of IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspF activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspF polypeptides, such as endogenous IspF polypeptides comprising sequences having 100% identity to SEQ ID NO:22. This can also be effected by engineering recombinant microorganisms to express heterologous IspF polypeptides, such as heterologous IspF polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:23.

[0082] In another approach, which can be used alone or in combination with others, Isoprenoid production can be increased by increasing the activity of IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspG activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspG polypeptides, such as endogenous IspG polypeptides comprising sequences having 100% identity to SEQ ID NO:24. This can also be effected by engineering recombinant microorganisms to express heterologous IspG polypeptides, such as heterologous IspG polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:25.

[0083] Additionally or alternatively, isoprenoid production can be increased by increasing the activity of IspH, 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspH activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspH polypeptides, such as endogenous IspH polypeptides comprising sequences having 100% identity to SEQ ID NO:26. This can also be effected by engineering recombinant microorganisms to express heterologous IspH polypeptides, such as heterologous IspH polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:27.

[0084] In another approach, which can be used alone or in combination with others, isoprenoid production can be increased by increasing the activity of Idi, isopentenyl-diphosphate Delta- isomerase (EC 5.3.3.2) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase Idi activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous Idi polypeptides, such as endogenous Idi polypeptides comprising sequences having 100% identity to SEQ ID NO:28. This can also be effected by engineering recombinant microorganisms to express heterologous Idi polypeptides, such as heterologous Idi polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:29.

[0085] Other heterologous enzymes of the DXP pathway that can be engineered into E. coli include those described in WO 2007 / 140339, WO 2008 / 128159, WO 2010 / 148150, WO 2012 / 088450, WO 2012 / 088462, WO 2012 / 135591, and WO 2018 / 140778, the contents of which are hereby incorporated herein by reference in their entireties.

[0086] In some embodiments, production of one or more isoprenoids, such as GPP; FPP; isoprene; limonene; β-myrcene; nerol; geraniol; linalool; β-ocimene;1,8-cineole; farnesene; or farnesol, is increased by modulating the activity of one or more enzymes downstream of Idi in FIG.2. For example, IspA, farnesyl diphosphate synthase (EC 2.5.1.10) activity can be increased relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase IspA activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous IspA polypeptides, such as endogenous IspA polypeptides comprising sequences having 100% identity to SEQ ID NO:30. This can also be effected by engineering recombinant microorganisms to express heterologous IspA polypeptides, such as heterologous IspA polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:31.

[0087] For another example, one or more activities of IspA, such as an activity to extend a C10isoprenoid to a C15isoprenoid, can be decreased relative to a wild-type microorganism or a non- wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, decrease IspA activity. Reducing this activity can increase the production of isoprenoids that are more readily synthesized from GPP than FPP. Reduction in this activity can be effected by engineering recombinant microorganisms to underexpress endogenous IspA polypeptides, such as endogenous IspA polypeptidescomprising sequences having 100% identity to SEQ ID NO:30. This can also be effected by engineering recombinant microorganisms to express heterologous IspA polypeptides which have reduced activity to extend C10isoprenoids relative to a wild-type microorganism or a non- wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, decrease IspA activity. Exemplary heterologous IspA polypeptides comprise sequences having at least 70% sequence identity to SEQ ID NO:31 and having mutations disruptive of binding of GPP and / or isopentenyl diphosphate, e.g., mutations at one or more of K46, R49, H78, R96, R97, K182, T183, Q220, and K237 of SEQ ID NO:31.

[0088] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing geranyl pyrophosphate (GPP) synthase (EC:2.5.1.10) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase GPP synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous ispA polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous GPP synthase polypeptides, such as heterologous gppS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:64.

[0089] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing farnesyl pyrophosphate (FPP) synthase (EC:2.5.1.10) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase FPP synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous ispA polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous ispA polypeptides, such as heterologous ispA polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:30 or SEQ ID NO:31.

[0090] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing isoprene synthase (EC 4.2.3.27 and EC 1.17.7.4) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase isoprene synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous ispH polypeptides. This can also be effected byengineering recombinant microorganisms to express heterologous isoprene synthase polypeptides, such as heterologous ispS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:65.

[0091] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing limonene synthase (EC 4.2.3.16 and EC 4.2.3.20) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase limonene synthase activity. This can be effected by engineering recombinant microorganisms to express heterologous limS polypeptides, such as heterologous limS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:66.

[0092] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing myrcene synthase activity (EC 4.2.3.15) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase myrcene synthase activity. This can be effected by engineering recombinant microorganisms to express heterologous beta-myrcene synthase, myrS, polypeptides, such as heterologous myrS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:67.

[0093] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing neryl-pyrophospate synthase, dimethylallylcistransferase (EC 2.5.1.28) activity relative to a wild-type microorganism or a non- wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase neryl-pyrophosphate synthase activity. This can be effected by engineering recombinant microorganisms to express heterologous neryl-pyrophospate synthase polypeptides, such as heterologous cpt1 polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:68.

[0094] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing neryl diphosphate phosphatase (EC 3.1.7.13 and EC 3.6.1) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase neryl diphosphate synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous non-specific phosphatases, for example, in E. coli recombinant microorganisms, nudJ (EC 3.6.1). This canalso be effected by engineering recombinant microorganisms to express heterologous neryl diphosphate diphosphatase polypeptides, such as heterologous nes polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:69.

[0095] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing nerol synthase (EC 3.1.7.13) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase nerol synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous nerol synthase polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous nerol synthase polypeptides.

[0096] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing geraniol synthase (gerS) activity (EC 3.1.7.11) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase gerS activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous non-specific geranyl-pyrophosphate pyrophosphatase polypeptides (e.g.,, in E. coli, nudJ or phoA, among others). This can also be effected by engineering recombinant microorganisms to express heterologous polypeptides, such as heterologous gerS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:70.

[0097] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing linalool synthases (linS) activity (EC 4.2.3.25 and EC 4.2.3.25) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase linS activity. This can be effected by engineering recombinant microorganisms to express heterologous linS polypeptides, such as heterologous linS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:71.

[0098] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing beta-ocimene synthase (ociS) (EC 4.2.3.106) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase osiS activity. This can be effected by engineering recombinant microorganisms toexpress heterologous polypeptides, such as heterologous ociS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:72.

[0099] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing 1,8-cineole synthase (cinS) activity (EC 4.2.3.108) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase cinS activity. This can be effected by engineering recombinant microorganisms to express heterologous cinS polypeptides, such as heterologous cinS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:73.

[0100] In another example, which can be used alone or in combination with others, isoprenoid production can be increased by increasing farnesene synthase (fnsS) (EC 4.2.3.47 and EC 4.2.3.46) activity relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase fnsS activity. This can be effected by engineering recombinant microorganisms to express heterologous fnsS polypeptides, such as heterologous fnsS polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:74.

[0101] For another example, isoprenoid production can be increased by increasing the activity of CrtEIB, CrtE, geranylgeranyl diphosphate synthase (EC 2.5.1.29) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase CrtEIB, CrtE, geranylgeranyl diphosphate synthase activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous CrtE polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous CrtE polypeptides, such as heterologous CrtE polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:32.

[0102] Additionally or alternatively, isoprenoid production can be increased by increasing the activity of CrtI, phytoene desaturase (EC 1.3.99.31) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase Crtl activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous CrtI polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous CrtIpolypeptides, such as heterologous CrtI polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:33.

[0103] In another approach, which can be used alone or in combination with others, isoprenoid production can be increased by increasing the activity of CrtB, 15-cis-phytoene synthase (EC 2.5.1.32) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase CrtB activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous CrtB polypeptides. This can also be effected by engineering recombinant microorganisms to express heterologous CrtB polypeptides, such as heterologous CrtB polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:34.

[0104] The recombinant microorganisms can also be engineered to increase isoprenoid production in part by being engineered to produce DXP from ribulose-5-P or xylulose-5-P. Such engineering can comprise increasing RibB, 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12) activity compared to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase RibB activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous RibB polypeptides, such as endogenous RibB polypeptides comprising sequences having 100% identity to SEQ ID NO:35. This can also be effected by engineering recombinant microorganisms to express heterologous RibB polypeptide, such as heterologous RibB polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:36.

[0105] Alternatively or additionally, engineering production of DXP from ribulose-5-P or xylulose-5-P can comprise increasing the activity of YajO, 1-deoxyxylulose-5-phosphate synthase (EC 1.1.-.-) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase YajO activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous YajO polypeptides, such as endogenous YajO polypeptides comprising sequences having 100% identity to SEQ ID NO:37. This can also be effected by engineering recombinant microorganisms to express heterologous YajO polypeptides, such as heterologous YajO polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:38.

[0106] As another addition or alternative, engineering production of DXP from ribulose-5-P or xylulose-5-P can comprise increasing the activity of XylB, xylulose kinase (EC 2.7.1.17) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, increase XylB activity. This can be effected by engineering recombinant microorganisms to overexpress endogenous XylB polypeptides, such as endogenous XylB polypeptides comprising sequences having 100% identity to SEQ ID NO:39. This can also be effected by engineering recombinant microorganisms to express heterologous XylB polypeptides, such as heterologous XylB polypeptides comprising sequences having at least 70% sequence identity to SEQ ID NO:40.

[0107] In some embodiments, isoprenoid production is increased by increasing the activity of one or more enzymes having activity in the conversion of glucose to gluconate. This can be combined with increasing isoprenoid production by increasing the activity of one or more enzymes having activity in the conversion of gluconate to KDG. For example, recombinant microorganisms can comprise nucleic acids comprising nucleotide sequences encoding gluconate dehydratases (EC 4.2.1.39), which catalyze the conversion of gluconate to KDG. Gluconate dehydratases that can be used include those comprising amino acid sequences having at least 70% sequence identity, such as at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92.5% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO:13.

