Metabolic manipulation for microbial production of terpenoid products

Abstract

The invention relates to methods and bacterial strains for manufacturing terpenes and terpenoid products, wherein the bacterial strains have improved carbon traction via the MEP pathway and for a downstream recombinant synthesis pathway.

Description

1 / 80 “Metabolic Manipulation for Microbial Production of Terpenoid Products” CROSS-REFERENCE TO REQUESTS RELATED

[0001] This application claims priority for U.S. Provisional Application No. 62 / 450,707, filed January 26, 2017, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated in its entirety herein by reference. The said ASCII copy, created on January 26, 2018, is named MAN-009PC_ST25 and is 125,103 bytes in size. FUNDAMENTALS

[0003] The food and beverage industries, as well as other industries such as perfumes, cosmetics, and healthcare, routinely use terpenes and / or terpenoid products, including for use as aromas and fragrances. However, factors such as: (i) the availability and high price of plant raw materials; (ii) the relatively low terpene content in the plant; and (iii) tedious and inefficient extraction processes to produce sufficient quantities of terpene products on an industrial scale have stimulated research into terpene biosynthesis using plant-independent systems. Consequently, effort has been spent on developing technologies to engineer microorganisms to convert renewable resources, such as glucose, into terpenoid products. Compared to traditional methods, microorganisms have the advantage of rapid growth without the need for land to sustain development.

[0004] There are two main biosynthetic pathways for the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl Petition 870260009364, dated 01 / 30 / 2026, page 14 / 187 2 / 80 diphosphate (DMAPP), the mevalonate pathway (MVA), and the methylerythritol phosphate (MEP) pathway. The MVA pathway is found in most eukaryotes, archaea, and some eubacteria. The MEP pathway is found in eubacteria, plant chloroplasts, cyanobacteria, algae, and apicomplexa parasites. E. coli and other Gram-negative bacteria use the MEP pathway to synthesize metabolic precursors of IPP and DMAPP. While the MEP pathway theoretically provides a better stoichiometric yield over the MVA pathway, the MEP pathway in E. coli and other bacteria has a variety of intrinsic regulatory mechanisms that control and / or limit carbon flux through the pathway. See Zhao et al., Methylerythritol Phosphate Path of Isoprenoid Biosynthesis, Annu Rev. Biochem. 2013; 82:497-530; Ajikumar PK, et al., Optimization of isoprenoid pathways for overproduction of Taxol precursors in Escherichia coli'. Science 2010; 330-70-74.

[0005] Microbial strains and methods to improve carbon flow through the MEP pathway and through downstream recombinant terpene and terpenoid synthesis pathways are needed for the industrial-scale production of terpenes and terpenoids in bacterial systems. SUMMARY OF THE INVENTION

[0006] In several respects, the invention relates to methods and bacterial strains for the production of terpenes and terpenoid products. In certain respects, the invention provides improved carbon flow to bacterial strains in the MEP pathway and to a downstream recombinant synthesis pathway, thereby increasing the production of terpenes and / or terpenoids by fermentation with inexpensive carbon sources (e.g., glucose).

[0007] In some respects, the invention relates to bacterial strains that overexpress IspG and IspH, so as to provide an increased carbon flux to the intermediate 1-hydroxy-2-methyl-2-(E)-butenyl-4-bisphosphate (HMBPP), but with balanced expression to avoid the accumulation of Petition 870260009364, dated 01 / 30 / 2026, p. 15 / 187 3 / 80 HMBPP is expressed in quantities that reduce cell growth or viability, or in quantities that inhibit MEP pathway flux and / or terpenoid production. Increased expression of IspG and IspH significantly increases terpene titers and terpenoid products. In contrast, overexpression of IspG alone results in growth defects, while overexpression of IspH alone does not significantly affect product titration. The HMBPP metabolite can act as a regulator or inhibitor of the MEP pathway and can be toxic to bacterial cells at certain levels. For example, in some embodiments, HMBPP does not accumulate to more than about 10 mg / g cell dry weight (DCW), or in some embodiments, it does not accumulate to more than about 5 mg / g DCW, or to more than about 2 mg / g DCW.Therefore, the balanced overexpression of IspG and IspH (e.g., favoring more IspH activity) is important for traversing carbon from downstream MEP through HMBP to IPP, while avoiding its imbalance and accumulation.

[0008] In several embodiments, the bacterial strain overexpresses a balanced MEP pathway to move carbon from MEP to the MEcPP intermediate, the substrate for IspG, and includes one or more genetic modifications to support the activities of IspG and IspH enzymes, which are Fe-sulfur clustering enzymes. Exemplary modifications include those that enhance electron delivery and transfer through the MEP pathway and / or to terpene or terpenoid products. These include recombinant expression of one or more oxidoreductase enzymes, including oxidoreductases that oxidize pyruvate and / or lead to the reduction of ferredoxin (which provides electrons to the MEP pathway). An exemplary oxidoreductase is E. coli YdbK and orthologs and derivatives thereof.

[0009] In several embodiments, the microbial strain comprises an overexpression or complementation with one or more of Petition 870260009364, dated 01 / 30 / 2026, p. 16 / 187 4 / 80 a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx) and ferredoxin reductase.

[0010] In other respects, the invention provides bacterial strains that overexpress PgpB or NudB, which dephosphorylate FPP to farnesol, and IPP and DMAPP to isoprenol and prenol, respectively. In these embodiments, the cell contains an additional product pull in the MEP pathway, while draining excess carbon in the MEP from the pathway outside the cell and thus avoiding intrinsic feedback inhibition mechanisms. Furthermore, since these products accumulate outside the cell, they can be used to track carbon flow through the MEP pathway, even without an established downstream terpenoid synthesis pathway. Therefore, bacterial strains that overexpress PgpB and / or NudB are convenient tools for balancing the expression of genes in the MEP pathway.Additionally or alternatively, in some embodiments, the bacterial strain overexpresses one or more strong synthases with sufficient product pull in the MEP pathway to evade intrinsic feedback inhibition mechanisms. For example, in some embodiments, the synthase is Artemisia annua farnesene synthase.

[0011] For the production of terpenes or terpenoid products, the bacterial cell will contain a recombinant downstream pathway that produces the terpenoid from IPP and DMAPP precursors. In certain embodiments, the bacterial cell produces one or more terpenoid compounds, such as monoterpenoids, sesquiterpenoids, triterpenoids, and diterpenoids, among others. These terpenoid compounds find use in perfumery (e.g., patchoulol), in the flavor industry (e.g., nootkatone), as sweeteners (e.g., steviol glycosides), as colorants, or as therapeutic agents (e.g., taxol).

[0012] The recovered terpene or terpenoid can be incorporated into a product (e.g., a consumer or industrial product). For example, the product could be a flavoring product, a Petition 870260009364, dated 01 / 30 / 2026, page 17 / 187 5 / 80 fragrance product, a sweetener, a cosmetic, a cleaning product, a detergent or soap, or a pest control product. The higher yields produced in embodiments of the invention can provide significant cost advantages, as well as sustainability and quality control of the terpene or terpenoid ingredient.

[0013] Other aspects and embodiments of the invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGURE 1 is a schematic for terpenoid production via the MEP pathway. A bacterial cell is represented, taking glucose as a carbon source. Glucose is converted into biomass via the TCA cycle or channeled through the MEP pathway to the desired terpenoid products. Glucose enters the cell and is converted to pyruvate (PYR) with glyceraldehyde-3-phosphate as an intermediate (GAP). PYR and GAP combine to make DOXP, which is converted to MEP and commits the pathway to FPP (via MEcPP). DOX and ME are dephosphorylated products of DOXP and MEP, respectively. DOX, ME, and MEcPP are found outside the cell. The more flux is forced into the MEP pathway, the more of these products are found extracellularly. These byproducts can be used as markers of bottlenecks in the MEP pathway and to identify targets for manipulation.Upon overexpressing nudB or pgpB, IPP and DMAPP or FPP are dephosphorylated to prenol and isoprenol or farnesol, respectively, which accumulate outside the cell and can be used to alleviate intermediate accumulation and activation of feedback inhibition of the MEP pathway. Black arrows show enzyme-mediated biochemical reactions related to terpenoids, light gray arrows show a competing byproduct, dark gray arrows show the transport of a product outside the cell, and white arrows show condensed pathways for simplification.

[0015] FIGURE 2 shows that the increase in expression Petition 870260009364, dated 01 / 30 / 2026, page 18 / 187 6 / 80 of ispH alone or ispH and ispG together improve terpenoid product titers in strains manipulated to increase the amount of carbon entering the MEP pathway; however, ispG alone decreases productivity. The control strain is an E. coli strain with additional copies of dxs, dxr, ispD, ispE, ispF, and idi (without additional copies of ispG or ispH), among other changes to improve MEP pathway flux. The strains include a 20 kb deletion, which was not manipulated.

[0016] FIGURE 3 shows that increased expression of ispG and / or ispH in production strains modified with enhanced MEP pathways affects the MEP product distribution pattern. The top panel (1x scale) shows all MEP pathway metabolites, with the majority of products being DOX, ME, and MEcPP. The middle panel (100x scale) shows MEP pathway metabolites with DOX, ME, and MEcPP not reported; in this case, DOXP and MEP are the most represented. The bottom panel (25000x scale) shows only the HMBPP concentration. In these panels, total extracellular and intracellular metabolites are shown from extracted cultures (broth plus cells), such that the reported concentration is relative to the extract volume.

[0017] FIGURE 4 shows the proportion of each individual MEP metabolite found inside or outside the cell ('Intra' vs 'Extra'). These values ​​do not reflect absolute abundance; for example, there is much more DOX in total than HMBPP. While DOX is 100% extracellular, HMBPP is 100% intracellular. The cell line profiled in Figure 4 is the high-performance 'Control + ispH / ispG' cell line. DOXP / DOX, MEP / ME, and MEcPP accumulate almost entirely, if not entirely, in the extracellular medium, while CDP-ME, CDP-MEP, HMBPP, IPP / DMAPP, and FPP are observed 100% intracellularly. The percentage of each metabolite found intracellularly is shown at the top of the graph.

[0018] FIGURE 5 shows that uncompensated upregulation of ispG causes a significant decrease in cell growth. Petition 870260009364, dated 01 / 30 / 2026, page 19 / 187 7 / 80 as determined by UV absorbance at 600 nm. Although some changes in final cell density are observed in strains compensated with ispH or ispH and ispG together, the variation is not significant.

[0019] FIGURE 6 shows that pgpB overexpression can triple farnesol titers in strains engineered to improve flux through the MEP pathway, but without an established downstream terpenoid product pathway. The control strain has additional copies of dxs, dxr, ispD, ispF, ispE, ispG, ispH, and idi under various levels of constitutive expression, and also has ydbK overexpression. The strain accumulates moderate amounts of farnesol, presumably as an overaccumulation of excess FPP, which feeds into the pathway and suffers from markedly slower growth compared to the wild type. When pgpB is overexpressed in this strain, the excess FPP is more efficiently converted to farnesol (preventing feedback control) and flux is effectively pulled through the MEP pathway.

[0020] FIGURE 7 shows that increasing and tuning the expression of ispG' and / or ispH in a farnesol-producing strain can improve product titration. The control strain has additional copies of dxs, dxr, ispD, ispF, ispE, ispG, ispH, and idi, as well as additional copies of ydbK and pgpB. Additional copies of ispH and / or ispG' are integrated into the strains under increasing promoter strength (+, ++, +++).

[0021] FIGURE 8 shows that the increase in the titration of the farnesol product (shown in Figure 7) is accompanied by a decrease in the MEcPP batch size, and depends on the ratio of ispG and ispH.

[0022] FIGURE 9 shows that idi overexpression increases product titration in a strain that does not overexpress ispGH and decreases titration in two strains that overexpress ispGH, indicating that the equilibrium between IPP and DMAPP controlled by Idi activity can be adjusted up or down depending on the needs of the downstream pathway.

[0023] FIGURE 10 illustrates the role of YdbK as a Petition 870260009364, dated 01 / 30 / 2026, p. 20 / 187 8 / 80 pyruvate oxidoreductase: flavodoxin and / or pyruvate synthase in increasing terpenoid biosynthesis.

[0024] FIGURE 11 shows that the expression of an additional copy of ydbK under increasing promoter resistance can improve terpenoid production. The control strain produces terpenoid product A and has additional copies of genes dxs, dxr, ispD, ispE, ispF, ispG', ispH, and idi of the MEP pathway under definite constitutive expression.

[0025] FIGURE 12 shows that enhancements in the titration of terpenoid products from ydbK overexpression require sufficient ispG and / or ispH. Control A has additional copies of dxs, ispD, ispF, and idi from the MEP pathway, an unmanipulated 20 kb deletion, as well as other modifications to improve the performance of iron-sulfur clustering proteins. Control B is Control A plus an additional integrated copy of ispG' and ispH in the operon configuration (G' first, such that the H / G ratio favors G), while Control C is Control A plus an additional integrated copy of ispH and ispG' in the operon configuration (H first, such that the H / G ratio favors H).

[0026] FIGURE 13 shows that the expression of fdx in addition to ydbK can improve terpenoid titration. The control strain produces the terpenoid product A and has additional copies of the dxs, dxr, ispD, ispE, ispF, ispG', ispH, and idi genes of the MEP pathway under defined constitutive expression. The strain also has an unmodified 20 kb deletion and other modifications to improve the performance of iron-sulfur cluster proteins.

[0027] FIGURE 14 illustrates the interface between glycolysis and the MEP pathway and illustrates opportunities to adjust cofactor availability by altering the expression of oxidoreductase enzymes.

[0028] FIGURE 15 is a diagram illustrating the three known reactions in E. coli to convert pyruvate (PYR) to acetyl-CoA (AcCoA) and illustrates how the reduction or elimination of the conversion mediated by Petition 870260009364, dated 01 / 30 / 2026, page 21 / 187 9 / 80 PDH of PYR in AcCoA results in increased conversion of PYR to AcCoA mediated by PFOR.

[0029] FIGURES 16A-D are graphs showing the fold change in terpenoid product production in bacterial strains with overexpressed YdbK and aceE knockout (ΔaceE), compared to the control. The control is the same strain without ΔaceE). ΔaceE prevents PDH-mediated conversion of PYR to AcCoA. Figure 16A shows the fold change in bacterial strains producing Terpenoid Product B. Figure 16B shows the fold change in bacterial strains producing Terpenoid Product C. Figure 16C shows the fold change in bacterial strains producing Terpenoid Product D. Figure 16D shows the fold change in bacterial strains producing Terpenoid Product E.

[0030] FIGURE 16E is a graph showing that bacterial strains that produce the terpenoid product D, overexpress Ydbk, and have ΔaceE show a reduction in MEcPP compared to the control (without ΔaceE).

[0031] FIGURES 17A-C are graphs showing the fold change in the terpenoid product in bacterial strains having overexpressed Ydbk and aceE (aceE mut), compared to the control (without aceE mut). aceE mut reduces the PDH-mediated conversion of PYR to AcCoA. Figure 17A shows the fold change in bacterial strains producing Terpenoid Product B. Figure 17B shows the modulation in bacterial strains producing Terpenoid Product C. Figure 17C shows the modulation in bacterial strains producing Terpenoid Product D.

[0032] FIGURE 17D is a graph showing that a bacterial strain that produces the terpenoid product D, overexpresses YdbK and expresses an aceE mut has a reduction in extracellular MEcPP compared to the control (without aceE mut).