[0108] In some embodiments, isoprenoid production is increased by increasing the activity of one or more enzymes having activity in the conversion of KDG to KDGP.

[0109] In some embodiments, isoprenoid production is increased by increasing the activity of one or more enzymes having activity in the conversion of KDGP to GAP and pyruvate.

[0110] As an additional approach to further increase isoprenoid production, recombinant microorganisms can be engineered to increase the activity of cytidylate kinases (EC 2.7.4.25) and / or nucleoside diphosphate kinases (EC 2.7.4.6) relative to parental microorganisms. These enzymatic activities in E. coli are provided by Cmk and Ndk, respectively. As shown in FIG.1, by overexpressing Cmk and / or Ndk, the yield and / or rate of regeneration of CTP from CMP may be increased.

[0111] In some embodiments, increasing the activity of cytidylate kinases and / or nucleoside diphosphate kinases comprises increasing the activity of an endogenous cytidylate kinase promoter or an endogenous nucleoside diphosphate kinase promoter.

[0112] In some embodiments, increasing the activity of cytidylate kinases and / or nucleoside diphosphate kinases comprises introducing nucleotide sequences encoding cytidylate kinases and / or nucleoside diphosphate kinases, optionally wherein the nucleotide sequences are codon- optimized for the recombinant microorganism.

[0113] In some embodiments, increasing the activity of cytidylate kinases and / or nucleoside diphosphate kinases comprises increasing the copy number of nucleic acids encoding the cytidylate kinases and / or the nucleoside diphosphate kinases. Increasing the copy number can comprise inserting heterologous nucleic acids into genomes of recombinant microorganisms.

[0114] Exemplary nucleotide sequences encoding cytidylate kinases include sequences having at least 80% sequence identity, such as at least 85% sequence identity, at least 90% sequence identity, at least 92.5% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO:9.

[0115] Exemplary amino acid sequences of cytidylate kinases include sequences having at least 80% sequence identity, such as at least 85% sequence identity, at least 90% sequence identity, at least 92.5% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO:10.

[0116] Exemplary nucleotide sequences encoding nucleoside diphosphate kinases include sequences having at least 80% sequence identity, such as at least 85% sequence identity, at least 90% sequence identity, at least 92.5% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO:11.

[0117] Exemplary amino acid sequences of nucleoside diphosphate kinases include sequences having at least 80% sequence identity, such as at least 85% sequence identity, at least 90% sequence identity, at least 92.5% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity, to SEQ ID NO:12.6.2.4. Measurement of Increased CTP Flux and / or Production

[0118] Increased CTP flux and / or production in an engineered microorganism relative to a parental microorganism or a wild-type microorganism resulting from the engineering described herein (e.g., reducing activity of one or more of, or any combination of, nucleoside monophosphate phosphohydrolases, ribonucleotide monophosphatases, nucleoside triphosphate pyrophosphohydrolases, and / or 5’-ribonucleotide phosphohydrolases) can be detected by, for example, comparing production by the engineered microorganism of a product produced by a pathway that utilizes (e.g., depends on or consumes) CTP to production of the product by a parental microorganism that lacks the engineering. For example, as described in Example 2 (Section 7.2), a parental microorganism capable of producing lycopene via the DXP pathway was engineered to delete various genes and combinations of genes. Comparing lycopene production by the engineered microorganisms to the parental microorganisms that did not have the gene deletions showed that engineered microorganisms had increased CTP flux and / or production. As another example, as described in Example 3 (Section 7.3), a parental microorganism that produced MEcPP as part of the DXP pathway was engineered to delete various genes and combinations of genes. Comparing MEcPP production by the engineered microorganisms to the parental microorganisms that did not have the gene deletions showed that engineered microorganisms had increased CTP flux and / or production.

[0119] In some embodiments, an engineered microorganism of the present disclosure has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% greater production of a product produced by a pathway that utilizes CTP (e.g., lycopene or MEcPP) as compared to production of the product by a parental microorganism. In some embodiments, the engineered microorganism has at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% greater production of a product produced by a pathway that utilizes CTP as compared to production of the product by a parental microorganism. In some embodiments, the engineered microorganism has 5% to 20%, 10% to 50%, 10% to 500%, 10% to 1000%, 20% to 100%, 20% to 200%, 20% to 500%, 20% to 1000%, 50% to 100%, 50% to 200%, 50% to 500%, 50% to 1000%, 100% to 200%, 100% to 500%, 100% to 1000%, 200% to 500%, 200% to 700%, 200% to 1000%, or 500% to 1000% greater production of a product produced by a pathway that utilizes CTP as compared to production of the product by a parental microorganism. In some embodiments, the increased production is measured according to methods described in the Examples (Section 7). In some embodiments, the product produced by a pathway that utilizes CTP is lycopene orMEcPP. In some embodiments, the pathway that utilizes CTP is the DXP pathway. In some embodiments, the pathway that utilizes CTP is an isoprenoid production pathway. In some embodiments, the increased production is measured according to the method of Section 7.2. In some embodiments, the increased production is measured according to the method of Section 7.3. 6.3. Engineering Recombinant Microorganisms 6.3.1. Parental Microorganisms

[0120] Any prokaryotes can be parental microorganisms engineered to yield recombinant microorganisms having reduced activity of endogenous nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, and / or endogenous 5’- ribonucleotide phosphohydrolases, and increased isoprenoid production. Optionally, the parental microorganisms can also be engineered to have increased activity of orotate phosphoribosyltransferases.

[0121] In some embodiments, the parental microorganism is E. coli. In particular aspects, the E. coli is E. coli strain K12 or a strain derived therefrom, such as E. coli K12 substr. MG1655.

[0122] Other E. coli strains from which recombinant microorganisms of the present disclosure can be engineered include, but are not limited to, E. coli K12 W3110, E. coli K12 DH5^, and non-K12 strains BL21 and W.

[0123] Recombinant microorganisms of the present disclosure can be engineered from bacteria other than E. coli. Examples of such bacteria include, but are not limited to, Rhodobacter capsulatus, Bacillus subtilis, Pantoea ananatis, Tatumella citrea, Pseudomonas fluorescens, and Pseudomonas putida. 6.3.2. Engineering Methods

[0124] Parental microorganisms can be engineered using techniques known in the art. For example, reductions or increases of activities of one or more enzymes as described herein can be engineered into parental microorganism via techniques known in the art.

[0125] In some embodiments, activities are reduced or increased by introducing into parental microorganisms nucleic acids comprising modified coding regions and / or modified regulatory sequences into cells of the parental microorganism.

[0126] In some embodiments, nucleic acids are introduced into microorganisms by any appropriate transformation technique. Nucleic acids can be extrachromosomal, on a vector(such as a plasmid or a phage), such as a low copy number vector, an intermediate copy number vector, or a high copy number vector. Nucleic acids can be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, nucleic acids can be integrated in one or more copies into the genome of the cell. Integration into the cell’s genome can occur at random by non-homologous recombination, or at selected locations by homologous recombination (e.g., to replace an endogenous coding region and / or regulatory sequence with a modified one, a replacement therefor, or a partial or complete deletion thereof), as is well known in the art.

[0127] 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 sequence to which it is not operably linked in a parental microorganism (which may change promoter strength, change whether a promoter is constitutive or inducible, or change which inducer molecule induces transcription of a coding region from an inducible promoter), to reduce or increase enzymatic activity of polypeptides encoded by those genes.

[0128] RNAi techniques can be used to reduce activity of enzymes in prokaryotes by regulating gene expression (Waters et al., 2009, Cell 136(4):615-628) or interfering with translation of RNAs encoding the enzymes. Nucleic acids can be introduced into or engineered in recombinant microorganisms to produce regulatory RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), antisense RNAs (asRNAs), and / or single guide RNAs (sgRNAs) for CRISPR interference.

[0129] The engineering methods can be applied in a number of ways to reduce activity of an endogenous nucleoside monophosphate phosphohydrolase (EC 3.1.3.5 or EC 3.1.3.6) and / or an endogenous ribonucleotide monophosphatase (EC 3.1.3.5) relative to a wild-type microorganism or a non-wild type parental microorganism, e.g., a parental microorganism that has mutations that do not and / or is engineered in a manner that does not, reduce activity of an endogenous nucleoside monophosphate phosphohydrolase and / or an endogenous ribonucleotide monophosphatase. For example, all or a portion of umpG coding sequences can be mutated, optionally wherein the mutation can be a deletion; all or a portion of umpG regulatory sequences can be mutated, optionally wherein the mutation can be a deletion; heterologous sequences can be introduced into umpG loci; interfering RNA (RNAi) systems thatreduce umpG activity can be engineered into recombinant microorganisms; or any two, any three, or all four thereof, among other techniques.

[0130] For another example, all or a portion of umpH coding sequences can be mutated, optionally wherein the mutation can be a deletion; all or a portion of umpH regulatory sequences can be mutated, optionally wherein the mutation can be a deletion; heterologous sequences can be introduced into umpH loci; interfering RNA (RNAi) systems that reduce umpH activity can be engineered into recombinant microorganisms; or any two, any three, or all four thereof, among other techniques. 6.4. Methods of Use

[0131] The present disclosure also relates to methods for producing isoprenoids, and methods for increasing recycling of cytidine monophosphate (CMP) to cytidine monophosphate (CTP), e.g., by culturing recombinant microorganisms as described herein. 6.4.1. Culture media

[0132] Generally, methods disclosed herein comprise growing cells of a recombinant microorganism (i.e., cells of a prokaryotic species into which nucleic acids disclosed herein have been introduced) in a growth medium suitable for growth to a desired cell concentration, and culturing the cells in a production medium suitable for production of a isoprenoids and / or recycling of CMP to CTP. Culturing can be in a batch mode or a continuous mode.

[0133] 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 CaCl2, 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).

[0134] In some embodiments, a culture medium comprises at least 0.1 w / v% sucrose, at least 0.2 w / v% sucrose, at least 0.3 w / v% sucrose, at least 0.4 w / v% sucrose, at least 0.5 w / v% sucrose, at least 0.6 w / v% sucrose, at least 0.7 w / v% sucrose, at least 0.8 w / v% sucrose, at least 0.9 w / v% sucrose, or at least 1 w / v% sucrose. A culture medium typically comprises less than 5 w / v% sucrose, more typically less than 2 w / v% sucrose (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% sucrose; from 0.1 w / v% to 2 w / v% sucrose; from 0.1 w / v% to 1 w / v% sucrose; or from 1 w / v% to 2 w / v% sucrose, among other possible ranges).

[0135] 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).

[0136] In some embodiments, culture media comprise at least 0.1 w / v% gluconate, at least 0.2 w / v% gluconate, at least 0.3 w / v% gluconate, at least 0.4 w / v% gluconate, at least 0.5 w / v% gluconate, at least 0.6 w / v% gluconate, at least 0.7 w / v% gluconate, at least 0.8 w / v% gluconate, at least 0.9 w / v% gluconate, or at least 1 w / v% gluconate. A culture medium typically comprises less than 5 w / v% gluconate, more typically less than 2 w / v% gluconate (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% gluconate; from 0.1 w / v% to 2 w / v% gluconate; from 0.1 w / v% to 1 w / v% gluconate; or from 1 w / v% to 2 w / v% gluconate, among other possible ranges). Gluconate can both provide a carbon source for recombinant microorganisms and induce expression of gluconate-inducible promoters.

[0137] In some embodiments, culture media comprise at least 0.1 w / v% cellulose-derived sugars, at least 0.2 w / v% cellulose-derived sugars, at least 0.3 w / v% cellulose-derived sugars, at least 0.4 w / v% cellulose-derived sugars, at least 0.5 w / v% cellulose-derived sugars, at least 0.6 w / v% cellulose-derived sugars, at least 0.7 w / v% cellulose-derived sugars, at least 0.8 w / v% cellulose-derived sugars, at least 0.9 w / v% cellulose-derived sugars, or at least 1 w / v% cellulose-derived sugars. A culture medium typically comprises less than 5 w / v% cellulose- derived sugars, more typically less than 2 w / v% cellulose-derived sugars (e.g., in some embodiments, culture media comprise from 0.1 w / v% to 5 w / v% cellulose-derived sugars; from 0.1 w / v% to 2 w / v% cellulose-derived sugars; from 0.1 w / v% to 1 w / v% cellulose-derived sugars; or from 1 w / v% to 2 w / v% cellulose-derived sugars, among other possible ranges). The concentrations of cellulose-derived sugars listed here are the sum of the concentrations of all cellulose-derived sugars (which may be one or more cellulose-derived sugars) in the media.

[0138] In some embodiments, a production medium comprises sucrose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises gluconate. In some embodiments, a production medium comprises one or more cellulose-derived sugars. In some embodiments, a production medium comprises any two, and three, or all four of sucrose, glucose, gluconate, or cellulose-derived sugars. The inclusion ofgluconate or precursors thereof in production media can induce expression of sequences of interest in recombinant microorganisms comprising nucleic acids comprising coding regions operably linked to gluconate-inducible promoters. Alternatively or additionally, induction of expression from gluconate-inducible promoters can occur if the cells produce gluconate from other sugars during culturing.

[0139] In some embodiments, a production medium comprises an inducer other than gluconate, i.e., a molecule other than gluconate which induces translation of a coding region regulated by an inducible promoter.

[0140] 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).

[0141] In some embodiments of some methods described herein, it may be desirable to allow growth of a recombinant microorganism without expression of one or more genes until a desired biomass of the recombinant microorganism has been reached. For example, such growth can be encouraged or effected by use of a growth medium comprising glycerol, such as at least 0.1 w / v% glycerol, at least 0.2 w / v% glycerol, at least 0.3 w / v% glycerol, at least 0.4 w / v% glycerol, at least 0.5 w / v% glycerol, at least 0.6 w / v% glycerol, at least 0.7 w / v% glycerol, at least 0.8 w / v% glycerol, at least 0.9 w / v% glycerol, or at least 1 w / v% glycerol. A growth medium typically comprises less than 5 w / v% glycerol, more typically less than 2 w / v% glycerol (e.g., in some embodiments, growth media comprise from 0.1 w / v% to 5 w / v% glycerol; from 0.1 w / v% to 2 w / v% glycerol; from 0.1 w / v% to 1 w / v% glycerol; or from 1 w / v% to 2 w / v% glycerol, among other possible ranges).

[0142] 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.

[0143] In some embodiments, a growth medium lacks added glucose and / or sucrose, i.e., one or both of these sugars is not intentionally included in a growth medium. In particular embodiments, a growth medium comprises no more than 0.1 w / v% each of glucose and / or sucrose.

[0144] The selection of particular concentrations of sucrose, glucose and / or glycerol to include in a production medium and / or a growth medium can be made by the person of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

[0145] For fed-batch and / or continuous mode culturing, the ranges of sucrose, glucose, gluconate, glycerol, or combinations thereof given above can be initially provided to the medium. The consumption of the carbon source(s) during culturing can be repeatedly or continuously monitored and additional carbon source(s) can be provided as needed to sustain a desired respiratory coefficient, growth rate, rate of expression of sequences of interest, and / or a rate of production of desired compound(s). The feed rate may be adjusted to avoid accumulation of carbon source(s), which may maximize output of desired compound(s) and minimize waste of carbon source(s). The person of ordinary skill in the art having the benefit of the present disclosure can select the medium composition and the amount of carbon source added thereto during the process to enable the expression of sequences of interest to a desired level and / or production of desired product(s) to a desired concentration, such as at least 20 g / L, at least 50 g / L, or at least 100 g / L. 6.4.2. Culture conditions

[0146] 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 astechniques 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.

[0147] 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.

[0148] Fermentation is typically carried out at a pH in the range of 4 to 8, in the range of 5 to 7, or the range of 5.5 to 6.5. For example, fermentation can be carried out for a period of time within the range of from 8 to 240 hours, from 12 hours to 168 hours, from 16 hours to 144 hours, from 20 hours to 120 hours, from 24 hours to 72 hours, or from 36 to 48 hours. 6.4.3. Methods for Producing Isoprenoids

[0149] The present disclosure also relates to methods for producing isoprenoids. In some embodiments, the methods comprising culturing recombinant microorganisms as described in Section 6.2 under conditions in which an isoprenoid is produced.

[0150] The conditions can include culturing recombinant microorganisms in appropriate media, e.g., media comprising glucose.

[0151] Cells can be grown to desired cell concentrations in appropriate media. After growth to desired cell concentrations, cells in which one or more genes encoding enzymes for which increased activity are operably linked to gluconate-inducible promoters can be cultured in media comprising gluconate and / or in which the cells produce gluconate during a production phase.

[0152] Isoprenoids produced in the methods of the disclosure can be recovered from media. This can comprise recovery of isoprenoids secreted by recombinant microorganisms into the media; lysis of cells in the media to release isoprenoids, followed by recovery; separation of cells from media, followed by lysis and isolation of isoprenoids; or a combination thereof. 6.4.4. Methods for Increasing Recycling of CMP to CTP

[0153] The present disclosure also relates to methods to improve the ability of a cell to recycle CMP to CTP, while decreasing its dependency for the de-novo synthesis of CTP. In some embodiments, the methods comprising culturing recombinant microorganisms as described in Section 6.2 under conditions in which CMP is recycled to CTP.

[0154] The conditions can include culturing recombinant microorganisms in appropriate media, e.g., media comprising glucose.

[0155] Cells can be grown to desired cell concentrations in appropriate media. After growth to desired cell concentrations, cells in which one or more genes encoding enzymes for which increased activity are operably linked to gluconate-inducible promoters can be cultured in media comprising gluconate and / or in which the cells produce gluconate during a production phase.

[0156] The increased recycling of CMP to CTP can increase the yield and / or production rate of isoprenoids produced via the DXP pathway, among other beneficial effects in other processes in which CTP is consumed. 6.5. Specific Embodiments