[0033] FIGURE 18 is a diagram illustrating the three known reactions in E. coli to convert pyruvate (PYR) to acetyl-CoA. Petition 870260009364, dated 01 / 30 / 2026, page 22 / 187 10 / 80 (AcCoA) and illustrates how expressing fdx or fldA homologs can increase the electron supply to IspG and / or IspH through the Fd redox reaction (shown in bold).

[0034] FIGURE 19A is a graph showing the modulation (compared to empty vector control (emp)) of terpenoid product B production in a bacterial strain modified to overexpress YdbK and overexpress an fdx or fldA homolog.

[0035] FIGURE 19B is a graph showing the modulation (compared to the control) of the terpenoid product D in a bacterial strain engineered to overexpress YdbK and Cv.fdx (an fdx homolog from Allochromatium vinosum).

[0036] FIGURE 19C is a graph showing that a bacterial strain that produces the terpenoid product D and overexpresses YdbK and Cv.fdx has a reduction in extracellular MEcPP compared to the control (without Cv.fdx overexpression).

[0037] FIGURE 20 is a graph showing the modulation in terpenoid product production in bacterial strains that produce the terpenoid product F and that overexpress one or more homologs of PFOR or fpr and, optionally, a homolog of fdx or fldA. DETAILED DESCRIPTION OF THE INVENTION

[0038] In several aspects, the invention relates to bacterial strains and methods for manufacturing terpenes and terpenoid products, wherein the bacterial strains have an improved carbon flow via the MEP pathway and a downstream recombinant synthesis pathway. In several embodiments, the invention provides an increase in the yield of terpene and / or terpenoid product by fermentation in the bacterial strains with carbon sources such as glucose, glycerol, sucrose, and others.

[0039] For example, in some respects, the invention provides a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway and converts IPP and Petition 870260009364, dated 01 / 30 / 2026, p. 23 / 187 11 / 80 DMAPP is converted to a terpene or terpenoid product via a downstream synthesis pathway. In the bacterial strain, IspG and IspH are overexpressed, so that IspG activity and IspH activity are enhanced to provide increased carbon flux to the intermediate 1-hydroxy-2-methyl-2-(E)-butenyl 4-bisphosphate (HMBPP), but balanced to avoid HMBPP accumulation in an amount that would significantly reduce cell growth, viability, MEP pathway flux, or product titration.

[0040] Increased expression of both IspG and IspH can significantly increase terpene and terpenoid product titers. Increasing the expression of only IspG or IspH alone does not significantly improve the titer. Furthermore, overexpression of IspG alone can result in growth defects, which may be related to the observation that HMBPP (the intermediate in the MEP pathway produced by IspG and consumed by IspH) is not found extracellularly, but is found 100% intracellularly. The metabolite HMBPP appears to act as an inhibitor of the MEP pathway and appears to be toxic to the bacterial cell at certain levels. Therefore, the balance of activity between IspG and IspH is important to prevent imbalance and accumulation of HMBPP.

[0041] The accumulation of HMBPP can be determined as an amount per dry cell weight (DCW). For example, in some embodiments, HMBPP does not accumulate to more than about 10 mg / g DCW, or in some embodiments it does not accumulate to more than about 8 mg / g DCW, or in some embodiments it does not accumulate to more than about 5 mg / g DCW, or in some embodiments it does not accumulate to more than about 4 mg / g DCW, or in some embodiments it does not accumulate to more than about 2 mg / g DCW. In some embodiments, HMBPP does not accumulate to more than about 1 mg / g DCW, or it does not accumulate to more than about 0.5 mg / g DCW, or more than about 0.2 mg / g DCW, or more than about 0.1 mg / g DCW. A Petition 870260009364, dated 01 / 30 / 2026, p. 24 / 187 12 / 80 balanced overexpression of IspG and IspH (e.g., favoring more IspH activities) is important to achieve carbon pull from downstream MEP through HMBP to IPP, while avoiding its imbalance and accumulation.

[0042] In some embodiments, IspG and IspH are overexpressed through the introduction of recombinant ispG and ispH genes into the bacterial strain. In other embodiments, endogenous genes can be overexpressed by modifying, for example, the endogenous promoter or ribosomal binding site. By introducing recombinant ispG and / or ispH genes, the genes may optionally comprise one or more beneficial mutations.

[0043] In some embodiments, the additional gene may be substantially identical to the wild-type enzyme (e.g., the wild-type enzyme of E. coli), or it may be modified to increase activity, or it may be an ortholog of IspG or IspH having similar, higher, or lower activity than the native bacterial enzyme (e.g., E. coli). For example, with respect to IspG, the amino acid sequence may have 50% or more sequence identity with SEQ ID NO: 1, or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 1.In some embodiments, between 1 and about 10, or between 1 and about 5 amino acid substitutions, deletions, and / or insertions are made to the amino acid sequence of IspG (SEQ ID NO:1) to alter the protein's activity, including substitutions to one or more of the substrate-binding or active sites. Modifications for E. coli or other IspG can be informed by constructing a homology model. For example, a suitable homolog for constructing an IspG homology model. Petition 870260009364, dated 01 / 30 / 2026, p. 25 / 187 E. coli 13 / 80 is disclosed in: Lee M, et al. Isoprenoid biosynthesis: crystal structure of the [4Fe-4S] IspG cluster protein. J Mol Biol. December 10, 2010; 404(4):600-10. An exemplary IspG mutant with improved activity has four amino acid substitutions compared to the wild-type E. coli enzyme (referred to in this document as IspG').

[0044] Furthermore, with respect to IspH, the amino acid sequence may have 50% or more sequence identity with SEQ ID NO: 2, or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO:2. In some embodiments, from 1 to about 10, or from 1 to about 5 amino acid substitutions, deletions, and / or insertions are made to the IspH amino acid sequence (SEQ ID NO:2) to alter the protein activity, including substitutions to one or more of the substrate binding site or active site. Modifications in the IspH enzyme can be informed by available IspH structures, including Grawert, T., et al.Structure of the active IspH enzyme from Escherichia coli provides mechanistic insights into substrate reduction 2009 Angew.Chem.Int.Ed.Engl. 48:5756-5759.

[0045] Table 1 provides a list of alternative enzymes useful for constructing bacterial strains and / or modifying IspG or IspH enzymes for enhanced expression in bacterial cells or enhanced physical properties, each of which can be modified by amino acid substitution, deletion, and / or insertion. For example, the amino acid sequence may have 50% or more sequence identity, or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity. Petition 870260009364, dated 01 / 30 / 2026, p. 26 / 187 14 / 80 sequence, or at least about 95% sequence identity, or at least about 98% sequence identity with an amino acid sequence described in Table 1. In some embodiments, from 1 to about 10, or from 1 to about 5 amino acid substitutions, deletions, and / or insertions are made to a sequence from Table 1 to alter the protein activity, including substitutions to one or more of the substrate binding site or active site. In some embodiments, the IspG and / or IspH enzyme is an ortholog of the E. coli enzyme having enhanced properties or activity under the conditions used for culture. TABLE 1 Gene Species Accession Number IspG Bacillus subtilis NP_390386.1 IspG Chloroboculum tepidum NP_661053.1 IspG Synechocystis sp. PCC 6803 WP_010872347.1 IspH Bacillus subtilis NP_390395.2 IspH Burkholderia sp. MSh1 WP_031398482.1 IspH Chloroboculum tepidum NP_661187.1 IspH Stevia rebaudiana ABB88836.2 IspH Stevia rebaudiana ALJ30091.1 IspH Synechocystis sp. PCC 6803 WP_010873388.1

[0046] The expression of recombinant IspG and IspH enzymes can be balanced, for example, by modifying promoter strength, gene copy number, gene position in an operon, and / or modifying the ribosome-binding site sequence of the recombinant ispG and / or ispH genes. When IspG and IspH expression and / or activity are balanced, the HMBPP intermediate does not accumulate in cells substantially more than in a source strain that does not include the recombinant or modified ispG and ispH genes. This occurs despite the substantial increase in carbon flux through the MEP pathway, which is Petition 870260009364, dated 01 / 30 / 2026, p. 27 / 187 15 / 80 is required for the commercial production of terpenes and terpenoids by fermentation. This result is shown in Figure 3, where strains that overexpress IspG and IspH, which can produce a 4-fold increase in product titration compared to a control strain that does not overexpress IspG and IspH (Figure 2), however, do not accumulate HMBPP intermediate above that of the control.

[0047] In some embodiments, recombinant IspH activity and / or expression is superior to recombinant IspG activity and / or expression. An IspG / IspH ratio that favors more H enzyme results in high flux through the MEP pathway compared to a strain that favors the IspG side of the ratio. IspG and IspH function sequentially to convert MEcPP to HMBPP and then to IPP. Increased spG accumulates a larger HMBPP pool (which may show inhibitory effects on strain growth), while increased IspH shrinks the HMBPP pool as it is converted to IPP. Therefore, the ideal balance between IspG and IspH increases the rate of HMBPP formation and consumption, preventing HMBPP accumulation, which significantly improves flux through the MEP pathway to the target terpenoid. A slight favoring of IspH over IspG can further improve productivity by 25%, to almost 4 times the titers of the original strain. See Figure 2.

[0048] Therefore, in some embodiments, the expression of recombinant IspH is superior to the expression of recombinant IspG. For example, recombinant IspH and IspG enzymes can be expressed from an operon, with IspH positioned before IspG in the operon. The gene positioned first in the operon will be slightly favored for expression, providing a sophisticated balancing mechanism for IspH and IspG. In some embodiments, ispG may be positioned first, optionally along with other modifications, such as mutations to the RBS to reduce expression or point mutations to one or both IspG and IspH that balance activity at the enzyme productivity level. In some Petition 870260009364, dated 01 / 30 / 2026, p. 28 / 187 In the 16 / 80 modalities, IspG and IspH are expressed in separate operons (e.g., monocistronic) and balanced expression is achieved using promoters or RBSs of different strengths.

[0049] In some embodiments, IspH and IspG are expressed together from an operon (with the ispH gene positioned before the ispG gene) and with the operon expressed under the control of a strong promoter. Although increasing promoter strength has a positive effect on productivity when ispH is positioned before ispG in the operon, increasing promoter strength can have a negative effect when ispG is positioned before ispH. See Figure 7, using farnesol production as a surrogate for the product.

[0050] Recombinant IspG and IspH enzymes can be expressed from a plasmid or the encoding genes can be integrated into the chromosome and can be present in single or multiple copies, in some embodiments, for example, about 2 copies, about 5 copies or about 10 copies per cell. The number of copies can be controlled by using plasmids with different copy numbers (as is well known in the art), or by incorporating multiple copies into the genome, for example, by serial gene duplication.

[0051] In some embodiments, the microbial strain has high flux through the MEP pathway, including, for example, the overexpression of one or more MEP enzymes (e.g., in addition to IspG and IspH). With glucose as the carbon source, the theoretical maximum for carbon entering the MEP pathway is about 30% in E. coli. Previous MEP carbon yields reported in the literature are less than 1%. See Zhou K, Zou R, Stephanopoulos G, Too HP (2012) Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production. PLoS UM 7 (11): e47513. doi: 10,1371 / journal.pone.0047513. The overexpression and balancing of MEP genes, in addition to other modifications described in this document, Petition 870260009364, dated 01 / 30 / 2026, p. 29 / 187 17 / 80 can perform carbon traction via the MEP pathway and in a downstream synthesis pathway to improve carbon flow to terpene and / or terpenoid products.

[0052] The host cell (the bacterial strain) expresses an MEP pathway that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Specifically, glucose enters the cell and is converted to pyruvate (PYR) with glyceraldehyde-3-phosphate as an intermediate (G3P or GAP). G3P and PYR combine to make 1-deoxy-D-xylulose-5-phosphate (DOXP), which is converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) and commits the pathway to IPP and DMAPP. DOX, ME, and MEcPP are found outside the cell. The greater the flux in the MEP pathway, the more of these products are found extracellularly in strains with unbalanced pathways. See Figure 1.

[0053] The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway is also called the MEP / DOXP (2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate) pathway or the non-mevalonate pathway or the mevalonic acid-independent pathway. The pathway typically involves the action of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr or IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH) and isopentenyl-diphosphate isomerase (Idi). The MEP pathway and the genes and enzymes that constitute the MEP pathway are described in US patent 8,512,988, which is incorporated herein by reference in its entirety. Therefore, the genes that make up the MEP pathway include dxs, dxr (or ispC), ispD, ispE, ispF, ispG, ispH, idi, and ispA.The amino acid sequences for the MEP pathway enzymes are shown in the attached Sequence Listing.

[0054] IPP and DMAPP (products of the MEP pathway) are precursors of terpenes and terpenoids, including monoterpenoids, Petition 870260009364, dated 01 / 30 / 2026, page 30 / 187 18 / 80 sesquiterpenoids, triterpenoids, and diterpenes, which have specific uses in the flavor, fragrance, cosmetics, and food industries. The synthesis of terpenes and terpenoids proceeds through the conversion of IPP and DMAPP precursors into geranyl diphosphate (GPP), farnesyl diphosphate (FPP), or geranylgeranyl diphosphate (GGPP), through the action of a prenyltransferase enzyme (e.g., GPPS, FPPS, or GGPPS). Such known enzymes are described, for example, in US documents 8,927,241, WO 2016 / 073740, and WO 2016 / 029153, which are incorporated herein by reference in their entirety.

[0055] In several embodiments, the invention results in substantial improvements in MEP carbon. As used in this document, the term MEP carbon refers to the total carbon present as an input, intermediate, metabolite, or product of the MEP pathway. Metabolites include byproducts such as degradation products and phosphorylation and dephosphorylation products. MEP carbon includes products and intermediates from downstream pathways, including terpenoid synthesis pathways. For the purposes of this disclosure, the MEP carbon includes the following entries, intermediates, and metabolites of the MEP pathway: D-glyceraldehyde 3-phosphate, pyruvate, 1-deoxy-D-xylulose-5-phosphate, 1-deoxy-D-xylulose, 2-C-methyl-D-erythritol-5-phosphate, 2-C-methyl-D-erythritol, 4-diphosphocytidyl-2-C-methyl-D-erythritol, 2-phospho-4-diphosphocytidyl-2-C-methyl-derythritol, 2,4-cyclodiphosphate of 2C-methyl-D-erythritol, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, isopentenyl diphosphate, and dimethylallyl diphosphate.MEP carbon additionally includes essential intermediates and metabolites in the downstream terpenoid synthesis pathway expressed by the cell. Although the identity varies based on the pathway and enzymes used, such products include: geranyl pyrophosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), or geranylfarnesyl diphosphate (FGPP); their monophosphorylated versions of geranyl phosphate, farnesyl phosphate, geranylgeranyl phosphate, or geranylfarnesyl phosphate; their alcohols geraniol, farnesol, geranylgeraniol, or geranylfarnesol; as well as downstream terpenes and terpenoid products. Petition 870260009364, dated 01 / 30 / 2026, page 31 / 187 19 / 80 carbon of MEP additionally includes compounds derived from FPP or pathways that use FPP, including squalene, undecaprenyl diphosphate (UPP), undecaprenyl phosphate, octaprenyl diphosphate (OPP), 4-hydroxybenzoate, 3-octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol, 3-octaprenylbenzene-1,2-diol, 2-methoxy-6-octaprenyl-2-methoxy-1,4-benzoquinol, 6-methoxy-3-methyloctaprenyl-1,4-benzoquinol, 3-desmethylubiquinol-8, ubiquinol-8, ubiquinone, 2-carboxy-1,4-naphthoquinol, demethylmenaquinol-8, menaquinol-8 and menaquinone. MEP carbon additionally includes the metabolites isoprenol, prenol, isopentenyl phosphate, and dimethylallyl phosphate. MEP carbon (the intermediates and metabolites mentioned above) can be quantified by mass spectrometry (MS), such as tandem mass spectrometry (MS / MS) via a triple quadrupole mass detector (QQQ).An example system is the Agilent 6460 QQQ; alternatively, with quantitative time-of-flight (QTOF), time-of-flight (TOF) or ion capture mass detectors.