[0157] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below. 1. A recombinant microorganism that: (a) has reduced activity, relative to a wild-type microorganism, of: (i) an endogenous nucleoside monophosphate phosphohydrolase (EC 3.1.3.5 or EC 3.1.3.6); (ii) an endogenous ribonucleotide monophosphatase (EC 3.1.3.5); (iii) an endogenous nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.56), (iv) an endogenous 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5),; or (v) any combination of two, three or all four of (i), (ii), (iii), and (iv). and (b) is engineered to increase activity of a pathway that utilizes cytidine triphosphate (CTP) as compared to a wild-type organism. 2. The recombinant microorganism of embodiment 1, which is an E. coli.3. The recombinant microorganism of embodiment 1 or embodiment 2, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous nucleoside monophosphate phosphohydrolase. 4. The recombinant microorganism of any one of embodiments 1 to 3, wherein the endogenous nucleoside monophosphate phosphohydrolase is encoded by eand optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:41. 5. The recombinant microorganism of embodiment 4, wherein the umpG gene in the recombinant microorganism has reduced activity as compared to a wild-type umpG gene. 6. The recombinant microorganism of embodiment 5, wherein all or a portion of the umpG coding sequence is mutated, optionally wherein the mutation is a deletion. 7. The recombinant microorganism of embodiment 5 or embodiment 6, wherein all or a portion of the umpG regulatory sequence is mutated, optionally wherein the mutation is a deletion. 8. The recombinant microorganism of any one of embodiments 5 to 7, wherein a heterologous sequence is introduced into the umpG locus. 9. The recombinant microorganism of any one of embodiments 5 to 8, which comprises an interfering RNA (RNAi) that reduces umpG activity. 10. The recombinant microorganism of any one of embodiments 1 to 9, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous ribonucleotide monophosphatase. 11. The recombinant microorganism of any one of embodiments 1 to 10, wherein the endogenous ribonucleotide monophosphatase is encoded by umpH and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:42. 12. The recombinant microorganism of embodiment 11, wherein the umpH gene in the recombinant microorganism has reduced activity as compared to a wild-type umpH gene. 13. The recombinant microorganism of embodiment 12, wherein all or a portion of the umpH coding sequence is mutated, optionally wherein the mutation is a deletion.14. The recombinant microorganism of embodiment 12 or embodiment 13, wherein all or a portion of the umpH regulatory sequence is mutated, optionally wherein the mutation is a deletion. 15. The recombinant microorganism of any one of embodiments 12 to 14, wherein a heterologous sequence is introduced into the umpH locus. 16. The recombinant microorganism of any one of embodiments 12 to 15, which comprises an interfering RNA (RNAi) that reduces umpH activity. 17. The recombinant microorganism of any one of embodiments 1 to 16, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous nucleoside triphosphate pyrophosphohydrolase. 18. The recombinant microorganism of any one of embodiments 1 to 17, wherein the endogenous nucleoside triphosphate pyrophosphohydrolase is encoded by nudG and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:43. 19. The recombinant microorganism of embodiment 18, wherein the nudG gene in the recombinant microorganism has reduced activity as compared to a wild-type nudG gene. 20. The recombinant microorganism of embodiment 19, wherein all or a portion of the nudG coding sequence is mutated, optionally wherein the mutation is a deletion. 21. The recombinant microorganism of embodiment 19 or embodiment 20, wherein all or a portion of the nudG regulatory sequence is mutated, optionally wherein the mutation is a deletion. 22. The recombinant microorganism of any one of embodiments 19 to 21, wherein a heterologous sequence is introduced into the nudG locus. 23. The recombinant microorganism of any one of embodiments 19 to 22, which comprises an interfering RNA (RNAi) that reduces nudG activity. 24. The recombinant microorganism of any one of embodiments 1 to 23, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous 5'-ribonucleotide phosphohydrolase.25. The recombinant microorganism of any one of embodiments 1 to 24, wherein the endogenous 5'-ribonucleotide phosphohydrolase is encoded by ushA and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:44. 26. The recombinant microorganism of embodiment 25, wherein the ushA gene in the recombinant microorganism has reduced activity as compared to a wild-type ushA gene. 27. The recombinant microorganism of embodiment 26, wherein all or a portion of the ushA coding sequence is mutated, optionally wherein the mutation is a deletion. 28. The recombinant microorganism of embodiment 26 or embodiment 27, wherein all or a portion of the ushA regulatory sequence is mutated, optionally wherein the mutation is a deletion. 29. The recombinant microorganism of any one of embodiments 26 to 28, wherein a heterologous sequence is introduced into the ushA locus. 30. The recombinant microorganism of any one of embodiments 26 to 29, which comprises an interfering RNA (RNAi) that reduces ushA activity. 31. The recombinant microorganism of any one of embodiments 1 to 30, wherein the activity of the pathway that utilizes CTP is increased by at least 10% as compared to a parental microorganism that does not have the reduced activity. 32. The recombinant microorganism of any one of embodiments 1 to 31, wherein the activity of the pathway that utilizes CTP is increased by at least 50% as compared to a parental microorganism that does not have the reduced activity. 33. The recombinant microorganism of any one of embodiments 1 to 32, wherein the activity of the pathway that utilizes CTP is increased by at most 500% as compared to a parental microorganism that does not have the reduced activity. 34. The recombinant microorganism of any one of embodiments 31 to 33, wherein the activity of the pathway that utilizes CTP is measured as described in the Examples below (Section 7). 35. The recombinant microorganism of any one of embodiments 31 to 34, wherein the activity of the pathway that utilizes CTP is measured as described in Section 7.3.36. The recombinant microorganism of any one of embodiments 1 to 35, which produces at least 10% greater amounts of a product of the pathway that utilizes CTP as compared to a wild-type microorganism or parental microorganism that does not have the reduced activity. 37. The recombinant microorganism of any one of embodiments 1 to 36, which produces at least 50% greater amounts of a product of the pathway that utilizes CTP as compared to a wild-type microorganism or parental microorganism that does not have the reduced activity. 38. The recombinant microorganism of any one of embodiments 1 to 37, which produces at most 500% greater amounts of a product of the pathway that utilizes CTP as compared to a wild-type microorganism or parental microorganism that does not have the reduced activity. 39. The recombinant microorganism of any one of embodiments 36 to 38, wherein the pathway that utilizes CTP is a DXP pathway. 40. The recombinant microorganism of any one of embodiments 36 to 39, wherein the product of the pathway that utilizes CTP is MEcPP. 41. The recombinant microorganism of any one of embodiments 1 to 40, wherein the recombinant microorganism is engineered to increase flux or production of an isoprenoid pathway component or precursor as compared to a wild-type microorganism or parental microorganism. 42. The recombinant microorganism of embodiment 41, wherein the isoprenoid pathway component or precursor is any of components (1) through (19) of FIG.2. 43. The recombinant microorganism of embodiment 41 or embodiment 42, which is engineered to recombinantly express one or more DXP pathway enzymes. 44. The recombinant microorganism of embodiment 43, which has increased Dxs, 1- deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7) activity as compared to a wild-type microorganism or parental microorganism.45. The recombinant microorganism of embodiment 44, which is engineered to overexpress an endogenous Dxs polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:14. 46. The recombinant microorganism of embodiment 44, which is engineered to express a heterologous Dxs polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:15. 47. The recombinant microorganism of embodiment 43, which has increased Dxr, 1- deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267) activity as compared to a wild- type microorganism or parental microorganism. 48. The recombinant microorganism of embodiment 47, which is engineered to overexpress an endogenous Dxr polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:16. 49. The recombinant microorganism of embodiment 47, which is engineered to express a heterologous Dxr polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:17. 50. The recombinant microorganism of embodiment 43, which has increased IspD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60) activity as compared to a wild-type microorganism or parental microorganism. 51. The recombinant microorganism of embodiment 50, which is engineered to overexpress an endogenous IspD polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:18. 52. The recombinant microorganism of embodiment 50, which is engineered to express a heterologous IspD polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:19. 53. The recombinant microorganism of embodiment 43, which has increased IspE, 4- (cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148) activity as compared to a wild-type microorganism or parental microorganism.54. The recombinant microorganism of embodiment 53, which is engineered to overexpress an endogenous IspE polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:20. 55. The recombinant microorganism of embodiment 53, which is engineered to express a heterologous IspE polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:21. 56. The recombinant microorganism of embodiment 43, which has increased IspF, 2- C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12) activity as compared to a wild-type microorganism or parental microorganism. 57. The recombinant microorganism of embodiment 56, which is engineered to overexpress an endogenous IspF polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:22. 58. The recombinant microorganism of embodiment 56, which is engineered to express a heterologous IspF polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:23. 59. The recombinant microorganism of embodiment 43, which has increased IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1) activity as compared to a wild-type microorganism or parental microorganism. 60. The recombinant microorganism of embodiment 59, which is engineered to overexpress an endogenous IspG polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:24. 61. The recombinant microorganism of embodiment 59, which is engineered to express a heterologous IspG polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:25. 62. The recombinant microorganism of embodiment 43, which has increased IspH, 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2) activity as compared to a wild-type microorganism or parental microorganism.63. The recombinant microorganism of embodiment 62, which is engineered to overexpress an endogenous IspH polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:26. 64. The recombinant microorganism of embodiment 62, which is engineered to express a heterologous IspH polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:27. 65. The recombinant microorganism of embodiment 43, which has increased Idi, isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2) activity as compared to a wild-type microorganism or parental microorganism. 66. The recombinant microorganism of embodiment 65, which is engineered to overexpress an endogenous Idi polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:28. 67. The recombinant microorganism of embodiment 65, which is engineered to express a heterologous Idi polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:29. 68. The recombinant microorganism of embodiment 43, which has increased IspA, farnesyl diphosphate synthase (EC 2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism. 69. The recombinant microorganism of embodiment 68, which is engineered to overexpress an endogenous IspA polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:30. 70. The recombinant microorganism of embodiment 68, which is engineered to express a heterologous IspA polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:31. 71. The recombinant microorganism of embodiment 43, which has decreased IspA, farnesyl diphosphate synthase (EC 2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism.72. The recombinant microorganism of embodiment 71, which is engineered to underexpress an endogenous IspA polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:30. 73. The recombinant microorganism of embodiment 71, which is engineered to express a heterologous IspA polypeptide having one or more mutations disruptive of binding of GPP and / or isopentenyl diphosphate, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:31. 74. The recombinant microorganism of any one of embodiments 1 to 73, which has increased gppS, geranyl pyrophosphate (GPP) synthase (EC:2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism. 75. The recombinant microorganism of embodiment 74, which is engineered to overexpress an endogenous IspA polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:30. 76. The recombinant microorganism of embodiment 74, which is engineered to express a heterologous gppS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:64. 77. The recombinant microorganism of any one of embodiments 1 to 76, which has increased farnesyl pyrophosphate (FPP) synthase (EC:2.5.1.10) activity as compared to a wild- type microorganism or parental microorganism. 78. The recombinant microorganism of embodiment 77, which is engineered to overexpress an endogenous IspA polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:30. 79. The recombinant microorganism of embodiment 77, which is engineered to express a heterologous ispA polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:30 or SEQ ID NO:31. 80. The recombinant microorganism of any one of embodiments 1 to 79, which has increased isoprene synthase (EC 4.2.3.27 and EC 1.17.7.4) activity as compared to a wild-type microorganism or parental microorganism.81. The recombinant microorganism of embodiment 80, which is engineered to overexpress an endogenous ispH polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:26. 82. The recombinant microorganism of embodiment 80, which is engineered to express a heterologous ispS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:65. 83. The recombinant microorganism of any one of embodiments 1 to 82, which has increased limonene synthase (EC 4.2.3.16 and EC 4.2.3.20) activity as compared to a wild-type microorganism or parental microorganism. 84. The recombinant microorganism of embodiment 83, which is engineered to express a heterologous limS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:66. 85. The recombinant microorganism of any one of embodiments 1 to 84, which has increased myrcene synthase (EC 4.2.3.15) activity as compared to a wild-type microorganism or parental microorganism. 86. The recombinant microorganism of embodiment 85, which is engineered to express a heterologous myrS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:67. 87. The recombinant microorganism of any one of embodiments 1 to 86, which has increased neryl-pyrophospate synthase, dimethylallylcistransferase (EC 2.5.1.28) activity as compared to a wild-type microorganism or parental microorganism. 88. The recombinant microorganism of embodiment 87, which is engineered to express a heterologous cpt1 polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:68. 89. The recombinant microorganism of any one of embodiments 1 to 88, which has increased neryl diphosphate phosphatase (EC 3.1.7.13 and EC 3.6.1) activity as compared to a wild-type microorganism or parental microorganism.90. The recombinant microorganism of embodiment 89, which is engineered to overexpress an endogenous non-specific phosphatase. 91. The recombinant microorganism of embodiment 89, which is engineered to express a heterologous neryl diphosphate diphosphatase polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:69. 92. The recombinant microorganism of any one of embodiments 1 to 91, which has increased nerol synthase (EC 3.1.7.13) activity as compared to a wild-type microorganism or parental microorganism. 93. The recombinant microorganism of embodiment 92, which is engineered to overexpress an endogenous nerol synthase polypeptide. 94. The recombinant microorganism of embodiment 92, which is engineered to express a heterologous nerol synthase polypeptide. 95. The recombinant microorganism of any one of embodiments 1 to 94, which has increased geraniol synthase (EC 3.1.7.11) activity as compared to a wild-type microorganism or parental microorganism. 96. The recombinant microorganism of embodiment 95, which is engineered to overexpress an endogenous non-specific geranyl-pyrophosphate pyrophosphatase polypeptide. 97. The recombinant microorganism of embodiment 95, which is engineered to express a heterologous gerS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:70. 98. The recombinant microorganism of any one of embodiments 1 to 97, which has increased linalool synthase (EC 4.2.3.25 and EC 4.2.3.25) activity as compared to a wild-type microorganism or parental microorganism. 99. The recombinant microorganism of embodiment 98, which is engineered to express a heterologous linS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:71.100. The recombinant microorganism of any one of embodiments 1 to 99, which has increased beta-ocimene synthase (EC 4.2.3.106) activity as compared to a wild-type microorganism or parental microorganism. 101. The recombinant microorganism of embodiment 100, which is engineered to express a heterologous ociS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:72. 102. The recombinant microorganism of any one of embodiments 1 to 101, which has increased 1,8-cineole synthase activity (EC 4.2.3.108) activity as compared to a wild-type microorganism or parental microorganism. 103. The recombinant microorganism of embodiment 102, which is engineered to express a heterologous cinS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:73. 104. The recombinant microorganism of any one of embodiments 1 to 103, which has increased farnesene synthase activity (EC 4.2.3.47) activity as compared to a wild-type microorganism or parental microorganism. 105. The recombinant microorganism of embodiment 104, which is engineered to express a heterologous fnsS polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:74. 106. The recombinant microorganism of any one of embodiments 1 to 105, wherein the recombinant microorganism is engineered to increase isoprenoid production in part by being engineered to produce 1-deoxyxylulose-5-phosphate (DXP) from 2-keto-3-deoxygluconate (KDG). 107. The recombinant microorganism of embodiment 106, wherein the recombinant microorganism comprises a nucleic acid comprising a nucleotide sequence encoding a gluconate dehydratase (EC 4.2.1.39). 108. The recombinant microorganism of embodiment 107, wherein the gluconate dehydratase comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:13.109. The recombinant microorganism of embodiment 107 or embodiment 108, wherein the gluconate dehydratase comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:13. 110. The recombinant microorganism of any one of embodiments 107 to 109, wherein the gluconate dehydratase comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:13. 111. The recombinant microorganism of any one of embodiments 107 to 110, wherein the gluconate dehydratase comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:13. 112. The recombinant microorganism of any one of embodiments 107 to 111, wherein the gluconate dehydratase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:13. 113. The recombinant microorganism of any one of embodiments 107 to 112, wherein the gluconate dehydratase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:13. 114. The recombinant microorganism of any one of embodiments 107 to 113, wherein the gluconate dehydratase comprises an amino acid sequence having 100% sequence identity to SEQ ID NO:13. 115. The recombinant microorganism of any one of embodiments 31 to 114, wherein the recombinant microorganism is engineered to increase isoprenoid production in part by being engineered to produce DXP from ribulose-5-P or xylulose-5-P. 116. The recombinant microorganism of embodiment 115, which has increased RibB, 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12) activity compared to a wild-type microorganism or parental microorganism. 117. The recombinant microorganism of embodiment 116, which is engineered to overexpress an endogenous RibB polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:35.118. The recombinant microorganism of embodiment 116, which is engineered to express a heterologous RibB polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:36. 119. The recombinant microorganism of any one of embodiments 31 to 118, wherein the recombinant microorganism is engineered to increase isoprenoid production in part by being engineered to produce DXP from xylulose via 1-deoxyxylulose (DX). 120. The recombinant microorganism of embodiment 119, which has increased activity of YajO, 1-deoxyxylulose-5-phosphate synthase (EC 1.1.-.-) compared to a wild-type microorganism or parental microorganism. 121. The recombinant microorganism of embodiment 120, which is engineered to overexpress an endogenous YajO polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:37. 122. The recombinant microorganism of embodiment 120, which is engineered to express a heterologous YajO polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:38. 123. The recombinant microorganism of embodiment 119, which has increased activity of XylB, xylulose kinase (EC 2.7.1.17) compared to a wild-type microorganism or parental microorganism. 124. The recombinant microorganism of embodiment 123, which is engineered to overexpress an endogenous XylB polypeptide, which optionally comprises a sequence having 100% identity to SEQ ID NO:39. 125. The recombinant microorganism of embodiment 123, which is engineered to express a heterologous XylB polypeptide, which optionally comprises a sequence having at least 70% sequence identity to SEQ ID NO:40. 126. The recombinant microorganism of any one of embodiments 1 to 125, which has increased Cmk, cytidylate kinase (EC 2.7.4.25) activity and / or increased Ndk, nucleoside diphosphate kinase (EC 2.7.4.6) activity relative to a wild-type microorganism or parental microorganism.127. The recombinant microorganism of embodiment 126, wherein the engineering the increasing the activity of cytidylate kinase and / or nucleoside diphosphate kinase comprises increasing the activity of an endogenous cytidylate kinase promoter or an endogenous nucleoside diphosphate kinase promoter. 128. The recombinant microorganism of embodiment 126 or embodiment 127, wherein the engineering the increasing the activity of cytidylate kinase and / or nucleoside diphosphate kinase comprises introducing nucleotide sequences encoding the cytidylate kinase and / or nucleoside diphosphate kinase, optionally wherein the nucleotide sequences are codon- optimized for the recombinant microorganism. 129. The recombinant microorganism of any one of embodiments 126 to 128, wherein the engineering the increasing the activity of cytidylate kinase and / or nucleoside diphosphate kinase comprises increasing the copy number of nucleic acids encoding the cytidylate kinase and / or the nucleoside diphosphate kinase. 130. The recombinant microorganism of embodiment 129, wherein the copy number of nucleic acids encoding the cytidylate kinase and / or the nucleoside diphosphate kinase is increased by integrating a heterologous nucleic acid encoding the cytidylate kinase and / or the nucleoside diphosphate kinase into the genome of the recombinant microorganism. 131. The recombinant microorganism of any one of embodiments 126 to 130, comprising a nucleotide sequence encoding a cytidylate kinase comprising a sequence having at least 80% sequence identity to SEQ ID NO:9. 132. The recombinant microorganism of embodiment 131, wherein a nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 85% sequence identity to SEQ ID NO:9. 133. The recombinant microorganism of embodiment 131 or embodiment 132, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 90% sequence identity to SEQ ID NO:9. 134. The recombinant microorganism of any one of embodiments 131 to 133, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 92.5% sequence identity to SEQ ID NO:9.135. The recombinant microorganism of any one of embodiments 131 to 134, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 95% sequence identity to SEQ ID NO:9. 136. The recombinant microorganism of any one of embodiments 131 to 135, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 96% sequence identity to SEQ ID NO:9. 137. The recombinant microorganism of any one of embodiments 131 to 136, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 97% sequence identity to SEQ ID NO:9. 138. The recombinant microorganism of any one of embodiments 131 to 137, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 98% sequence identity to SEQ ID NO:9. 139. The recombinant microorganism of any one of embodiments 131 to 138, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having at least 99% sequence identity to SEQ ID NO:9. 140. The recombinant microorganism of any one of embodiments 131 to 139, wherein the nucleotide sequence encoding the cytidylate kinase comprises a sequence having 100% sequence identity to SEQ ID NO:9. 141. The recombinant microorganism of embodiment 131, wherein a nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 80% sequence identity to SEQ ID NO:11. 142. The recombinant microorganism of embodiment 141, wherein a nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 85% sequence identity to SEQ ID NO:11. 143. The recombinant microorganism of embodiment 141 or 142, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 90% sequence identity to SEQ ID NO:11.144. The recombinant microorganism of any one of embodiments 141 to 143, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 92.5% sequence identity to SEQ ID NO:11. 145. The recombinant microorganism of any one of embodiments 141 to 144, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 95% sequence identity to SEQ ID NO:11. 146. The recombinant microorganism of any one of embodiments 141 to 145, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 96% sequence identity to SEQ ID NO:11. 147. The recombinant microorganism of any one of embodiments 141 to 146, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 97% sequence identity to SEQ ID NO:11. 148. The recombinant microorganism of any one of embodiments 141 to 147, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 98% sequence identity to SEQ ID NO:11. 149. The recombinant microorganism of any one of embodiments 141 to 148, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having at least 99% sequence identity to SEQ ID NO:11. 150. The recombinant microorganism of any one of embodiments 141 to 149, wherein the nucleotide sequence encoding the nucleoside diphosphate kinase comprises a sequence having 100% sequence identity to SEQ ID NO:11. 151. The recombinant microorganism of any one of embodiments 1 to 150, wherein activity is increased by operably linking a nucleotide sequence to an inducible promoter, optionally a gluconate-inducible promoter. 152. The recombinant microorganism of any one of embodiments 1 to 151, which comprises a coding sequence for a non-mutant orotate phosphoribosyltransferase (EC 2.4.2.10).153. The recombinant microorganism of any one of embodiments 1 to 152, which has increased activity of an orotate phosphoribosyltransferase (EC 2.4.2.10) relative to a wild-type microorganism or parental microorganism. 154. The recombinant microorganism of embodiment 152 or embodiment 153, wherein the orotate phosphoribosyltransferase is encoded by pyrE. 155. The recombinant microorganism of any one of embodiments 152 to 154, wherein the orotate phosphoribosyltransferase comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:59. 156. The recombinant microorganism of any one of embodiments 152 to 154, wherein the orotate phosphoribosyltransferase comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:59. 157. The recombinant microorganism of any one of embodiments 152 to 156, wherein the orotate phosphoribosyltransferase comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO:59. 158. The recombinant microorganism of any one of embodiments 152 to 157, wherein the orotate phosphoribosyltransferase comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO:59. 159. The recombinant microorganism of any one of embodiments 152 to 158, wherein the orotate phosphoribosyltransferase comprises the amino acid sequence of SEQ ID NO:59. 160. The recombinant microorganism of any one of embodiments 152 to 159, in which the orotate phosphoribosyltransferase coding region is operably linked to a heterologous promoter. 161. The recombinant microorganism of embodiment 160, which is an E. coli strain. 162. The recombinant microorganism of embodiment 161, in which the E. coli rph coding sequence does not comprise a frameshift mutation. 163. The recombinant microorganism of embodiment 162, whose parental cell is an E. coli K12 substrain.164. The recombinant microorganism of embodiment 163, wherein the K12 substrain is MG1655. 165. The recombinant microorganism of any one of embodiments 162 to 164, which has been engineered to eliminate a frameshift mutation in the rph coding sequence. 166. A method for producing an isoprenoid, comprising culturing the recombinant microorganism of any one of embodiments 1 to 165conditions in which an isoprenoid is produced. 167. The method of embodiment 166, wherein the conditions comprise culturing the recombinant microorganism in a medium comprising glucose. 168. The method of embodiment 166 or embodiment 167, wherein the conditions comprise adding gluconate to the medium during a production phase. 169. The method of any one of embodiments 166 to 168, wherein the recombinant microorganism produces gluconate during the production phase. 170. The method of any one of embodiments 166 to 169, further comprising recovering the isoprenoid from the medium. 171. The method of any one of embodiments 166 to 170, wherein the isoprenoid is geranyl pyrophosphate (GPP). 172. The method of any one of embodiments 166 to 170, wherein the isoprenoid is farnesyl pyrophosphate (FPP). 173. The method of any one of embodiments 166 to 170, wherein the isoprenoid is isoprene. 174. The method of any one of embodiments 166 to 170, wherein the isoprenoid is limonene. 175. The method of any one of embodiments 166 to 170, wherein the isoprenoid is ^- myrcene.176. The method of any one of embodiments 166 to 170, wherein the isoprenoid is nerol. 177. The method of any one of embodiments 166 to 170, wherein the isoprenoid is geraniol. 178. The method of any one of embodiments 166 to 170, wherein the isoprenoid is linalool. 179. The method of any one of embodiments 166 to 170, wherein the isoprenoid is ^- ocimene. 180. The method of any one of embodiments 166 to 170, wherein the isoprenoid is 1,8-cineole. 181. The method of any one of embodiments 166 to 170, wherein the isoprenoid is farnesene. 182. The method of any one of embodiments 166 to 170, wherein the isoprenoid is farnesol. 183. A method for increasing recycling of cytidine monophosphate (CMP) to cytidine monophosphate (CTP), comprising culturing the recombinant microorganism of any one of embodiments 1 to 165 under conditions in which CMP is recycled to CTP. 184. The method of embodiment 183, wherein the conditions comprise culturing the recombinant microorganism in a medium comprising glucose. 185. The method of embodiment 183 or embodiment 184, wherein the conditions comprise culturing the recombinant microorganism in a medium comprising sucrose. 186. The method of any one of embodiments 183 to 185, wherein the conditions comprise culturing the recombinant microorganism in a medium comprising cellulose-derived sugars. 187. The method of embodiment 183 or embodiment 184, wherein the conditions comprise adding gluconate to the medium during a production phase.188. The method of any one of embodiments 183 to 185, wherein the recombinant microorganism produces gluconate during the production phase. 7. EXAMPLES 7.1. Example 1: In Silico Modeling of CTP Utilization