[0056] In some embodiments, the microbial strain has at least one additional copy of the dxs, ispD, ispF, and / or idi genes, which may be rate-limiting, and which may be expressed from an operon or module, on a plasmid, or integrated into the bacterial chromosome. In some embodiments, the bacterial strain has at least one additional copy of dxs and idi expressed as an operon / module; or dxs, ispD, ispF, and idi expressed as an operon or module. In some embodiments, the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which are optionally expressed as 1, 2, or 3 individual operons or modules. The recombinant genes of the MEP pathway are expressed from one or more plasmids or are integrated into the chromosome. In these embodiments, the strain provides increased flux through the MEP pathway compared to the wild type.

[0057] The amino acid sequences for the wild-type E. coli enzymes Dxs, Dxr, IspD, IspE, IspF, and Idi are shown in this document as SEQ ID NOS: 3 to 8. In various embodiments, they may be Petition 870260009364, dated 01 / 30 / 2026, p. 32 / 187 20 / 80 used enzymes having structural or sequence homology and comparable functionality (including bacterial homologs). For example, the amino acid sequence may have 50% or more sequence identity with SEQ ID NOs: 3-8, or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 3-8. In some embodiments, from 1 to about 10, or from 1 to about 5 amino acid substitutions, deletions, and / or insertions are made to the amino acid sequence (SEQ ID NO: 3-8) to alter the protein activity, including substitutions to one or more of the substrate binding site or active site.Modifications in enzymes can be informed by constructing a homology model. Such mutants can be informed by enzyme structures available in the art, including Yajima S, et al., Structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin, Acta Cryst. F63, 466-470 (2007).

[0058] In some embodiments, MEP complementation increases the conversions of DOXP and MEP pools to MEcPP, from substrate to IspG. See Figure 1. The bottlenecks in the MEP pathway from dxs to IspF can be determined in relation to the levels of DOX, ME, and MEcPP, which can be detected extracellularly. The complementation and expression of MEP pathway enzymes can be balanced to shift the carbon flux to the MEcPP intermediate, as determined by the metabolite profile. In some embodiments, the expression or activity of IspG and IspH is balanced in relation to the expression or activity of Dxr, Dxs, IspD, IspE, and IspF to drive the MEcPP metabolite to IPP and DMAPP precursors. MEcPP can be transported to the extracellular medium and, therefore, large MEcPP pools can result in loss of Petition 870260009364, dated 01 / 30 / 2026, p. 33 / 187 21 / 80 carbon MEP.

[0059] In some embodiments, the expression or activity of a recombinant idi gene is adjusted to increase the production of terpenes or terpenoids. The Idi enzyme catalyzes the reversible isomerization of IPP to DMAPP. Since each desired terpenoid product or undesired MEP byproduct (e.g., UPP) uses one DMAPP and several IPPs, the ratio between the two precursors can have an effect on strain productivity. Variation in the available IPP:DMAPP ratio, for example, variation in Idi expression or activity, can impact the production of desired terpenoids relative to other undesired products from the MEP pathway.For example, as shown in Figure 9, while Idi overexpression slightly increases product titration in a strain that does not overexpress IspGH (Strain 1), it decreases titration in two strains that do (Strains 2 and 3), indicating that the equilibrium between IPP and Idi-controlled DMAPP can be adjusted up or down depending on the needs of the downstream pathway. However, strain 4 (Figure 9), which has a different equilibrium of enzyme expression from the MEP pathway, more than doubles titrations with Idi complementation. The expression of the recombinant idi gene can be adjusted in several ways by modifying promoter strength, gene copy number, position in an operon or ribosomal binding site, as well as point mutations to increase or decrease enzyme productivity.

[0060] The microbial strain provides substantial increases in MEP carbon, including substantial increases in IPP and DMAPP precursor flux, without substantial effect on strain growth and viability, for example, as determined by optical density (OD) in culture, peak OD, and / or growth rate. For example, despite the increased flux through the MEP pathway, which is tightly controlled in bacterial cells, the microbial strain does not have a peak OD drop greater than about 20%, or in some embodiments, does not have a peak OD drop of more than Petition 870260009364, dated 01 / 30 / 2026, p. 34 / 187 22 / 80 approximately 15%, or more than approximately 10%, or more than approximately 5%. In some embodiments, the strain does not exhibit a measurable effect on strain growth or viability, as determined, for example, by measuring growth rate or peak OD.

[0061] In some embodiments, the bacterial strain contains one or more genetic modifications that enhance electron supply and transfer through the MEP pathway and / or to terpenes or terpenoid products. In some embodiments, the enhanced electron supply and transfer through the MEP pathway is by recombinant expression of one or more oxidoreductase enzymes, including oxidoreductases that oxidize pyruvate and / or lead to ferredoxin reduction. Ferredoxin supplies electrons to the MEP pathway and supports the activity of IspG and IspH (which are Fe-S clustering enzymes). See Figure 10. In several embodiments, the microbial strain comprises an overexpression or complementation with one or more of a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), and ferredoxin reductase.

[0062] For example, in some embodiments, pyruvate oxidoreductase:flavodoxin oxidoreductase (PFOR). In some embodiments, PFOR is YdbK. In some embodiments, YdbK is Ydbk from E. coli, or orthologs and derivatives thereof.

[0063] In some embodiments, the strain contains a complementation or overexpression of YdbK. YdbK is predicted to function as pyruvate:flavodoxin oxidoreductase and / or pyruvate synthase. The oxidoreductase is believed to oxidize pyruvate to acetyl-CoA, reducing ferredoxin, which can then provide electrons to the MEP pathway, especially to support the strongly upregulated IspG and IspH enzymes containing Fe-S clusters. In some embodiments, the expression of a recombinant YdbK is balanced with the expression of IspG and IspH, which can be determined by product titration (or farnesol titration, as described below). In some embodiments, the YdbK gene is under the Petition 870260009364, dated 01 / 30 / 2026, page 35 / 187 23 / 80 control of a weak or intermediate strength promoter. Additionally, extra electron transport or transfer cofactors may be expressed in the overexpression of YdbK. See, for example, Akhtar, et al., Metabolic Engineering, 11 (3): 139-147 (2009). In some experiments, the YdbK is overexpressed with fdx (ferredoxin) from Clostridium pasteurianum (SEQ ID NO: 10) and / or E. coli (Ec.ydhY) (SEQ ID NO: 34), or an enzyme having at least 80% or at least 90% sequence identity. The bacterial strain may comprise a recombinant YdbK gene, which may be integrated into the chromosome or expressed from a plasmid. The amino acid sequence of the E. coli YdbK enzyme is shown in this document as SEQ ID NO: 9. In various embodiments, enzymes having structural or sequence homology and comparable functionality may be used.For example, the amino acid sequence may have 50% or more sequence identity with any of SEQ ID NO: 9, or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least 97% sequence identity, or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, from 1 to about 10, or from 1 to about 5 amino acid substitutions, deletions, and / or insertions are made to the amino acid sequence (SEQ ID NO: 9) to alter the protein activity.

[0064] In some embodiments, the strain comprises one or more P450 enzymes for the production of a terpenoid compound. Overexpression of YdbK and potentially other oxidoreductases may support higher levels of P450 oxidative chemistry.

[0065] In some embodiments, including embodiments in which the bacterial strain overexpresses or has increased pyruvate:flavodoxin oxidoreductase (PFOR) activity, the strain exhibits Petition 870260009364, dated 01 / 30 / 2026, p. 36 / 187 24 / 80 Reduced conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH). In some embodiments, the conversion of pyruvate to acetyl-CoA by PDH is reduced by deletion or inactivation of PDH, or by reduced expression or activity of PDH. In some embodiments, PDH is deleted. Alternatively, PDH activity may be reduced by one or more amino acid modifications. An exemplary mutation to reduce PDH activity is a mutation of G267C to aceE.

[0066] In some embodiments, the conversion of pyruvate to acetyl-CoA by PDH is reduced by modifying the aceE-aceF-lpd complex of PDH. In some embodiments, the aceE-aceF-lpd complex is modified by deletion, inactivation, or reduced expression or activity of aceE, aceF, lpd, or a combination thereof. By way of example, in some embodiments, aceE is deleted (e.g., by knockout). Alternatively, in some embodiments, the aceE-aceF-lpd complex is modified by one or more mutations of aceE, aceF, lpd, or a combination thereof.

[0067] By reducing the conversion of pyruvate to acetyl-CoA by PDH, the bacterial strain will become more dependent on PFOR (e.g., YdbK) for the conversion of pyruvate to acetyl-CoA. See Figure 15. This dependence increases the activity of IspG and IspH.

[0068] In some embodiments, the supply and transfer of electrons to IspG and IspH is enhanced by overexpression or supplementation with one or more oxidoreductases, such as PFOR. For example, in some embodiments, the PFOR, or a homologue thereof, is selected from YdbK (SEQ ID NO: 9), Scy.pfor (Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor (Kluyvera intermedia) (SEQ ID NO: 30), Da.pfor (Desulfovibrio africanus) (SEQ ID NO: 31), Ns.pfor (Nostoc sp.) (SEQ ID NO: 32), Ec.ydV (E. coli) (SEQ ID NO: 33), Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35), and Sco.pfor (Synechococcus sp.). In some embodiments, the PFOR is YdbK.

[0069] In some modalities, PFORs comprise Petition 870260009364, dated 01 / 30 / 2026, p. 37 / 187 25 / 80 a sequence that is at least 60% identical to any of the SEQ ID Nos. 29-35. For example, PFOR may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID Nos. 29-35.

[0070] In some embodiments, overexpression or complementation with PFOR such as, for example, YdbK, can result in improved performance through the expression of electron carriers with a redox potential of about 400 to 550 mV, or in some embodiments, in the range of about 400 to 500 mV, or in the range of about 400 to 475 mV. In some embodiments, the electron carrier is ferredoxin, flavodoxin, or NADPH. As an example, in some embodiments, the electron carrier is Cv.fdx (Allochromatium vinosum).

[0071] In some embodiments, the bacterial strain has overexpression or complementation with one or more fpr homologs. As an example, in some embodiments, the fpr homolog is selected from Ns.fpr (Nostoc sp.) (SEQ ID NO: 36), Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37) and Ec.fpr (E. coli) (SEQ ID NO: 38).

[0072] In some embodiments, fldA homologues comprise a sequence that is at least 60% identical to any of the SEQ ID Nos. 36-38. For example, fpr may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID Nos: 36-38.

[0073] In some embodiments, the bacterial strain that overexpresses YdbK or its homolog or derivative, additionally expresses a non-native electron acceptor / donor, such as one or more non-native homologs of fdx and / or fldA. As an example, the homolog of fdx may be Petition 870260009364, dated 01 / 30 / 2026, p. 38 / 187 26 / 80 selected from Hm.fdxl (Heliobacterium modesticaldum) (SEQ ID NO: 15), Pa.fdx (Pseudomonas aeruginosa) (SEQ ID NO: 16), Cv.fdx (Allochromatium vinosum) (SEQ ID NO: 17), Cv.fdx_C57A (synthetic) (SEQ ID NO: 18), Ec.yfhL (E. Coli) (SEQ ID NO: 19), Ca.fdx (Clostridium acetobutylicum) (SEQ ID NO: 20), Cp.fdx (Clostridium pasteurianum) (SEQ ID NO: 10), Ec.fdx (E. Coli) (SEQ ID NO: 21), Ev2.fdx (Ectothiorhodospira shaposhnikovii) (SEQ ID NO: 22), Pp1.fdx (Pseudomonas putida) (SEQ ID NO: 23) and Pp2.fdx (Pseudomonas putida) (SEQ ID NO: 24). In some embodiments, the fldA homolog includes one or more selected from Ec.fldA (E. coli) (SEQ ID NO: 27), Ac.fldA2 (Azotobacter chroococcum) (SEQ ID NO: 26), Av.fldA2 (Azotobacter vinelandii) (SEQ ID NO: 25) and Bs.fldA (B. subtilis) (SEQ ID NO: 28).The expression of a non-native fdx homolog and / or fldA homolog results in an increased supply of electrons to IspG and / or IspH, an increase in IspG / H activity, and an increase in terpenoid production. See Figures 19A-C.

[0074] In some embodiments, non-native fdx homologs comprise a sequence that is at least 60% identical to any of the SEQ IDs NOS: 10 and 15-24. For example, non-native fdx homologs may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ IDs NOS. 10 and 15-24.

[0075] In some embodiments, non-native fldA homologs comprise a sequence that is at least 60% identical to any of the SEQ ID NOs: 25-28. For example, non-native fldA homologs may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID NOs. 25-28. Petition 870260009364, dated 01 / 30 / 2026, p. 39 / 187 27 / 80

[0076] In some embodiments, the bacterial strain has overexpression or complementation with one or more PFOR and / or fpr and, optionally, one or more of a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), and ferredoxin reductase. As an example, in some embodiments, the bacterial strain includes Ec.ydhV (E. coli) (SEQ ID NO: 33) and Ec.ydhY (E. coli) (SEQ ID NO: 34); Ec.ydbK (E. coli) (SEQ ID NO: 9) and Cp.fdx (Clostridium pasteurianum) (SEQ ID NO: 10); Ec.fpr (E. coli) (SEQ ID NO: 38) and Ec.fdx (E. coli) (SEQ ID NO: 21); or Ec.fpr (E. Coli) (SEQ ID NO: 38) and Ec.fldA (E. coli) (SEQ ID NO: 27).

[0077] In other respects, the invention provides bacterial strains that overexpress PgpB or NudB enzymes to increase the carbon pull of MEP. The installation of this alternative 'product' pull by overexpression genes such as pgpB and nudB further enhances the pull on the flux through the MEP pathway (albeit for non-target products) and minimizes the accumulation of feedback inhibitory or toxic intermediates (e.g., IPP, DMAPP, FPP). In some embodiments, the overexpression of PgpB or NudB is in the absence of a downstream terpenoid pathway, thus creating a universal chassis; that is, a strain that can have any downstream terpenoid transformed downstream and be rapidly optimized for commercial production.

[0078] More specifically, carbon can undergo pull-through via the MEP pathway to create alternative products that will cluster on the outside of the cell. PgpB dephosphorylates FPP to farsenol (FOH), and NudB dephosphorylates IPP and DMAPP to isoprenol (3-methyl-3-buten-1-ol) and prenol (3-methyl-2-buten-1-ol), respectively (see Figure 1). Improving the transport of these products out of the cell prevents the accumulation of IPP, DMAPP, and FPP; which, like HMBPP, can provide feedback and exert control over the MEP pathway. IPP inhibits growth, and feedback inhibits Dxs. See Cordoba, Salmi & Leon (2009) J. Exp. Bot. 60, 10, 2933-2943. FPP inhibits growth, and feedback inhibits the ispF-MEP complex, which is formed when MEP binds and Petition 870260009364, dated 01 / 30 / 2026, p. 40 / 187 28 / 80 increases IspF activity in an anticipated manner. Bitok and Meyers (2012) ACS Chem. Biol. 2012, 7, 1702-1710. Farnesol, isoprenol, and prenol accumulate outside the cell and, as intermediates in the MEP pathway, can be used to track carbon flux through the MEP pathway via LC / MS or GC / MS quantification.

[0079] In several embodiments, enzymes having structural or sequence homology and comparable functionality may be used. For example, the amino acid sequence may have 50% or more sequence identity with SEQ ID NO: 11 (PgpB) or 12 (NudB), or at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, from 1 to about 10, or from 1 to about 5 amino acid substitutions, deletions, and / or insertions are made to the amino acid sequence (SEQ ID NO: 11 or 12) to alter the protein activity, including substitutions to one or more of the substrate binding site or active site.

[0080] Therefore, by constitutively expressing an additional copy of pgpB or nudB, carbon flux through the MEP pathway can be enhanced and a slow-growing phenotype is improved. In cases where IspG and IspH are balanced and pgpB or nudB are overexpressed, the increase or decrease in farnesol product is inversely correlated with the MEcPP level (Figures 7 and 8).

[0081] Meanwhile, excessive expression of PgpB or NudB can negatively affect the total flux through farnesol, resulting in lower titer and less modulation. See Figure 6. Therefore, in several embodiments, the expression of PgpB and / or NudB is adjusted to provide a higher terpene or terpenoid product titer, optionally by varying the promoter strength, gene copy number, position in an operon and / or site of Petition 870260009364, dated 01 / 30 / 2026, p. 41 / 187 29 / 80 ribosomal linkage. In some embodiments, the recombinant pgpB and / or nudB genes are expressed under the control of a weak or intermediate-strength promoter. The recombinant pgpB or nudB may be integrated into the chromosome or expressed from a plasmid.

[0082] In some embodiments, bacterial strains overexpress one or more synthases to increase the carbon pull of MEP. For example, in some embodiments, the synthase is selected from Artemisia annua farnesene synthase and valencene synthase.

[0083] In some embodiments, the bacterial strain has one or more additional modifications to increase cofactor availability or turnover, including NADH and NADPH cofactors, thus leading to increases in MEP carbon. See Figure 14. In some embodiments, the bacterial strain expresses a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), for example, from Methanococcus maripaludis (SEQ ID NO: 14) or another mesophilic organism. Expression of a GAPOR would provide electrons to ferredoxins in central carbon metabolism and could provide electrons to IspG, IspH, or P450 enzymes. In some embodiments, the bacterial strain overexpresses one or more genes of the ydh operon, such as ydhV or ydhY (e.g., complementing the wild-type gene or potentiating the expression of the endogenous bacterial gene). YdhV or other bacterial genes with GAPOR-like activity may increase cofactor availability to further increase MEP carbon.Other gene modifications include downregulation, inactivation, or deletion of gshA or expression of CHAC1 and / or CHAC2 (e.g., in Homo sapiens). These modifications can alter glutathione levels, thus indirectly increasing NADPH availability.

[0084] Although various bacterial species may be modified according to the disclosure, in some embodiments, the bacterial strain is a bacterium selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp. and Petition 870260009364, dated 01 / 30 / 2026, page 42 / 187 30 / 80 Pseudomonas spp. In some embodiments, the bacterial strain is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterial strain is E. coli.

[0085] According to the modalities described in this document, several strategies can be used to manipulate the expression or activity of recombinant genes and enzymes, including, for example, modifications or substitution of promoters of different strengths, modifications to the ribosomal binding sequence, modifications to the order of genes in an operon or module, use of gene codon, RNA or protein stability, RNA secondary structure and gene copy number, among others.

[0086] In some embodiments, the ribosomal binding site sequence can be altered to adjust mRNA translation. The Shine-Dalgarno (SD) sequence is the ribosomal binding site in bacteria and is generally located around 8 bases upstream of the AUG initiation codon. The RNA sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the initiation codon. The six-base consensus sequence is AGGAGG (SEQ ID NO: 13) in Escherichia coli. Mutations in the consensus sequence can be screened for improvements in product titration (including farnesol titration in some embodiments), or screened by MEP carbon metabolic analysis.

[0087] For gene complementation, wild-type genes can be used and, in some embodiments, the gene is a wild-type E. coli gene. Alternatively, several orthologs can be used, which may show nucleotide or amino acid homology to the E. coli gene. Exemplary genes can be derived from orthologs of Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Petition 870260009364, dated 01 / 30 / 2026, p. 43 / 187 31 / 80 Vibrio spp., Pseudomonas spp., Chloroboculum spp., Synechocystis sp., Burkholderia spp. and Stevia rebaudiana, for example.

[0088] The similarity of nucleotide and amino acid sequences, that is, the percentage of sequence identity, can be determined using sequence alignments. Such alignments can be performed with several algorithms known in the art, such as the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or with the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res.). The degree of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated in the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. BLAST polynucleotide searches can be performed with the BLASTN program, score = 100, word length = 12.BLAST protein searches can be performed using the BLASTP program, score = 50, word length = 3. To obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs can be used. Sequence matching analysis can be supplemented by established homology mapping techniques such as Shuffle-Lagan random fields (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov.

[0090] Conservative substitutions can be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: Petition 870260009364, dated 01 / 30 / 2026, page 44 / 187 32 / 80 (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acids: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatics: Trp, Tyr, Phe.

[0091] As used in this document, “conservative substitutions” are defined as exchanges of one amino acid for another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp for Glu maintains a negative charge in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Some preferred conservative substitutions within the six groups above are exchanges within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (iii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe.

[0092] As used in this document, non-conservative substitutions are defined as exchanges of one amino acid for another amino acid listed in a group other than the six standard amino acid groups (1) to (6) shown above.

[0093] Enzyme modifications as described in this document may include conservative and / or non-conservative mutations.

[0094] In some embodiments, rational design is involved in constructing specific mutations in enzymes. Rational design refers to incorporating knowledge of the enzyme, or related enzymes, such as the thermodynamics and kinetics of the reaction, its three-dimensional structure, its active site(s), its substrate(s), and / or enzyme-substrate interaction, into the design of the specific mutation. Based on a rational design approach, mutations can be created in an enzyme that can then be traced to increased production of a terpene or Petition 870260009364, dated 01 / 30 / 2026, page 45 / 187 33 / 80 terpenoid relative to parent strain levels, or metabolite profile corresponding to improvements in MEP carbon. In some modalities, mutations can be rationally designed based on homology modeling. “Homology modeling” refers to the process of constructing an atomic resolution model of a protein from its amino acid sequence, using the three-dimensional structure of a related homologous protein.

[0095] Amino acid modifications can be made to enzymes to increase or decrease the activity of the enzyme or enzyme complex. Genetic mutations can be performed using any gene mutation method known in the art. In some embodiments, a gene knockout eliminates a gene product in whole or in part. Gene knockouts can be performed using any elimination method known in the art.

[0096] Manipulation of gene and / or protein expression, including genetic modules, can be achieved through various methods. For example, gene or operon expression can be regulated by selecting promoters, such as inducible or constitutive promoters, with different intensities (e.g., strong, intermediate, or weak). Several non-limiting examples of promoters include Trc, T5, and T7. Additionally, gene or operon expression can be regulated by manipulating the copy number of the gene or operon in the cell. In some embodiments, gene or operon expression can be regulated by manipulating the order of genes within a module, where genes transcribed first are generally expressed at a higher level. In some embodiments, gene or operon expression is regulated by the integration of one or more genes or operons into the chromosome.

[0097] In some modes, gene expression equilibrium includes the selection of plasmids with a high number of Petition 870260009364, dated 01 / 30 / 2026, page 46 / 187 34 / 80 copies, or plasmids with simple, low, or medium coding numbers. In other modes, the transcription termination step can also be directed toward gene expression regulation through the introduction or elimination of stem-loop structures.

[0098] Expression vectors containing all the elements necessary for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically manipulated by introducing heterologous DNA into the cells. The heterologous DNA is placed under operable control of transcriptional elements to allow expression of the heterologous DNA in the host cell.

[0099] In some modalities, endogenous genes are edited instead of undergoing gene complementation. Editing can modify endogenous promoters, ribosomal binding sequences, or other expression control sequences, and / or, in some modalities, modifies trans- and / or cis-acting factors in gene regulation. Genome editing can be performed using CRISPR / Cas genome editing techniques, or similar techniques employing zinc finger nucleases and TALENs. In some modalities, endogenous genes are replaced by homologous recombination.

[0100] In some embodiments, genes are overexpressed, at least in part, by controlling the gene copy number. Although gene copy number can be conveniently controlled using variable copy number plasmids, gene duplication and chromosomal integration can also be employed. For example, a process for genetically stable serial gene duplication is described in US 2011 / 0236927, which is incorporated herein by reference in its entirety.

[0101] In certain forms, the bacterial cell produces Petition 870260009364, dated 01 / 30 / 2026, p. 47 / 187 35 / 80 one or more terpene or terpenoid compounds. A terpenoid, also known as an isoprenoid, is an organic chemical compound derived from a five-carbon (C5) isoprene unit. Several non-limiting examples of terpenoids, classified based on the number of isoprene units they contain, include: hemiterpenoids (1 isoprene unit), monoterpenoids (2 isoprene units), sesquiterpenoids (3 isoprene units), diterpenoids (4 isoprene units), sesterterpenoids (5 isoprene units), triterpenoids (6 isoprene units), tetraterpenoids (8 isoprene units), and polyterpenoids with a higher number of isoprene units. In one embodiment, the bacterial host cell produces a terpenoid selected from a monoterpenoid, a sesquiterpenoid, a diterpenoid, a sesterpenoid, or a triterpenoid.Terpenoids represent a diverse class of molecules that provide numerous commercial applications, including in the food and beverage industries, as well as in the perfume, cosmetics, and health industries. For example, terpenoid compounds find use in perfumery (e.g., patchoulol), in the flavoring industry (e.g., nootkatone), as sweeteners (e.g., steviol), colorants, or therapeutic agents (e.g., taxol), and many are conventionally extracted from plants. However, terpenoid molecules are found in ppm levels in nature and therefore require massive harvesting to obtain sufficient quantities for commercial applications.

[0102] The bacterial cell will generally contain a downstream recombinant pathway that produces the terpenoid from IPP and DMAPP precursors. Terpenes such as Monoterpenes (C10), Sesquiterpenes (C15), Diterpenes (C20), Sesterterpenes (C25), and Triterpenes (C30) are derived from the substrates prenyl diphosphate, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (FGPP), and two FPPs, respectively, through the action of a very large group of enzymes called terpene (terpenoid) synthases. These enzymes are Petition 870260009364, dated 01 / 30 / 2026, page 48 / 187 36 / 80 are frequently referred to as terpene cyclases, since the product of the reactions is cyclized into various carbon skeleton products: monoterpene, sesquiterpene, diterpene, sesterterpene, and triterpene. Many of the resulting carbon skeletons undergo subsequent oxygenation by cytochrome P450 enzymes to give rise to large families of derivatives.

[0103] Examples of terpenes or terpenoid products that can be produced according to the invention are described in US 8,927,241, which is incorporated herein by reference, and include: farnesene, amorphadiene, artemisinic acid, artemisinin, bisabolol, bisabolene, alpha-sinensal, beta-thujone, camphor, carveol, carvone, cineole, citral, citronellal, cubebol, geraniol, limonene, menthol, menthone, myrcene, nootkatone, patchouli, piperitone, rose oxide, sabinene, steviol, steviol glycoside (including rebaudioside D or rebaudioside M), taxadiene, thymol, and valencene. Enzymes for recombinant pathway construction in E. coli are described in US patents 8,927,241, WO 2016 / 073740, and WO 2016 / 029153, which are incorporated herein by reference.

[0104] Exemplary P450 enzymes that are operative on sesquiterpene supports to produce oxygenated terpenoids are described in WO 2016 / 029153, which is incorporated herein by reference. In addition, P450 reductase proteins that find utility in the bacterial strains described in this document are described in WO 2016 / 029153, as well as in WO 2016 / 073740.

[0105] As used in this document, the term oxygenated terpene refers to a terpene skeleton possessing one or more oxygenation events, producing a corresponding alcohol, aldehyde, carboxylic acid and / or ketone. In some embodiments, the bacterial cell produces at least one terpenoid selected from Abietadiene, Abietic Acid, alpha-Sinensal, beta-Thujone, Camphor, Carveol, Carvone, Celastrol, Ceroplastol, Cineole, Citral, Citronellal, Cubebol, Cucurbitane, Forskolin, Gascardic Acid, Geraniol, Haslene, Levopimaric Acid, Limonene, Lupeol, Menthol, Menthone, Petition 870260009364, dated 01 / 30 / 2026, p. 49 / 187 37 / 80 Mogroside, Nootkatona, Nootkatol, Ophiobolin A, Patchouli, Piperitone, Rebaudioside D, Rebaudioside M, Sabinene, Steviol, Steviol Glycoside, Taxadiene, Thymol and Ursolic Acid.

[0106] In some embodiments, the terpenoid synthase enzyme is enhanced to increase the kinetics, stability, product profile and / or temperature tolerance of the enzyme, as disclosed, for example, in documents WO 2016 / 029153 and WO 2016 / 073740, which are hereby incorporated by reference.

[0107] In other embodiments, the bacterial cell produces valencene and / or nootkatone. In such an embodiment, the bacterial cell may express a biosynthetic pathway that also includes a farnesyl diphosphate synthase, a valencene synthase, and a valencene oxidase. Farnesyl diphosphate synthases (FPPS) produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) farnesyl diphosphates. An exemplary farnesyl diphosphate synthase is ERG20 from Saccharomyces cerevisiae (NCBI accession P08524) and E. coli ispA. Valencenosynthase produces sesquiterpene structures and is described, for example, in US 2012 / 0107893, US 2012 / 0246767 and US 7,273,735, which are incorporated herein by reference in their entirety. The genes and host cells for the production of terpenoid product comprising valencene and / or nootkatone are described in WO 2016 / 029153, which is incorporated herein by reference.

[0108] In one embodiment, the bacterial cell produces steviol or steviol glycoside (e.g., RebD or RebM). Steviol is produced from kaurene by the action of two P450 enzymes, kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). After steviol production, various steviol glycoside products can be produced through a series of glycosylation reactions, which can occur in vitro or in vivo. The pathways and enzymes for the production of steviol and steviol glycosides are disclosed in documents US 2013 / 0171328, US 2012 / 0107893, WO 2012 / 075030, WO 2014 / 122328, which are incorporated herein by Petition 870260009364, dated 01 / 30 / 2026, page 50 / 187 38 / 80 reference in its entirety. Document WO 2016 / 073740 also discloses enzymes and bacterial host cells for the production of RebM.

[0109] Other biosynthetic pathways for the production of terpene or terpenoid compounds are disclosed in US 8,927,241, which is incorporated herein by reference in its entirety.