[0158] FIG.1 schematically depicts three pathways leading to CTP in E. coli.

[0159] The “de novo” pathway requires both phosphoribosyl pyrophosphate (PRPP), produced from glucose via the pentose phosphate pathway, and orotate, produced from glucose via the TCA cycle, to produce orotidine monophosphate (OMP), which is converted to uridine monophosphate (UMP) and subsequently to CTP. The de novo pathway involves numerous steps and is energetically expensive, consuming five adenosine triphosphate (ATP) molecules to produce one CTP molecule. The pyrimidine ribonucleosides degradation pathway recycles CMP to CTP via cytosine and uracil. The ribonucleoside degradation pathway involves six or eight enzymatic steps and consumes four ATP molecules. The ribonucleoside degradation pathway shares with the de novo pathway any chokepoints that may exist in regulation of the expression or activity of PyrH, Ndk, or PyrG. In particular, regulatory control of Ndk, or nucleoside diphosphate (NDP) kinase (EC 2.7.4.6), is important for cells to maintain balance of intracellular nucleotide pools and inhibit spontaneous mutation (Lu et al., 1995, J. Mol. Biol. 254:337–341). The salvage pathway recycles CMP to CTP by phosphorylation. The salvage pathway requires only two enzymatic steps and consumes only two ATP molecules, and thus has reduced energy consumption and fewer potential regulatory chokepoints compared to the pyrimidine ribonucleosides degradation pathway.

[0160] Consideration of FIG.1 shows that CMP recycling via the pyrimidine ribonucleoside degradation pathway begins with conversion of CMP to cytidine. This conversion can be catalyzed by nucleoside monophosphate phosphohydrolases, endogenous ribonucleotide monophosphatases, and / or endogenous 5’-ribonucleotide phosphohydrolases. Reduction of a cell’s endogenous nucleoside monophosphate phosphohydrolase activity, endogenous ribonucleotide monophosphatase activity, and / or endogenous 5’-ribonucleotide phosphohydrolase activity would thus be expected to increase CMP recycling via the salvage pathway and thereby increase the yield of products of pathways which consume CMP.

[0161] It was hypothesized that by reducing the activity of endogenous nucleoside monophosphate phosphohydrolases, e.g., umpG in E. coli, endogenous ribonucleotide monophosphatases, e.g., umpH in E. coli, and / or 5-ribonucleotide phosphohydrolases, e.g.ushA in E. coli, the ability of recombinant microorganisms to recycle CMP to CTP via pyrimidine ribonucleoside degradation would be relatively reduced and recycle via the salvage pathway would be relatively increased. Further, because the salvage pathway requires fewer steps and less ATP consumption than the pyrimidine ribonucleoside degradation pathway, it was expected that the yield and / or production rate of products of metabolic pathways which consume CTP would be increased if recombinant microorganisms were forced to use the salvage pathway preferentially to the pyrimidine ribonucleoside degradation pathway.

[0162] This question was first tested by in silico genome-scale models of isoprenoid production in E. coli in minimal media, aerobic conditions, and no growth. The theoretical maximum mole / mole yield of C10isoprenoids from glucose in a model in which CTP was only recycled via the ribonucleoside degradation pathway was 0.3299. In contrast, the theoretical maximum yield of C10isoprenoids from glucose in a model in which CTP was only recycled via the salvage pathway was 0.3611, roughly 9.5% greater. 7.2. Example 2: Effect of umpG and / or umpH, and / or ushA deletion on growth and lycopene production 7.2.1. Materials and Methods

[0163] An E. coli strain capable of producing lycopene via the DXP pathway, with induction of lycopene synthesis by expression of heterologous crtEIB integrated downstream of the lacZp promoter in place of wild-type lacZAY coding regions, was used as a base strain. Five study strains were prepared by deletion of endogenous umpG, umpH, ushA, both umpG and umpH, or both umpG and ushA using the CRISPR / MAD7-associated Lambda-RED recombineering technique known for use in genome editing, to yield study strains ΔumpG, ΔumpH, ΔumpGΔumpH, ΔushA, and ΔumpGΔushA, respectively. A single gene of interest was knocked out by deleting almost its entire coding sequence without disrupting adjacent coding and / or regulatory regions. Double knock-out strains were prepared by conducting single gene deletions sequentially.