[0110] The bacterial strain can be cultivated in batch culture, continuous culture, or semi-continuous culture. In some embodiments, the bacterial strain is cultivated using a fed-batch process comprising a first phase in which bacterial biomass is created, followed by a terpene or terpenoid production phase. Fed-batch culture is a process in which nutrients are fed to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the operation. Generally, a base medium supports the initial cell culture and a feed medium is added to prevent nutrient depletion. The controlled addition of nutrients directly affects the culture growth rate and helps prevent overflow metabolism and the formation of secondary metabolites.

[0111] An exemplary batch medium for the growth of the bacterial strain (biomass production) comprises, without limitation, yeast extract. In some embodiments, carbon substrates, such as C1, C2, C3, C4, C5 and / or C6 carbon substrates, are fed to the culture for the production of the terpene or terpenoid product. In exemplary embodiments, the carbon source is glucose, sucrose, fructose, xylose and / or glycerol. Culture conditions are generally selected from aerobic, microaerobic and anaerobic.

[0112] In some embodiments, the culture is maintained under aerobic or microaerobic conditions. For example, when using a fed-batch process, the biomass production phase may occur under aerobic conditions, followed by a reduction in oxygen levels for the product production phase. For example, the culture may be Petition 870260009364, dated 01 / 30 / 2026, page 51 / 187 39 / 80 changed to microaerobic conditions after about 10 to about 20 hours. In this context, the term “microaerobic conditions” means that the cultures are maintained just below detectable dissolved oxygen. See, Partridge JD, et al., Transition of Escherichia coli from Aerobic to Micro-aerobic Conditions Involves Fast and Slow Reacting Regulatory Components, J. Biol. Chem. 282(15):11230-11237 (2007).

[0113] The production phase includes feeding a nitrogen source and a carbon source. For example, the nitrogen source may comprise ammonium (e.g., ammonium hydroxide). The carbon source may contain C1, C2, C3, C4, C5, and / or C6 carbon sources, such as, in some embodiments, glucose, sucrose, or glycerol. Nitrogen and carbon feeding can be initiated when a predetermined amount of batch media is consumed, a process that provides ease of scaling. In some embodiments, the nitrogen feed rate is from about 8 L per hour to about 20 L per hour, but will depend in part on the product, strain, and scale.

[0114] In several embodiments, the bacterial host cell can be cultured at a temperature between 22°C and 37°C. While commercial biosynthesis in bacteria such as E. coli may be limited by the temperature at which overexpressed and / or foreign enzymes become stable, recombinant enzymes (including terpenoid synthase) can be manipulated to allow cultures to be maintained at higher temperatures, resulting in higher yields and greater overall productivity. In some methods, the crop is grown at approximately 22°C or higher, approximately 23°C or higher, approximately 24°C or higher, approximately 25°C or higher, approximately 26°C or higher, approximately 27°C or higher, approximately 28°C or higher, approximately 29°C or higher, approximately 30°C or higher, approximately 31°C or higher, approximately 32°C or higher, approximately 33°C or higher, approximately 34°C or higher, approximately 35°C or higher, approximately 36°C or higher, or approximately 37°C.In some methods, the culture is maintained at a temperature of 22 to 37 °C, or one. Petition 870260009364, dated 01 / 30 / 2026, p. 52 / 187 40 / 80 temperature of 25 to 37 °C, or a temperature of 27 to 37 °C, or a temperature of 30 to 37 °C.

[0115] In some embodiments, the bacterial strain is grown on a commercial scale. In some embodiments, the culture size is at least about 100 L, or at least about 200 L, or at least about 500 L, or at least about 1,000 L, or at least about 10,000 L, or at least about 100,000 L, or at least about 500,000 L. In some embodiments, the culture is from about 300 L to about 1,000,000 L.

[0116] In several embodiments, the methods also include recovering the terpene or terpenoid product from cell culture or cell lysates. In some embodiments, the culture yields at least about 100 mg / L, at least about 150 mg / L, or at least about 200 mg / L, or at least about 500 mg / L, or at least about 1 g / L, or at least about 5 g / L, or at least about 10 g / L, or at least about 15 g / L of the terpene or terpenoid product.

[0117] In some embodiments, indole production is used as a surrogate marker for terpenoid production and / or indole accumulation in the culture is controlled to increase production. For example, in several embodiments, indole accumulation in the culture is controlled below about 100 mg / L, or below about 75 mg / L, or below about 50 mg / L, or below about 25 mg / L, or below 10 mg / L. Indole accumulation can be controlled by balancing enzyme expression (and in particular, balancing upstream and downstream pathways) and activity using the multivariate modular approach as described in US 8,927,241 (which is incorporated herein by reference). In some embodiments, indole accumulation is controlled by chemical means.

[0118] Other markers for efficient terpene and terpenoid production include the accumulation of DOX or ME in the culture medium. Generally, the bacterial strains described in this document do not accumulate Petition 870260009364, dated 01 / 30 / 2026, p. 53 / 187 41 / 80 large quantities of these chemical species, which accumulate in the crop at less than about 5 g / L, or less than about 4 g / L, or less than about 3 g / L, or less than about 2 g / L, or less than about 1 g / L, or less than about 500 mg / L, or less than 100 mg / L.

[0119] In some embodiments, MEcPP is the predominant MEP metabolite in culture media, although its accumulation is limited by genetic modifications in the bacterial strain, which perform the traction of the downstream MEP carbon to IPP and DMAPP precursors. In several embodiments, MEcPP accumulates in the culture at less than about 30 g / L, or less than about 20 g / L, or less than about 2 g / L, or less than about 1 g / L, or less than about 500 mg / L, or less than about 100 mg / L.

[0120] Optimizing terpene or terpenoid production by manipulating MEP pathway genes, as well as upstream and downstream pathways, is not expected to be a simple linear or additive process. Instead, through combinatorial analysis, optimization is achieved by balancing components of the MEP pathway, as well as upstream and downstream pathways. Indole accumulation (including prenylated indole) and MEP metabolite accumulation (e.g., DOX, ME, MEcPP, HMBPP, farnesol, prenol, and isoprenol) in culture or cells can be used as surrogate markers to guide this process.

[0121] The terpene or terpenoid product can be recovered by any suitable process. Generally, recovery includes separating the material comprising the product from the culture or cells, followed by extraction and purification. For example, recovery may include partitioning the desired product into an organic phase or a hydrophobic phase. Alternatively, the aqueous phase may be recovered, or the entire cell biomass may be recovered, for further processing.

[0122] For example, in some embodiments, the product is a volatile terpene or terpenoid product. In such embodiments, the terpene or terpenoid product can be recovered from an organic phase or Petition 870260009364, dated 01 / 30 / 2026, page 54 / 187 42 / 80 hydrophobic, which is mechanically separated from the culture. Alternatively or in addition, the terpene or terpenoid product is harvested from the liquid and / or solid phase. In some embodiments, the product is purified by sequential extraction and purification. For example, the product may be purified by chromatography-based separation and recovery, such as supercritical fluid chromatography. The product may be purified by distillation, including simple distillation, steam distillation, fractional distillation, film distillation, or continuous distillation.

[0123] In some embodiments, the product is a non-volatile terpene or terpenoid product, which in some embodiments is an extracellular product recovered from the culture medium. Alternatively, the product is an intracellular product recovered from harvested cell material. When the product is poorly soluble, it can be recovered by filtration and, optionally, by solvent extraction (e.g., ethanol extraction). Alternatively, or additionally, the product is recovered by chromatography-based separation, such as liquid chromatography. In some embodiments, the product is purified by sequential extraction and purification. In still other embodiments, the product is crystallized from the solution.

[0124] The production of the desired product can be determined and / or quantified, for example, by gas chromatography (e.g., GC-MS). Product production, recovery, and / or product analysis can be performed as described in US patent 2012 / 0246767, which is incorporated herein by reference in its entirety. For example, in some embodiments, the product oil is extracted from the aqueous reaction medium using an organic solvent, such as an alkane like heptane or dodecane, followed by fractional distillation. In other embodiments, the product oil is extracted from the aqueous reaction medium using a hydrophobic phase, such as a vegetable oil, followed by organic solvent extraction and fractional distillation. The terpene and terpenoid components of the fractions can be quantitatively measured by GC / MS, followed by mixing. Petition 870260009364, dated 01 / 30 / 2026, page 55 / 187 43 / 80 of fractions to generate a desired product profile.

[0125] In several embodiments, the recovered terpene or terpenoid is incorporated into a product (e.g., a consumer or industrial product). For example, the product may be a flavoring product, a fragrance product, a sweetener, a cosmetic, a cleaning product, a detergent or soap, or a pest control product. For example, in some embodiments, the recovered product comprises nootkatone, and the product is a flavoring product selected from a beverage, a chewing gum, a candy, or a flavoring additive, or the product is an insect repellent. In some embodiments, the oxygenated product is steviol or a steviol glycoside (e.g., RebM), which is supplied as a sweetener, or is incorporated into ingredients, flavorings, beverages, or food products.

[0126] The invention further provides methods for manufacturing products such as foods, beverages, texturizers (e.g., starches, fibers, gums, fats and mimetics and emulsifiers), pharmaceuticals, tobacco products, nutraceutical products, oral hygiene products and cosmetic products, incorporating the terpene or terpenoids produced in this document. The higher yields of such species produced in embodiments of the invention can provide significant cost advantages as well as sustainability.

[0127] In other aspects, the invention provides bacterial cells, such as E. coli, possessing one or more genetic modifications that increase the products of IPP and DMAPP precursors. In various embodiments, the bacterial cells produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway and convert the IPP and DMAPP into a terpene or terpenoid product via a downstream synthesis pathway. The downstream synthesis pathway is generally a recombinant pathway and may comprise a prenyltransferase, one or more terpene synthases and, optionally, one or more P450 enzymes and P450 reductase enzymes (by Petition 870260009364, dated 01 / 30 / 2026, page 56 / 187 44 / 80 example, each as described above). For example, the product may be a diterpene or diterpene synthase, with the sequential action of a recombinant Type II diterpene synthase (DiTPS) on GGPP followed by a recombinant Type I DiTPS, or alternatively, a single recombinant synthase performs both steps.

[0128] In addition, to improve the MEP carbon available for product biosynthesis, the bacterial strain has one or more of the following genetic modifications:

[0129] (a) overexpression of IspG and IspH enzymes, with IspG and IspH enzymes having balanced expression to avoid the accumulation of HMBPP intermediates,

[0130] (b) recombinant or modified gene encoding an enzyme that enhances electron supply and / or transfer via the MEP pathway and / or to terpene or terpenoid products, which is optionally an overexpression of a YdbK gene and optionally with a non-native fdx and / or fldA homolog,

[0131] (c) inactivation or deletion, or reduction in the expression or activity, of aceE or aceE enzyme complex and, optionally

[0132] (d) recombinant or modified idi gene to adjust activity for increased production of terpene or terpenoids.

[0133] Genes can be overloaded by complementation with recombinant genes, or endogenous genes can be modified to alter expression, as revealed elsewhere in this document.

[0134] A bacterial strain is a bacterium selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. For example, a bacterial strain is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter Petition 870260009364, dated 01 / 30 / 2026, p. 57 / 187 45 / 80 sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterial strain is E. coli.

[0135] In several embodiments, after cultivation, HMBPP does not accumulate to more than about 10 mg / g DCW, or in some embodiments it does not accumulate to more than about 8 mg / g DCW, or in some embodiments it does not accumulate to more than about 5 mg / g DCW, or in some embodiments it does not accumulate to more than about 4 mg / g DCW, or in some embodiments it does not accumulate to more than about 2 mg / g DCW. In some embodiments, HMBPP does not accumulate to more than about 1 mg / g DCW, or it does not accumulate to more than about 0.5 mg / g DCW, or more than about 0.2 mg / g DCW, or more than about 0.1 mg / g DCW.

[0136] In some embodiments, the bacterial strain expresses dxs, ispD, ispF, and idi as recombinant genes (e.g., as a complement to wild-type MEP pathway enzymes) and which are optionally expressed as an operon. In some embodiments, the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which are optionally expressed as 1, 2, or 3 individual operons. The recombinant MEP pathway genes are expressed from one or more plasmids or are integrated into the chromosome, and the expressions are balanced to enhance MEP carbon flux. Specifically, the bacterial cell can produce MEcPP as the predominant MEP metabolite in the extracellular medium.

[0137] Recombinant IspG and IspH genes may comprise one or more beneficial mutations, or may be an IspG or IspH ortholog possessing enhanced properties or activity, as described in this document. Furthermore, in several embodiments, the expression of recombinant IspH is superior to the expression of recombinant IspG, which can optionally be achieved, at least in part, by positioning IspH before IspG in an operon. Thus, the bacterial strain may express both IspH and IspG at Petition 870260009364, dated 01 / 30 / 2026, p. 58 / 187 46 / 80 of the same operon (with ispH positioned first) and under the control of a strong promoter. The recombinant IspG and IspH genes are expressed from a plasmid or are integrated into the chromosome.

[0138] In some embodiments, the bacterial strain expresses a recombinant idi gene, which is tuned to increase the product, optionally modifying the promoter strength, gene copy number, position in an operon, or ribosome binding site.

[0139] In some embodiments, the bacterial strain expresses a recombinant YdbK gene, which is integrated into the chromosome or expressed from a plasmid. The bacterial strain may also comprise an overexpression of one or more of a flavodoxin, flavodoxin reductase, ferredoxin, and ferredoxin reductase, such as Clostridium pasteurianum ferredoxin (Cp.fdx). In some embodiments, the strain expresses one or more non-native fdx and / or fldA homologs. For example, the fdx homolog may be selected from Hm.fdx1 (Heliobacterium modesticaldum), Pa.fdx (Pseudomonas aeruginosa), Cv.fdx (Allochromatium vinosum), Ca.fdx (Clostridium acetobutylicum), Cp.fdx (Clostridium pasteurianum). ), Ev2.fdx (Ectothiorhodospira shaposhnikovii), Pp1.fdx (Pseudomonas putida) and Pp2.fdx (Pseudomonas putida). In some embodiments, the fldA homolog includes one or more selected from Ec.fldA (E. coli), Ac.fldA2 (Azotobacter chromeococcum), Av.fldA2 (Azotobacter vinelandii) and Bs.fldA (B.subtle).

[0140] In some embodiments, fdx homologs comprise a sequence that is at least 60% identical to any of the SEQ ID Nos. 10 and 15-24. For example, non-native fdx homologs may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID Nos. 10 and 15-24. Petition 870260009364, dated 01 / 30 / 2026, p. 59 / 187 47 / 80

[0141] In some embodiments, fldA homologs comprise a sequence that is at least 60% identical to any of the SEQ ID Nos. 25-28. For example, non-native fldA homologs may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID Nos. 25-28.

[0142] In some embodiments, the bacterial strain has overexpression or complementation with one or more PFOR and / or fpr and, optionally, one or more of a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), and ferredoxin reductase. As an example, in some embodiments, the bacterial strain includes Ec.ydhV (E. coli) (SEQ ID NO: 33) and Ec.ydhY (E. coli) (SEQ ID NO: 34); Ec.ydbK (E. coli) (SEQ ID NO: 9) and Cp.fdx (Clostridium pasteurianum) (SEQ ID NO: 10); Ec.fpr (E. coli) (SEQ ID NO: 38) and Ec.fdx (E. coli) (SEQ ID NO: 21); or Ec.fpr (E. Coli) (SEQ ID NO: 38) and Ec.fldA (E. coli) (SEQ ID NO: 27).

[0143] In some embodiments, the bacterial strain has overexpression or complementation with one or more PFORs, or a homolog thereof. For example, in some embodiments, the PFOR is selected from YdbK (SEQ ID NO: 9), Scy.pfor (Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor (Kluyvera intermedia) (ID SEQ NO: 30), Da.pfor (Desulfovibrio africanus) (ID SEQ NO: 31), Ns.pfor (Nostoc sp.) (SEQ ID NO: 32), Ec.ydV (E. coli) (SEQ ID NO: 33), Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35) and Sco.pfor (Synechococcus sp.). In some embodiments, the PFOR is YdbK.

[0144] In some embodiments, fldA homologs comprise a sequence that is at least 60% identical to any of the SEQ ID Nos. 29-35. For example, PFOR may comprise a sequence that is at least about 60%, at least about 70%, at least Petition 870260009364, dated 01 / 30 / 2026, p. 60 / 187 48 / 80 less approximately 80%, at least approximately 90%, at least approximately 95%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical to any of the SEQ ID Nos. 29-35.

[0145] In some embodiments, overexpression or complementation with PFOR such as, for example, YdbK, can result in improved performance through the expression of electron carriers with a redox potential of about 400 to 550 mV, or in some embodiments, in the range of about 400 to 500 mV, or in the range of about 400 to 475 mV. In some embodiments, the electron carrier is ferredoxin, flavodoxin, or NADPH. As an example, in some embodiments, the electron carrier is Cv.fdx (Allochromatium vinosum).

[0146] In some embodiments, the bacterial strain has overexpression or complementation with one or more fpr homologs. As an example, in some embodiments, the fpr homolog is selected from Ns.fpr (Nostoc sp.) (SEQ ID NO: 36), Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37) and Ec.fpr (E. coli) (SEQ ID NO: 38).

[0147] In some embodiments, fldA homologues comprise a sequence that is at least 60% identical to any of the SEQ ID Nos. 36-38. For example, fpr may comprise a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the SEQ ID Nos: 36-38.

[0148] In some embodiments, E. coli contains a deletion of one or more genes selected from: pgrR, mppA, ynaI, insH-4, ynaJ, uspE, fnr, ogt, abgT, abgB, abgA, abgR, mcaS, isrA, smrA, ydaM, ydaN, fnrS, C0343, dbpA, REP115, ttcA, intR, ydaQ, ydaC, ralA, ralR, recT, recE, racC, ydaE and kilR.

[0149] The expression of recombinant pgpB and / or nudB can be adjusted to provide a higher product titer, Petition 870260009364, dated 01 / 30 / 2026, p. 61 / 187 49 / 80 optionally varying the promoter strength, gene copy number, position in an operon and / or ribosome binding site. In some embodiments, recombinant pgpB and / or nudB is expressed under the control of a weak or intermediate strength promoter. Recombinant pgpB or nudB is integrated into the chromosome or expressed from a plasmid.

[0150] In various embodiments, the bacterial strain produces a terpene or terpenoid product comprising at least one of Amorfadiene, Artemisinic Acid, Artemisinin, Bisabolol, Bisabolene, alpha-Sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Farnesene, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Rose Oxide, Sabinene, Steviol, Steviol Glycoside (including Rebaudioside D or Rebaudioside M), Taxadiene, Thymol and Valencene.

[0151] Aspects and embodiments of the invention are further demonstrated below with reference to the following Examples. EXAMPLES Example 1: Adjustment of IspG / IspH Expression Conclusions

[0152] Overexpression and balancing of MEP pathway genes can result in more carbon entering the MEP pathway and can change this carbon 'downstream' of DOXP and MEP to MEcPP. Modifying the expression of ispG and / or ispH can convert even more MEcPP into HMBPP in IPP.

[0153] Increased expression of IspG and IspH significantly increases the titrations of terpene and terpenoid products. However, increasing the expression of only ispG or ispH alone did not improve the titration. Overexpression of ispG alone resulted in growth defects, and overexpression of ispH alone did not significantly improve the titration but converted HMBPP to IPP. The effects of ispG overexpression may be related to the observation that the Petition 870260009364, dated 01 / 30 / 2026, p. 62 / 187 50 / 80 HMBPP is not found extracellularly, but is found 100% intracellularly. Since the molecule does not appear to be transported out of the cell, it may act as a feedback molecule, providing a firm halt to the MEP pathway. For example, if the HMBPP pool gets above a certain size, the pathway will be blocked. Alternatively, or additionally, HMBPP may be toxic at certain levels, which is consistent with the observed impact of IspG overexpression on cell growth.

[0154] Thus, the balance of activity between ispG and ispH is important to prevent imbalance and accumulation of HMBPP. In some situations, less is more; that is, stronger overexpression of MEP genes may begin to impair productivity.

[0155] In summary, overexpressing ispG and ispH together, when one or both ispG or ispH are wild-type or mutated / manipulated, in a suitably balanced configuration, prevents the accumulation of HMBPP from becoming toxic and pushing carbon through the MEP pathway to IPP, DMAPP, and downstream terpene and terpenoid products. Description of Experimental Results

[0156] Figure 2 shows that increased expression of ispH alone or ispH and ispG together enhances terpenoid product titrations in strains engineered to increase the amount of carbon entering the MEP pathway, however ispG alone decreases productivity. In this example, the control strain is an E. coli strain with balanced overexpression of MEP pathway genes (but no additional copies of ispG or ispH), increasing the amount of carbon entering the MEP pathway. As shown, IspG overexpression decreases product titration by about 15%, while IspH overexpression at the same expression strength increases product titration by 17%. IspG and IspH overexpression more than triples product titration.

[0157] The data also show that a ratio of Petition 870260009364, dated 01 / 30 / 2026, page 63 / 187 51 / 80 The IspG / IspH ratio, which favors the H enzyme more, further improves flux through the MEP pathway compared to a strain that favors the IspG side of the ratio. IspG and ispH are expressed here in operon format, and therefore the second gene in the operon will have a lower expression level than the first. Thus, the ispH / ispG operon showed significantly more product titrations than ispG / ispH.

[0158] IspG and IspH function sequentially to convert MEcPP to HMBPP and then to IPP. Increased ispG will accumulate a larger HMBPP pool, while increased ispH will reduce the HMBPP pool as it is converted to IPP. The fact that IspG alone decreases productivity, while ispH alone increases it, strongly suggests that HMBPP accumulation has a negative feedback effect on the MEP pathway. When both IspG and IspH are overexpressed, we increase the rate of HMBPP formation and consumption, which significantly improves flux through the MEP pathway to the target terpenoid. However, even in this enhanced flux regime, the balance of IspG to IspH is critical, since a slight favoring of ispH over ispG can further improve productivity by 25%, to almost 4x the titers of a parental strain with wild-type expression of both IspG and IspH.

[0159] The increased expression of IspG and / or IspH in production strains modified with enhanced MEP pathways impacts the distribution pattern of the MEP product (Figure 3). Most products are DOX, ME, and MEcPP (Figure 3, top panel), followed by DOXP and MEP (Figure 3, middle panel) and HMBPP (Figure 3, bottom panel). In these panels, total extracellular and intracellular metabolites are shown from extracted cultures (broth plus cells), such that the reported concentration is relative to the extract volume.

[0160] Increasing IspG or IspH alone increases the MEcPP conversion rate and decreases the pool size (top panel), although IspH increases the product titration and IspG loses the titration of Petition 870260009364, dated 01 / 30 / 2026, page 64 / 187 52 / 80 product. However, a significant difference between the two variants is apparent with the concentration of HMBPP (lower panel), which IspG alone increases 2.5x compared to the control, while IspH alone decreases by 20%. This accumulation of HMBPP could be fed back into the MEP pathway and interrupting the flux enhancement. HMBPP accumulates at very low levels (nM concentration) and 100% of it is found intracellularly.

[0161] It can be seen that increasing the expression of ispG and ispH together, in any of the operon orders, allows the complete conversion of the remaining DOX and decreases the size of the ME pool. Furthermore, an IspG / IspH ratio that favors more IspH is able to improve MEcPP conversion (and improved product titration) compared to a strain that favors IspG.

[0162] The proportion of each individual MEP metabolite found inside or outside the cell ('Intra' vs 'Extra') is shown in Figure 4. These values ​​do not reflect absolute abundance, as Figure 3 shows, there is much more DOX in total than there is HMBPP. While DOX is 100% extracellular, HMBPP is 100% intracellular. The strain shown here is the high-performance 'Control + ispH / ispG' strain from Figures 2 and 3. DOXP / DOX, MEP / ME, and MEcPP accumulate almost entirely, if not entirely, in the extracellular medium, while CDP-ME, CDP-MEP, HMBPP, IPP / DMAPP, and FPP are observed 100% in the intracellular medium. The percentage of each metabolite found intracellularly is shown at the top of the graph.

[0163] Uncompensated upregulation of ispG causes a significant drop in cell growth, as determined by UV absorption at 600 nm (Figure 5). While some changes in final cell density are observed in a strain compensated with ispH or ispH and ispG together, the variation is not significant.

[0164] To determine HMBPP accumulation, HMBPP can be expressed in terms of cell dry weight (DCW). For example, using a strain with balanced ispGH expression: Petition 870260009364, dated 01 / 30 / 2026, p. 65 / 187 53 / 80 [HMBPP] = 0.42 ug / mL in 0.35 mL of sampled culture [OD600] = 12.69 Assumption: 1 OD600 = 0.4 g-DCW / L = 0.4 mg-DCW / mL HMBPP yield = [(0.42 ug / mL) * (0.35 mL)] / [(12.69 * 0.4 mg-DCW / mL) * 0.35 mL)]

[0165] In this example, HMBPP = 0.0827 ug / mg DCW or 0.0827 mg / g DCW. Example 2: Overexpression of pgpB and nudB Conclusions

[0166] Installing an alternative 'product' pull by overexpressing genes like pgpB and nudB could achieve even more flow pull through the MEP pathway (albeit for non-target products), or it could even replace the various downstream terpenoid pathways to create a tool for designing a 'universal chassis' (i.e., a strain that can have any downstream terpenoid transformed into it and be rapidly optimized for commercial production).

[0167] More specifically, carbon can undergo transport via the MEP pathway to create alternative products that will cluster outside the cell. PgpB dephosphorylates FPP into farsenol (FOH), and NudB dephosphorylates IPP and DMAPP into isoprenol (3-methyl-3-buten-1-ol) and prenol (3-methyl-2-buten-1-ol), respectively. Improving the transport of these products out of the cell prevents the accumulation of IPP, DMAPP, and FPP; which, like HMBPP, can provide feedback and exert control over the MEP pathway. IPP inhibits growth, and feedback inhibits Dxs. See Cordoba, Salmi & Leon (2009) J. Exp. Bot. 60, 10, 2933-2943. FPP feedback inhibits the ispF-MEP complex, which is formed when MEP binds and increases IspF activity in an anticipatory manner. Bitok & Meyers (2012) ACS Chem. Biol. 2012, 7, 1702-1710. These products accumulate outside the cell and, as intermediates in the MEP pathway, can be used to track C flux through the MEP pathway via LC / MS metabolomic quantification. Petition 870260009364, dated 01 / 30 / 2026, page 66 / 187 54 / 80

[0168] By constitutively expressing an additional copy of pgpB, carbon flow through the MEP pathway can be improved, and a slow-growing phenotype enhanced in a strain that has overexpressed MEP genes but no additional downstream pathway to carry all the carbon to the product. In fact, the downstream 'pull' becomes the conversion of FPP to farnesol, which is exported out of the cell. Similarly, constitutive expression of nudB should result in pools of IPP and DMAPP being increasingly redirected to extracellular isoprenol and prenol products.

[0169] Further modulation of MEP pathway gene expression levels in the presence of overexpressed pgpB or nudB can significantly impact MEP flux and carbon distribution through the pathway. Increases or decreases in farnesol, prenol, or isoprenol products can be inversely correlated with MEcPP levels. Description of Experimental Results

[0170] Overexpression of PgpB can triple farnesol titers in strains engineered to improve flux through the MEP pathway, but without an established downstream terpenoid product pathway (Figure 6). The control strain has additional copies of dxs, dxr, ispD, ispF, ispE, ispG, ispH, and idi under various levels of constitutive expression, and also has YdbK overexpression (Example 4). The strain accumulates moderate amounts of farnesol, presumably as an 'overflow' due to excess FPP accumulation, which feeds back into the pathway and suffers from noticeably slower growth compared to the wild type. When pgpB is overexpressed in this strain, the excess FPP is more efficiently converted to farnesol (preventing feedback control) and flux effectively gains traction through the MEP pathway.

[0171] However, excess PgpB expression (the '+++' condition) appears to negatively impact total flux through farnesol, with lower titration and lower modulation observed, on average. Petition 870260009364, dated 01 / 30 / 2026, page 67 / 187 55 / 80 Some possible reasons for this result include: (1) an overly strong pull of PgpB is overloading the capacity of the MEP pathway to keep up with the demand for FPPs, especially from necessary competing products; or (2) since PgpB is known to dephosphorylate multiple targets in vivo, including an essential membrane phospholipid, a high level of expression for PgpB could have unintended negative consequences on cellular health.

[0172] Increasing and tuning the expression of IspG' and / or IspH in a farnesol-producing strain can improve product titration (Figure 7). In this example, the IspG' enzyme is a manipulated version with higher activity than the wild type. The control strain has additional copies of dxs, dxr, ispD, ispF, ispE, ispG, ispH, and idi, as well as additional copies of ydbK and pgpB. Additional copies of ispH and / or ispG' are integrated into the strains under increasing promoter strength (+, ++, +++).

[0173] When IspH is overexpressed (Figure 7, Panel A), no significant change in product titration is observed. Although the increased amount of IspH improves the conversion of HMBPP to IPP, there is no additional IspG' to provide this additional HMBPP. The MEcPP pool mediated by IspG activity becomes the rate-limiting step in the pathway. However, when IspG' is overexpressed in addition to IspH (Figure 7, Panel B), we see a significant increase in farnesol product titration. In this situation, the additional HMBPP enabled by the additional copy of IspG' is rapidly converted by the increased IspH level to IPP, preventing HMBPP from accumulating and feeding back into the pathway.

[0174] Clearly, the balance between IspG and IspH is critical. The data show that IspG / H is expressed in operon format, so the second gene in the operon will have a lower expression level than the first. When the gene order in the operon for ispG' and ispH is switched (i.e., ispH + ispG' versus ispG' + ispH), thus altering the expression rate of IspG' / IspH, we see opposite trends in the data. When the Petition 870260009364, dated 01 / 30 / 2026, p. 68 / 187 56 / 80 ratio favors IspH over IspG' (B), an increase in promoter strength results in a steady increase in product titration. However, when the ratio favors IspG' over IspH (C), the excess HMBPP that can be created by this unbalanced pathway accumulates continuously as promoter strength increases, resulting in less and less product improvement and slower growth.