[0164] Briefly, each deletion was facilitated by an exogenous double-stranded repair DNA fragment comprised of two regions homologous to 49-50 bp upstream and downstream of the deleted region. Donor DNA fragments were constructed using known overlap-extension PCR method from two oligonucleotides with 18 bp to 21 bp complementary sequence at their 3’-ends. Guide RNAs (gRNA) were constructed using two 25-bp complementary oligonucleotides comprised of a 4-bp ligation region at 5’-ends followed by a 21-bp region homologous to target gene’s coding sequence. Forward (F) and reverse (R) oligonucleotide (oligo) sequences usedfor construction of gRNAs and donor DNAs for ΔumpG, ΔumpH, and ΔushA are presented as SEQ ID NO:47-SEQ ID NO:58. Matured gRNA, MAD7 endonuclease, and Lambda-RED proteins were introduced into the cells using two expression plasmids. The plasmids were then eliminated from the mutant strain following successful genomic modification.

[0165] Cell growth was assessed by inoculating defined media (3 g / L potassium phosphate dibasic; 2 g / L ammonium chloride; 0.2 g / L magnesium chloride hexahydrate; 1.5 g / L potassium sulfate; 1 g / L sodium citrate dihydrate; 1.92 g / L Drop-out Mix Synthetic Minus Uracil w / o Yeast Nitrogen Base, USBiological Life Sciences, Catalog No. D9535; 1X trace metals, Teknova, Catalog No. T1001; 1X Thomas Vitamins, Teknova, Catalog No.2T1200; pH=7.0) comprising 1% gluconate with the base strain and the five study strains prepared from overnight seed cultures in the same media, followed by measuring the quantity of back-scattered light from the cell particles (excitation light = 620 nm; gain=1) in a BioLector Pro Microbioreactor System where quantity of scattered light was proportional to the biomass concentration of the cell suspension culture. Measurements were conducted every 2.5 min from 0 hr to 20 hr after inoculation while pH of cell culture was maintained at 7.0.

[0166] To induce lycopene biosynthesis, cell cultures were spiked with isopropyl ß-D-1- thiogalactopyranoside (IPTG) (1 mM final concentration) when the biomass concentration of the cell cultures reached about 4.0 (a.u.) (7 hr to 7.5 hr post inoculation). Because UmpH, UmpG, and UshA have relatively wide specificity for 5’ nucleotides, and may impact intracellular nucleotide pools, assessment of cell growth was of interest in determining whether either of the single-deletant strains and / or the double deletant strain would have a negative impact on cell survival.

[0167] Lycopene production was assessed by measuring lycopene concentration (wt / vol) and normalizing by the biomass concentration (measured as scattered light) of the strain assessed. Generally, lycopene is bound to cellular membranes. To measure lycopene concentration, briefly, cells harvested at the end of the fermentation were incubated in acetone at 55°C for 15 min to extract intracellular lycopene from heat-lysed cells in acetone. Lycopene level in acetone was then quantified by measuring OD475nmin a 284 QS 10mm quartz cuvette by a spectrophotometer. OD475nmvalues were then converted to wt / vol using a calibration curve. 7.2.2. Results

[0168] Growth curves are shown in FIG.3. Growth was essentially the same between the base strain and the five study strains. The single-deletant strains and the double deletant strain allpresented no evidence of negative impact on cell survival by altering intracellular nucleotide pools (Table 1). Relative specific lycopene levels (lycopene titers normalized to corresponding biomass level) are shown in Table 1. The results suggested that study strains ΔumpHΔumpG, ΔushA, and ΔumpGΔushA had significantly higher specific lycopene level than the base strain. Table 1 Strain Fold change – biomass Fold change – specific lycopene Table 1 to thebase strain at the end of fermentation. Statistical significances shown inside parathesis are calculated using Student’s t test (one-tailed, two-sample unequal variance) from two biological replicates; ns: p-value>0.05, *: 0.01<p-value<0.05, **: p-value < 0.01. 7.3. Example 3: Effect of umpG and / or umpH and / or ushA deletion on 2-C- methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) production

[0169] The base strain and the ΔumpH, ΔumpG, ΔumpHΔumpG, ΔushA, and ΔumpGΔushA study strains of Example 2 produced MEcPP as part of the DXP pathway. In these strains, MEcPP was typically produced more rapidly than it could be converted to HMBPP by the activity of IspG and was secreted into media. Accordingly, the effect of deletion of one, any two, any three, or all four of umpG, umpH, nudG, and / or ushA is assessed by quantifying MEcPP levels secreted into media and / or accumulated within cells. 7.3.1. Materials and Methods

[0170] MEcPP production is assessed by measuring MEcPP concentration (wt / vol) using liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis on a LCMSMS Triple Quad TQ QQQ Systems (Agilent Technologies) and normalizing by the biomass concentration (measured as scattered light) of the strain assessed. 7.3.2. Results Relative specific MEcPP levels (MEcPP titers normalized to corresponding biomass levels) of study strains are shown on Table 2. Consistent with relative specific lycopene levels (Table 1), these results suggested that study strains ΔumpHΔumpG, ΔushA, and ΔumpGΔushA hadhigher specific MEcPP level than the base strain. In particular, increased MEcPP production is more pronounced for ΔumpG ΔushA strain. Table 2 Strain Fold change – Specific MEcPP B 1 Table 2. Fold change the base strain at the end of fermentation.7.4. Example 4: Reduction of Direct CTP to CMP Conversion

[0171] Consideration of FIG.1 shows that reduction of a cell’s endogenous nucleoside triphosphate pyrophosphohydrolase activity would be expected to reduce direct conversion of CTP to CMP and thereby increase the availability of CTP to other processes.

[0172] It was hypothesized that by reducing the activity of endogenous nucleoside triphosphate pyrophosphohydrolases, e.g., nudG in E. coli, the yield and / or production rate of products of metabolic pathways which consume CTP would be increased if recombinant microorganisms were prevented from directly converting CTP to CMP. 7.4.1. Materials and Methods

[0173] Single deletions of nudG and one or more multiple deletions ΔnudGΔumpG, ΔnudGΔumpH, ΔnudGΔumpGΔumpH, ΔnudGΔushA, and ΔnudGΔumpGΔushA are prepared using the methods of Example 2. Lycopene and / or MEcPP production is assessed using the methods of Examples 2 and 3. 7.5. Example 5: Increased pyrE Expression

[0174] To increase pyrE expression in certain E. coli K12 substrains, such as MG1655 or W3110, the frameshift deletion in the rph coding region of these strains is corrected using CRISPR gene editing. 7.5.1. Materials and Methods

[0175] Briefly, the rph-1 allele of E. coli K12 substrains MG1655 and W3110 has a 1 bp deletion at nucleotide position ~668 relative to the corresponding allele in strain K12, where two glycines are coded for 5’-GGG GGA. A deletion of one of these Gs creates a frameshift and an early stop codon, characteristic of the rph-1 allele. To repair this frameshift mutation, an A wasinserted at position 669, yielding 5’-GGA GGA, thus encoding two glycines and reverting the polypeptide sequence to the K12 rph polypeptide sequence. The insertion was performed using the CRISPR / MAD7-associated Lambda-RED recombineering technique.

[0176] Concurrently, the neighboring protospacer adjacent motif (PAM) sequence was eliminated by a silent mutation (CTTG->CCTG) while preserving the W1485 codon as part of genome editing process. Both mutations were facilitated with the help of a double-stranded repair donor DNA fragment constructed from two oligonucleotides complementary at their 3’- ends. gRNA homologous to rph-1 coding region was constructed using the method described at Example 2. Forward (F) and reverse (R) oligonucleotide (oligo) sequences used for construction of gRNA and donor repair DNA are presented as SEQ ID NO:75-SEQ ID NO:78.

[0177] An E. coli K12 strain engineered to include the repaired rph-pyrE operon is prepared. Also prepared are study strains comprising the repaired rph-pyrE operon and having one or more deletions ΔumpG, ΔumpH, ΔushA, and / or ΔnudG. Strains are prepared using the methods of Example 2 and the present example. Lycopene and / or MEcPP production is assessed using the methods of Examples 2 and 3. 8. SEQUENCES

[0178] Exemplary sequences referred to herein are provided in Table 1 below (where “SEQ” refers to the SEQ ID NO). Table 1 QTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQTable 1 Description Sequence SEQ9. INCORPORATION BY REFERENCE

[0179] 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.

[0180] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed anywhere before the priority date of this application.

Claims

WHAT IS CLAIMED IS:

1. A recombinant microorganism that: (a) has reduced activity, relative to a wild-type microorganism or parental microorganism, of: (i) an endogenous nucleoside monophosphate phosphohydrolase (EC 3.1.3.5 or EC 3.1.3.6); (ii) an endogenous ribonucleotide monophosphatase (EC 3.1.3.5); (iii) an endogenous nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.56), (iv) an endogenous 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5),; or (v) any combination of two, three or all four of (i), (ii), (iii), and (iv). and (b) is engineered to increase activity of a pathway that utilizes cytidine triphosphate (CTP) as compared to a wild-type organism or parental microorganism.

2. The recombinant microorganism of claim 1, which is an E. coli.

3. The recombinant microorganism of claim 1 or claim 2, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous nucleoside monophosphate phosphohydrolase.

4. The recombinant microorganism of any one of claims 1 to 3, wherein the endogenous nucleoside monophosphate phosphohydrolase is encoded by umpG and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:

41.