[0175] The increase in farnesol product titration can be accompanied by a decrease in the size of the MEcPP pool, although it depends on the IspG and IspH ratio (Figure 8). As seen in Figure 7, additional copies of IspG' and IspH in farnesol-producing strains can improve farnesol product titration by up to 2.5 times. When only IspH is upregulated without additional IspG (Figure 8, Panel A), the titration did not change significantly, nor did MEcPP. MEcPP decreases moderately, probably due to MEcPP undergoing downstream pull as HMBPP is more efficiently converted to IPP by the additional IspH enzyme. When relatively more IspH was expressed than IspG' (Figure 8, Panel B), as the promoter strength increases, the farnesol product titration increased. MEcPP, in turn, decreases as it is consumed by a balanced pathway that distributes flux to the desired end product.

[0176] However, although a non-ideal ratio favoring IspG' over IspH may improve the conversion of MEcPP from HMBPP to IPP and improve the titration of the farnesol product, eventually the imbalance is too severe for the E. coli strain to tolerate and the product improvement disappears, while more MEcPP accumulates and becomes trapped in the intermediate carbon pool of the MEP pathway. Example 3: Idi Expression Adjustment Conclusions

[0177] The Idi enzyme catalyzes the reversible isomerization of IPP to DMAPP. Once each desired terpenoid product or Petition 870260009364, dated 01 / 30 / 2026, p. 69 / 187 57 / 80 unwanted MEP byproduct (e.g., UPP) uses DMAPP and various IPP numbers; the ratio between the two precursors can have an effect on strain productivity. For example, 1 FPP = 1 DMAPP + 2 IPP, while 1 UPP = 1 FPP + 8 IPP (or 1 DMAPP + 10 IPP). Therefore, an ideal ratio for FPPS to produce FPP is 2:1 IPP:DMAPP, but 10:1 for UPP. Thus, varying the IPP:DMAPP ratio by varying Idi expression can impact the production of desired terpenoids relative to other unwanted byproducts from the MEP pathway. Description of Experimental Results

[0178] Idi was supplemented in different strains producing product A or B. Cells were cultured in 96-well round culture plates at 37°C for 48 hours at 280 RPM in a customized medium with glucose as a carbon source. Idi was expressed from a pBAC under an IPTG-inducible promoter. Strain 1 already has overexpression of dxs, dxr, ispD, ispF, ispE, idi, FPPS, and YdbK, while strains 2 and 3 also have ispH and a mutant version of IspG is overexpressed. On the other hand, strain 4 has the same enzymes overexpressed, but under a very different expression regime.

[0179] While Idi overexpression slightly increases product titration in a strain that does not overexpress IspGH, it decreases titration in two strains that do, indicating that the balance between IPP and Idi-controlled DMAPP can be adjusted up or down depending on the needs of the downstream pathway (Figure 9). However, the titration of Strain 4 more than doubles with idi complementation. The same genes are overexpressed in this strain, but the balance between the expression of the MEP genes is very different. Example 4: Superexpression of YdbK Conclusions

[0180] YdbK is predicted to function as a pyruvate:flavodoxin oxidoreductase and / or pyruvate synthase. It is believed that the Petition 870260009364, dated 01 / 30 / 2026, p. 70 / 187 58 / 80 oxidoreductase oxidizes pyruvate to acetyl-CoA, reducing ferredoxin, which can then provide electrons to the MEP pathway, especially to support the strongly positively regulated IspG and IspH enzymes containing Fe-S clusters. Overexpression of YdbK has been shown to produce hydrogen (H2) (Akhtar MK & Jones PR (2014), Cofactor engineering for enhancing the flux of metabolic pathways. Frontiers in Bioeng. and Biotech.), but not terpenoid production.

[0181] The product titration of Terpene Product A doubled in these strains. The Fe-S clusters are better supported by the extra YdbK cofactor, and their activity improves. The product titration rises, and when the MEP metabolites are profiled, we see an increased conversion of MEcPP, similar to what is observed when the control strain adds one more copy of the ispH-ispG' operon.

[0182] On the other hand, when a Product B strain that did not have IspG / H overexpressed relative to WT was supplemented with YdbK, the Product B titration decreased. When IspG / H was increased in this strain, YdbK supplementation improved the Product B titration, suggesting that YdbK expression must be carefully balanced with IspG / H expression (which, in turn, must be carefully balanced for the H / G ratio).

[0183] In addition, extra transport or electron transfer cofactors were added in addition to YdbK overexpression to see if we can further improve the titrations. In some experiments, YdbK plus fdx (ferredoxin) from Clostridium pasteurianum slightly improved productivity. Description of Experimental Results

[0184] An additional copy of the E. coli YdbK gene is integrated into the chromosome or expressed on a plasmid (specifically a single-copy pBAC or multi-copy plasmids), under the control of constitutive or inducible promoters. Additionally, copies of Petition 870260009364, dated 01 / 30 / 2026, p. 71 / 187 Native or non-native recombinant electron acceptors / donors (59 / 80) can also be overexpressed with YdbK, to capitalize on and more efficiently utilize the additional electrons made available for biosynthesis.

[0185] Expression of an additional copy of YdbK under increased promoter strength can enhance terpenoid production. In this example, the control strain produces terpenoid product A and has additional copies of the dxs, dxr, ispD, ispE, ispF, ispG', ispH, and idi genes of the MEP pathway under defined constitutive expression.

[0186] In this strain, adding an extra copy of ispH and ispG' in operon format (such that the H / G' ratio favors H) further increases the Product A titration, indicating that these steps are limiting (Figure 11, Panel A). Increasing these genes containing Fe-S clusters clearly increases MEcPP conversion and decreases the concentration observed in culture (Figure 11, Panel C).

[0187] When YdbK is supplemented in the control strain, we see a gradual response to upregulation, where increasing expression increases terpenoid production, up to a point – the shift to stronger expression results in 50% less Product A in the +++ YdbK strain (Figure 11, Panel B). We see the same MEcPP conversion occurring for these strains (Figure 11, Panel D), suggesting that YdbK is supporting enhanced IspG and / or IspH activity. It is noteworthy that the MEP metabolite profile of the ++ vs +++ strains does not change significantly, but has ~3x less product titration, suggesting that some kind of feedback mechanism has been activated. Given the observations from the work with IspG and IspH, it is possible that this feedback is due to HMBPP accumulation.

[0188] The improvement in terpenoid product titration from increased YdbK expression requires sufficient IspG and / or IspH to manifest (Figure 11). In this example, Control A has additional copies of dxs, ispD, ispF, and idi from the MEP pathway, as well as deletion of rhyB and Petition 870260009364, dated 01 / 30 / 2026, p. 72 / 187 60 / 80 operon changes. Control B is Control A plus an additional integrated copy of ispG' and ispH in the operon configuration (G' first, so the H / G ratio favors G), while Control C is Control A plus an additional integrated copy of ispH and ispG' in the operon configuration (H first, so the H / G ratio favors H).

[0189] In Panel A, we see that YdbK supplementation in the absence of IspG / H upregulation decreases the titration of Terpenoid Product B by about 25%. However, when you supplement YdbK in strains with additional copies of ispG'-ispH or ispHispG', we observe an 18% and 27% improvement in terpenoid titrations. Clearly, IspG and IspH must be overexpressed relative to the MEP WT pathway for the benefit of YdbK to be seen.

[0190] Furthermore, these data again highlight the importance of the balance of expression between IspG and IspH for MEP pathway flux and terpenoid productivity. In control B versus C, the same enzymes are upregulated under the same promoter strength – the difference lies in the order of the genes in the operon. Genes closer to the promoter will be expressed more strongly than subsequent genes in the operon, such that the H / G ratio of the enzymes favors IspG in Control B or IspH in Control C. Given this, we observe that a ratio favoring H further improves the titration than one favoring G. In addition, the improvement made possible by YdbK is increased in a strain favoring H. Thus, the balance between IspH and IspG is very important for strain productivity.

[0191] The expression of fdx in addition to YdbK can further enhance terpenoid titrations (Figure 12). In this example, the control strain produces the terpenoid product A and has additional copies of the dxs, dxr, ispD, ispE, ispF, ispG', ispH, and idi genes of the MEP pathway under defined constitutive expression.

[0192] As shown in Figure 10, the expression of Petition 870260009364, dated 01 / 30 / 2026, p. 73 / 187 61 / 80 an additional copy of YdbK under constitutive expression increases the production of Product A. Complementation was attempted with three other electron acceptor / donor molecules, fldA, fldA and erpA (each from E. coli), or fdx from Clostridium pasteurianum (which can support 4Fe-4S clusters, as they are found in IspG and IspH, instead of 2Fe-2S clusters).

[0193] Adding another copy of fldA (flavodoxin) or fldA and erpA (essential respiratory protein A) in addition to YdbK did not further improve the Product A titration, but adding Clostridium pasteurianum fdx did improve Product A titrations. Interestingly, while the addition of YdbK results in total downstream conversion of DOX / DOXP to ME / MEP, adding further fldA causes some carbon to accumulate upstream in the MEP pathway as DOX / DOXP. Adding erpA to the mix restores the profile. However, the MEP metabolite profile for the additional ydbK+fdx strain is more similar to ydbK+fldA, suggesting that optimal MEP flux will result from the coordinated balancing of MEP pathway gene expression as well as critical electron donor / acceptor expression. Example 5: Reducing the PDH conversion of pyruvate to acetyl-CoA increases the YdbK conversion of pyruvate to acetyl-CoA.

[0194] The increased dependence of YdbK for the conversion of pyruvate to acetyl-CoA may improve the production of terpene and / or terpenoid products by the manipulated microbial strain, since YdbK has a lower redox potential (higher absolute number in Table 4) than the hydroquinone / semiquinone pair of FMN in fldA. Thus, YdbK is the preferred electron source (not fpr / NADPH) for IspG and IspH.

[0195] Iron-sulfur clusters (e.g., Fe4S4) in enzymes (such as IspG and IspH) utilize a wide range of reduction potentials, e.g., -200 to -800 mV. Blachly et al., Inorganic Chemistry, 54(13): 6439-6461 (2015).

[0196] The reduction potentials for charging the electron carriers YdbK and fpr are shown in Tables 2 and 3, Petition 870260009364, dated 01 / 30 / 2026, p. 74 / 187 62 / 80 respectively, and the reduction potentials for discharging electron carriers (e.g., YdbK and fpr) for IspG and IspH are given in Table 4. See McIver, et al., FEBS J, 257(3):577-85 (1998) and Lupton, et al., J Bacteriol, 159:843-9 (1984). TABLE 2: Electron Carrier Charge YdbK ε° (mV) AG° (pyruvate kcal / mol) Oxidation half-reaction of YdbK: pyruvate + CoA → acetyl-CoA + CO2 + 2H+ + 2e- 540 (D. africanus) Reduction half-reactions of potential electron carriers: 2 fldA oxidized + 2e- + 2H+ → 2 semiquinone fldA -254 -13.2 2fdx 2Fe(lll)2+ + 2e- 2 fdx Fe(lll)2 + Fe(ll)1+ -380 -7.4 2 semiquinone fldA + 2e- + 2H+ → 2 hydroquinone fldA -433 -4.9 TABLE 3: Electron Carrier Charging Potential (e°) ε° (mV) ΔG° (kcal / mol NADPH) Step 1 NADPH oxidation half-reaction: NADPH → NADP+ + 2e- + H+ 320 (370 for NADPH / NADP+ = 60) Potential reduction half-reactions: 2 oxidized e- + 2e- + 2H+ → 2 semiquinone e- + -308 -0.6 Petition 870260009364, dated 01 / 30 / 2026, page 75 / 187 63 / 80 2 semiquinone fpr + 2e~ + 2H+—> 2 hydroquinone fpr -268 -2.4 Step 2 Half-reaction of potential fpr oxidation: semiquinone fpr → oxidized fpr + e- + H+ 308 hydroquinone fpr → semiquinone fpr + e- + H+ 268 Half-reactions of potential electron carrier reduction: oxidized fldA + e- + H+ → semiquinone fldA -254 -2.5 or 0.6 TABLE 4: Discharging Electron Carriers for IspG and IspH Potential half-reactions: oxidation of carriers ε° (mV) Source 2 semiquinone fldA 2 oxidized fldA + 2e- + 2H+ 254 Fpr, YdbK 2 fdx Fe(lll)2+Fe(ll)1+ 2fdx2Fe(lll)2+ + 2e- 380 YdbK 2 hydroquinone fldA 2H+ —>2 semiquinone fldA + 2e- + 433 YdbK

[0197] The optimal activity of IspG was tested in vitro using a range of redox dyes. Xiao, et al., Biochemistry, 48(44):10483-10485 (2009). The optimal activity of IspG was tested with externally fed methyl viologen (ε° = 446 mV). The activity of IspG using fed methyl viologen (ε° = 446 mV) was 20x greater than an fpr-fldA system in vitro.

[0198] IspH activity was 50x higher with methyl viologen (ε° = 446 mV) and 100x higher with externally fed dithionite-MDQ (ε° = 490 mV). Xiao et al., Journal of the American Chemical Petition 870260009364, dated 01 / 30 / 2026, page 76 / 187 64 / 80 Society, 131(29): 9931-9933 (2009).

[0199] It is hypothesized that the semiquinone / hydroquinone pair of fldA, which is accessible by YdbK but not fpr, is the preferred in vivo reduction system for IspG and IspH.

[0200] In order to increase the dependence of a microbial strain on PFOR-mediated conversion (e.g., YdbK) of pyruvate to acetyl-CoA, PDH-mediated conversion of pyruvate to acetyl-CoA was reduced. See Figure 15.

[0201] There are three known reactions in E. coli to convert pyruvate (PYR) to acetyl-CoA (AcCoA): pflB, PDH and PFOR or YdbK.

[0202] Of the three enzymes, PDH predominates and is a multienzyme complex (aceE-aceF-lpd), consisting of 24 pyruvate dehydrogenase (aceE) subunits, 24 lipoate acetyltransferase (aceF) subunits, and 12 dihydrolipoate dehydrogenase (lpd) subunits. The net reaction of the PDH system, in addition to reducing NAD+, is the conversion of pyruvate to AcCoA and CO2, a key reaction of central metabolism because it links glycolysis I, which generates pyruvate, to the TCA cycle, in which AcCoA flows. During aerobic growth, PDH is an essential source of AcCoA to fuel the TCA cycle and thus to meet cellular requirements for the precursor metabolites it forms. Mutant strains defective in the PDH complex require an exogenous source of acetate to meet this requirement.

[0203] pflB is only active under anaerobic conditions. Therefore, it is not a primary reaction to convert PYR to AcCoA under aerobic and microaerobic conditions.

[0204] In microbial strains with at least YdbK overexpression, PDH (see, for example, Example 4) is no longer essential, as YdbK can be used to provide AcCoA. To ensure that the PYR step for AcCoA is primarily catalyzed by YdbK, which in turn provides electrons to IspG and IspH, PDH activity has been reduced or Petition 870260009364, dated 01 / 30 / 2026, page 77 / 187 65 / 80 eliminated through gene knockouts or knockdowns (e.g., by mutation).

[0205] Elimination of PDH via knockout of aceE

[0206] Four different strains of E. coli manipulated to produce four different terpenoid products (designated as Product B, Product C, Product D, and Product E) were further manipulated to eliminate aceE (AaceE), which eliminated PDH activity. The control strains were the same, but without the aceE knockout.

[0207] The data show an increase in the titration of each of the four terpenoid products through the exclusion of aceE compared to the control. See Figures 16A-D. The differences in modulation improvement can be largely attributed to the biochemical characteristics (e.g., Km and kcat) of the different terpenoid synthase enzymes employed for the downstream pathway. Specifically, enzymes with lower synthase activity compared to Product D synthase (the most catalytically efficient) had slightly smaller modulation improvements, presumably due to the accumulation of the substrate FPP, which can accumulate and lead to cellular toxicity or feedback regulation on upstream components of the MEP pathway.