5. The recombinant microorganism of claim 4, wherein the umpG gene in the recombinant microorganism has reduced activity as compared to a wild-type umpG gene.

6. The recombinant microorganism of claim 5, wherein all or a portion of the umpG coding sequence is mutated, optionally wherein the mutation is a deletion.

7. The recombinant microorganism of claim 5 or claim 6, wherein all or a portion of the umpG regulatory sequence is mutated, optionally wherein the mutation is a deletion.

8. The recombinant microorganism of any one of claims 5 to 7, wherein a heterologous sequence is introduced into the umpG locus.

9. The recombinant microorganism of any one of claims 5 to 8, which comprises an interfering RNA (RNAi) that reduces umpG activity.

10. The recombinant microorganism of any one of claims 1 to 9, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous ribonucleotide monophosphatase.

11. The recombinant microorganism of any one of claims 1 to 10, wherein the endogenous ribonucleotide monophosphatase is encoded by umpH and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:

42.

12. The recombinant microorganism of claim 10, wherein the umpH gene in the recombinant microorganism has reduced activity as compared to a wild-type umpH gene.

13. The recombinant microorganism of claim 12, wherein all or a portion of the umpH coding sequence is mutated, optionally wherein the mutation is a deletion.

14. The recombinant microorganism of claim 12 or claim 13, wherein all or a portion of the umpH regulatory sequence is mutated, optionally wherein the mutation is a deletion.

15. The recombinant microorganism of any one of claims 12 to 14, wherein a heterologous sequence is introduced into the umpH locus.

16. The recombinant microorganism of any one of claims 12 to 15, which comprises an interfering RNA (RNAi) that reduces umpH activity.

17. The recombinant microorganism of any one of claims 1 to 16, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous nucleoside triphosphate pyrophosphohydrolase.

18. The recombinant microorganism of any one of claims 1 to 17, wherein the endogenous nucleoside triphosphate pyrophosphohydrolase is encoded by nudG and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:

43.

19. The recombinant microorganism of claim 17, wherein the nudG gene in the recombinant microorganism has reduced activity as compared to a wild-type nudG gene.

20. The recombinant microorganism of claim 19, wherein all or a portion of the nudG coding sequence is mutated, optionally wherein the mutation is a deletion.

21. The recombinant microorganism of claim 19 or claim 20, wherein all or a portion of the nudG regulatory sequence is mutated, optionally wherein the mutation is a deletion.

22. The recombinant microorganism of any one of claims 19 to 21, wherein a heterologous sequence is introduced into the nudG locus.

23. The recombinant microorganism of any one of claims 19 to 22, which comprises an interfering RNA (RNAi) that reduces nudG activity.

24. The recombinant microorganism of any one of claims 1 to 23, which has reduced activity, relative to a wild-type microorganism or parental microorganism, of the endogenous 5'-ribonucleotide phosphohydrolase.

25. The recombinant microorganism of any one of claims 1 to 24, wherein the endogenous 5'-ribonucleotide phosphohydrolase is encoded by ushA and optionally comprises an amino acid sequence having 100% identity to SEQ ID NO:

44.

26. The recombinant microorganism of claim 24, wherein the ushA gene in the recombinant microorganism has reduced activity as compared to a wild-type ushA gene.

27. The recombinant microorganism of claim 26, wherein all or a portion of the ushA coding sequence is mutated, optionally wherein the mutation is a deletion.

28. The recombinant microorganism of claim 26 or claim 27, wherein all or a portion of the ushA regulatory sequence is mutated, optionally wherein the mutation is a deletion.

29. The recombinant microorganism of any one of claims 26 to 28, wherein a heterologous sequence is introduced into the ushA locus.

30. The recombinant microorganism of any one of claims 26 to 29, which comprises an interfering RNA (RNAi) that reduces ushA activity.

31. The recombinant microorganism of any one of claims 1 to 30, wherein the pathway that utilizes cytidine triphosphate is an isoprenoid pathway.

32. The recombinant microorganism claim 31, wherein the recombinant microorganism is engineered to increase flux or production of a component or precursor in the isoprenoid pathway as compared to a wild-type microorganism or parental microorganism.

33. The recombinant microorganism of claim 32, wherein the component or precursor in the isoprenoid pathway comprises any of components (1) through (19) of FIG.

2.

34. The recombinant microorganism of claim 31 or claim 33, which is engineered to recombinantly express one or more DXP pathway enzymes.

35. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise Dxs, wherein the recombinant microorganism has increased Dxs, 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7), activity as compared to a wild- type microorganism or parental microorganism and is engineered to overexpress an endogenous Dxs polypeptide or to express a heterologous Dxs polypeptide.

36. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise Dxr, wherein the recombinant microorganism has increased Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267), activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous Dxr polypeptide or to express a heterologous Dxr polypeptide.

37. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise IspD, wherein the recombinant microorganism has increased IspD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60), activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspD polypeptide or to express a heterologous IspD polypeptide.

38. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise IspE, wherein the recombinant microorganism has increased IspE, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148), activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspE polypeptide or to express a heterologous IspE polypeptide.

39. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise IspF, wherein the recombinant microorganism has increased IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12) activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspF polypeptide or to express a heterologous IspF polypeptide.

40. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise IspG, wherein the recombinant microorganism has increased IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1) activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspG polypeptide or to express a heterologous IspG polypeptide.

41. The recombinant microorganism of claim 34, wherein the one or more DXP pathway enzymes comprise IspH, wherein the recombinant microorganism has increased IspH, 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2), activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspH polypeptide or to express a heterologous IspH polypeptide.

42. The recombinant microorganism of claim 34, which has increased Idi, isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2), activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous Idi polypeptide or to express a heterologous Idi polypeptide.

43. The recombinant microorganism of claim 34, which has increased IspA, farnesyl diphosphate synthase (EC 2.5.1.10), activity as compared to a wild-type microorganism or parental microorganism.

44. The recombinant microorganism of claim 34, which has decreased IspA, farnesyl diphosphate synthase (EC 2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism and is engineered to underexpress an endogenous IspA polypeptide or to express a heterologous IspA polypeptide having one or more mutations disruptive of binding of GPP and / or isopentenyl diphosphate.

45. The recombinant microorganism of any one of claims 1 to 44, which has increased gppS, geranyl pyrophosphate (GPP) synthase (EC:2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspA polypeptide or to express a heterologous gppS polypeptide.

46. The recombinant microorganism of any one of claims 1 to 45, which has increased farnesyl pyrophosphate (FPP) synthase (EC:2.5.1.10) activity as compared to a wild-type microorganism or parental microorganism and is engineered to overexpress an endogenous IspA polypeptide or to express a heterologous IspA polypeptide.

47. The recombinant microorganism of any one of claims 1 to 46, which has increased isoprene synthase (EC 4.2.3.27 and EC 1.17.7.4) activity; limonene synthase (EC4.2.3.16 and EC 4.2.3.20) activity; myrcene synthase (EC 4.2.3.15) activity; neryl- pyrophospate synthase, dimethylallylcistransferase (EC 2.5.1.28) activity; neryl diphosphate phosphatase (EC 3.1.7.13 and EC 3.6.1) activity; nerol synthase (EC 3.1.7.13) activity; geraniol synthase (EC 3.1.7.11) activity; linalool synthase (EC 4.2.3.25 and EC 4.2.3.25) activity; beta-ocimene synthase (EC 4.2.3.106) activity; 1,8-cineole synthase activity (EC 4.2.3.108) activity; or farnesene synthase activity (EC 4.2.3.47) activity as compared to a wild-type microorganism or parental microorganism.

48. The recombinant microorganism of any one of claims 1 to 47, wherein the recombinant microorganism is engineered to increase isoprenoid production in part by being engineered to produce 1-deoxyxylulose-5-phosphate (DXP) from 2-keto-3-deoxygluconate (KDG); to produce DXP from ribulose-5-P or xylulose-5-P; to produce DXP from xylulose via 1-deoxyxylulose (DX).

49. The recombinant microorganism of any one of claims 1 to 48, which has increased Cmk, cytidylate kinase (EC 2.7.4.25) activity and / or increased Ndk, nucleoside diphosphate kinase (EC 2.7.4.6) activity relative to a wild-type microorganism or parental microorganism.

50. The recombinant microorganism of any one of claims 1 to 49, wherein the recombinant microorganism is engineered to increase activity of a pathway that utilizes CTP by operably linking a nucleotide sequence to an inducible promoter, optionally a gluconate- inducible promoter.

51. The recombinant microorganism of any one of claims 1 to 50, which comprises a coding sequence for a non-mutant orotate phosphoribosyltransferase (EC 2.4.2.10).

52. The recombinant microorganism of any one of claims 1 to 51, which has increased activity of an orotate phosphoribosyltransferase (EC 2.4.2.10) relative to a wild- type microorganism or parental microorganism.

53. A method for producing an isoprenoid, comprising culturing the recombinant microorganism of any one of claims 1 to 52 under conditions in which an isoprenoid is produced.

54. The method of claim 53, wherein the conditions comprise culturing the recombinant microorganism in a medium comprising glucose and adding gluconate to the medium during a production phase.

55. The method of claim 53 or 54, wherein the isoprenoid is geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isoprene, limonene, ^-myrcene, nerol, geraniol, linalool, ^-ocimene, 1,8-cineole, farnesene, or farnesol.

56. A method for increasing recycling of cytidine monophosphate (CMP) to cytidine monophosphate (CTP), comprising culturing the recombinant microorganism of any one of claims 1 to 52 under conditions in which CMP is recycled to CTP.

57. The method of claim 56, wherein the conditions comprise: culturing the recombinant microorganism in a medium comprising glucose, sucrose, cellulose-derived sugars, or any combination thereof.

58. The method of claim 57, wherein the conditions comprise adding gluconate to the medium during a production phase.

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