[0208] The data also show a reduction in MEcPP concentrations in the extracellular broth (Figure 16E) compared to the control, which confirms that carbon flux was pushed through the IspG / H steps to the product. See Figure 1. Knockdown of PDH via mutated aceE

[0209] Three strains of E. coli, each of which was manipulated to produce three different terpenoid products (shown as Product B, Product C, and Product D), were further manipulated to express a mutated aceE (G267C; aceE mut), which resulted in reduced PDH activity. The control strains were the same, but lacked a Petition 870260009364, dated 01 / 30 / 2026, p. 78 / 187 66 / 80 aceE mutated.

[0210] Similar to the aceE knockout results, the data show an increase in the titer of each of the three terpenoid products in microbial strains expressing mutated aceE compared to the control. See Figures 17A-C.

[0211] The data also show a reduction in MEcPP concentrations in the extracellular broth (Figure 17D) compared to the control, which confirms that carbon flux was pushed through the IspG / H steps to the product. See Figure 1. Example 6: Non-native electron acceptors / donors increase the production of YdbK-dependent isoprenoids.

[0212] When YdbK was overexpressed in E. coli, native ferredoxin (fdx) or flavodoxin (fldA) transported electrons to IspG and IspH (PYR / YdbK / fldA or fdx). E. coli manipulated to produce Product B and overexpress YdbK was further manipulated to overexpress one of the following fdx homologs in Table 5 or fldA homologs in Table 6. The first seven fdx homologs are 2[4Fe-4S]ferredoxins, i.e., they contain two 4Fe-4S iron-sulfur clusters that may have the same or different redox potentials. For ferredoxin, where the clusters differ in redox potential, given the redox potential of YdbK, we predict that in most cases cluster 1 will be the relevant cluster. The remaining fdx homologs are a high-potency 2Fe-2S ferredoxin and a high-potency 4Fe-4S ferredoxin, both containing a single cluster. The control E. coli did not express any fdx or fldA homologs. TABLE 5: Fdx Homologues fdx Cluster 1 (mV) Cluster 2 (mV) Organism Hm.fdx1 -480 -524 Heliobacterium modesticaldum Pa.fdx -475 -655 Pseudomonas aeruginosa Petition 870260009364, dated 01 / 30 / 2026, p. 79 / 187 67 / 80 Cv.fdx -467 -640 Allochromatium vinosum Cv.fdx_C57A -451 -590 Synthetic Ec.yfhL 418 675 E. coli Ca.fdx -400 -400 Clostridium acetobutylicum Cp.fdx -390 -390 Clostridium pasteurianum Ev2.fdx +50 - Ectothiorhodospira shaposhnikovii Pp1 .fdx - - Pseudomonas putida Pp2.fdx - - Pseudomonas putida TABLE 6: FldA Homologues fldA Semiquinone-> oxidized (mV) Hydroquinone-> semiquinone (mV) Organism Ac.fldA2 -522 -133 Azotobacter chroococcum Av.fldA2 -483 -187 Azotobacter vinelandii Ec.fldA -433 -254 E. coli Bs.fldA -382 -105 B. subtilis

[0213] The data show that overexpression of certain fdx or fldA homologs in E. coli and overexpression of YdbK had increased terpenoid product titers (Product B in this example) compared with the empty vector control (emp) (e.g., H.fdx, Cv.fdx, Cv.fdxC57A and Pa.fdx). Figure 19A.

[0214] E. coli manipulated to produce Product D and overexpress YdbK were further manipulated to overexpress Cv.fdx. Similar to previous results, the data show that overexpression of Cv.fdx in E. coli manipulated to produce a terpenoid product (Product D in this example) and overexpress YdbK resulted in increased terpenoid product titers compared to the control. Figure Petition 870260009364, dated 01 / 30 / 2026, p. 80 / 187 68 / 80 19B. The data also show a reduction in MEcPP concentrations in the extracellular broth (Figure 19C) compared to the control, which confirms that carbon flux was pushed through the IspG / H steps to the product. See Figure 1.

[0215] Example 7: Superexpression or complementation with homologs of PFOR, homologs of fpr and / or homologs of fdx or fldA

[0216] E. coli manipulated to produce the F product were further manipulated to overexpress at least one PFOR homolog or fpr homolog and, optionally, an fdx or fldA homolog as shown in Table 7. TABLE 7 Homologous Group SEQ ID NO(s) 1 Scy.pfor (Synechocystis sp.) SEQ ID NO: 29 2 Ki.pfor (Kluyvera intermedia) SEQ ID NO: 30 3 Da.pfor (Desulfovibrio africanus) SEQ ID NO: 31 4 Sco.pfor (Synechococcus sp.) - 5 Ec.ydhV and Ec.ydhY (E. Coli) SEQ ID NO: 33 and SEQ ID NO: 34 6 Ga.pfor (Gilliamella apicola) SEQ ID NO: 35 7 Ec.ydbK (E. Coli) SEQ ID NO: 9 8 Ec.ydbK and Cp.fdx (E. Coli and C. pasteurianum) SEQ ID NO: 9 and 10 9 Ns.fpr (Nostoc sp.) SEQ ID NO: 36 10 Sco.fpr (Synechococcus sp.) SEQ ID NO: 37 11 Ec.fpr and Ec.fdx (E. Coli) SEQ ID NO: 38 and SEQ ID NO: 21 12 Ec.fpr and Ec.fldA (E. Coli) SEQ ID NO: 38 and SEQ ID NO: 27

[0217] The data show that some bacterial strains manipulated to express PFOR and fpr homologs had increased titers of terpenoid product (Product F in this example) compared to the empty vector control (CTRL) (e.g., Da.pfor (Desulfovibrio)). Petition 870260009364, dated 01 / 30 / 2026, p. 81 / 187 69 / 80 africanus) (SEQ ID NO: 31); Sco.pfor (Synechococcus sp.); Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35); Ec.ydbk (E. Coli) (SEQ ID NO: 9); and Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37). See Figure 20.

[0218] The data also show that bacterial strains manipulated to overexpress at least one PFOR homolog and one fdx had increased titers of terpenoid product (Product F) compared to the empty vector control (CTRL) (e.g., Ec.ydhV / Ec.ydhY; E. coli (SEQ ID NO: 33 and SEQ ID NO: 34, respectively) and Ec.ydbK / Cp.fdx; E. coli (SEQ ID NO: 9 and 10, respectively)). See Figure 20.

[0219] Additionally, the data show that bacterial strains manipulated to overexpress at least one homolog of fpr and / or fdx or fldA had increased titers of terpenoid product (Product F) compared to the empty vector control (CTRL) (e.g., Ec.fpr / Ec.fdx E. coli (SEQ ID NO: 38 and SEQ ID NO: 21, respectively) and Ec.fpr / Ec.fldA; E. coli (SEQ ID NO: 38 and SEQ ID NO: 27, respectively)). See Figure 20. SEQUENCES SEQ ID NO: 1 (E. coli IspG) MHNQAPIQRRKSTRIYVGNVPIGDGAPIAVQSMTNTRTT DVEATVNQIKALERVGADIVRVSVPTMDAAEAFKLIKQQVNVPLVADIHFDYRIA LKVAEYGVDCLRINPGNIGNEERIRMVVDCARDKNIPIRIGVNAGSLEKDLQEK YGEPTPQALLESAMRHVDHLDRLNFDQFKVSVKASDVFLAVESYRLLAKQIDQ PLHLGITEAGGARSGAVKSAIGGLGLLLSEGIGDTLRVSLAADPVEEIKVGFDILK SLRIRSRGINFIACPTCSRQEFDVIGTVNALEQRLEDIITPMDVSIIGCVVNGPGE ALVSTLGVTGGNKKSGLYEDGVRKDRLDNNDMIDQLEARIRAKASQLDEARRI DVQQVEK SEQ ID NO: 2 (E. coli IspH) MQILLANPRGFCAGVDRAISIVENALAIYGAPIYVRHEVV HNRYVVDSLRERGAIFIEQISEVPDGAILIFSAHGVSQAVRNEAKSRDLTVFDAT Petition 870260009364, 01 / 30 / 2026, pág. 82 / 187 70 / 80 CPLVTKVHMEVARASRRGEESILIGHAGHPEVEGTMGQYSNPEGGMYLVESP DDVWKLTVKNEEKLSFMTQTTLSVDDTSDVIDALRKRFPKIVGPRKDDICYATT NRQEAVRALAEQAEVVLVVGSKNSSNSNRLAELAELAQRMGKRAFLIDDAKDIQE EWVKEVKCVGVTAGASAPDILVQNVVARLQQLGGGEAIPLEGREENIVFEVPK ELRVDIREVD SEQ ID NO: 3 (E. coli Dxs) MSFDIAKYPTLALVDSTQELRLLPKESLPKLCDELRRYLL DSVSRSSGHFASGLGTVELTVALHYVYNTPFDQLIWDVGHQAYPHKILTGRRD KIGTIRQKGGLHPFPWRGESEYDVLSVGHSSTSISAGIGIAAVAAEKEGKNRRTV CVIGDGAITAGMAFEAMNHAGDIRPDMLVILNDNEMSISENVGALNNHLAQLLS GKLYSSLREGGKKVFSGVPPIKELLKRTEEHIKGMVVPGTLFEELGFNYIGPVD GHDVLGLITTLKNMRDLKGPQFLHIMTKKGRGYEPAEKDPITFHAVPKFDPSS GCLPKSSGGLPSYSKIFGDWLCETAAKDNKLMAITPAMREGSGMVEFSRKFP DRYFDVAIAEQHAVTFAAGLAIGGYKPIVAIYSTFLQRAYDQVLHDVAIQKLPVL FAIDRAGIVGADGQTHQGAFDLSYLRCIPEMVIMTPSDENECRQMLYTGYHYN DGPSAVRYPRGNAVGVELTPLEKLPIGKGIVKRRGEKLAILNFGTLMPEAAKVA ESLNATLVDMRFVKPLDEALILEMAASHEALVTVEENAIMGGAGSGVNEVLMA HRKPVPVLNIGLPDFFIPQGTQEEMRAELGLDAAGMEAKIKAWLA SEQ ID NO: 4 (E. coli Dxr) MKQLTILGSTGSIGCSTLDVVRHNPEHFRVVALVAGKNV TRMVEQCLEFSPRYAVMDDEASAKLLKTMLQQQGSRTEVLSGQQQAACDMAA LEDVDQVMAAIVGAAGLLPTLAAIRAGKTILLANKESLVTCGRLFMDAVKQSKA QLLPVDSEHNAIFQSLPQPIQHNLGYADLEQNGVVSILLTGSGGPFRETPLRDL ATMTPDQACRHPNWSMGRKISVDSATMMNKGLEYIEARWLFNASASQMEVLI HPQSVIHSMVRYQDGSVLAQLGEPDMRTPIAHTMAWPNRVNSCKGPLDFKLDVK SALTFAAPDYDRYPCLKLAMEAFEQGQAATTALNAANEITVAFLAQQIRFTDI AALNLSVLEKMDMREPQCVDDVLSVDANAREVARKEVMRLAS SEQ ID NO: 5 (E. coli IspD) MATTHLDVCAVVPAAGFGRRMQTECPKQYLSIGNQTIL EHSVHALLAHPRVKRVVIAISPGDSRFAQLPLANHPQITVVDGGDERADSVLA Petition 870260009364, of 30 / 01 / 2026, p. 83 / 187 71 / 80 GLKAAGDAQWVLVHDAARPCLHQDDLARLLALSETSRTGGILAAPVRDTMKR AEPGKNAIAHTVDRNGLWHALTPQFFPRELLHDCLTRALNEGATIDEASALE YCGFHPQLVEGRADNIKVTRPEDLALAEFYLTRTIHQENT SEQ ID NO: 6 (E. coli IspE) MRTQWPSPAKLNLFLYITGQRADGYHTLQTLFQFLDYG DTISIELRDDGDIRLLTPVEGVEHEDNLIVRAARLLMKTAADSGRLPTGSGANIS IDKRLPMGGGLGGGSSNAATVLVALNHLWQCGLSMDELAEMGLTLGADVPV FVRGHAAFAEGVGEILTPVDPPEKWYLVAHPGVSIPTPVIFKDPELPRNTPKRS IETLLKCEFSNDCEVIARKRFREVDAVLSWLLEYAPSRLTGTGACVFAEFDTES EARQVLEQAPEWLNGFVAKGANLSPLHRAML SEQ ID NO: 7 (E. coli IspF) MRIGHGFDVHAFGGEGPIIIGGVRIPYEKGLLAHSDGDV ALHALTDALLGAAALGDIGKLFPDTDPAFKGADSRELLREAWRRIQAKGYTLG NVDVTIIAQAPKMLPHIPQMRVFIAEDLGCHMDDVNVKATTTEKLGFTGRGEGI ACEAVALLIKATK SEQ ID NO: 8 (E. coli Idi) MQTEHVILLNAQGVPTGTLEKYAAHTADTRLHLAFSSWL FNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGV EITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDL ADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK SEQ ID NO: 9 (E. coli YdbK) MITIDGNGAVASVAFRTSEVIAIYPITPSSTMAEQADAWA GNGLKNVWGDTPRVVEMQSEAGAIATVHGALQTGALSTSFTSSQGLLLMIPTL YKLAGELTPFVLHVAARTVATHALSIFGDHSDVMAVRQTGCAMLCAANVQEA QDFALISQIATLKSRVPFIHFFDGFRTSHEINKIVPLADDTILDLMPQVEIDAHRA RALNPEHPVIRGTSANPDTYFQSREATNPWYNAVYDHVEQAMNDFSAATGR QYQPFEYYGHPQAERVIILMGSAIGTCEEVVDELLTRGEKVGVLKVRLYRPFS AKHLLQALPGSVRSVAVLDRTKEPGAQAEPLYLDVMTALAEAFNNGERETLP RVIGGRYGLSSKEFGPDCVLAVFAELNAAKPKARFTVGIYDDVTNLSLPLPENT LPNSAKLEALFYGLGSDGSVSATKNNIKIIGNSTPWYAQGYFVYDSKKAGGLT Petição 870260009364, de 30 / 01 / 2026, pág. 84 / 187 72 / 80 VSHLRVSEQPIRSAYLISQADFVGCHQLQFIDKYQMAERLKPGGIFLLNTPYSA DEVWSRLPQEVQAVLNQKKARFYVINAAKIARECGLAARINTVMQMAFFHLTQ ILPGDSALAELQGAIAKSYSSKGQDLVERNWQALALARESVEEVPLQPVNPHS ANRPPVVSDAAPDFVKTVTAAMLAGLGDALPVSALPPDGTWPMGTTRWEKR NIAEEIPIWKEELCTQCNHCVAACPHSAIRAKVVPPEAMENAPASLHSLDVKSR DMRGQKYVLQVAPEDCTGCNLCVEVCPAKDRQNPEIKAINMMSRLEHVEEEK INYDFFLNLPEIDRSKLERIDIRTSQLITPLFEYSGACSGCGETPYIKLLTQLYGD RMLIANATGCSSIYGGNLPSTPYTTDANGRGPAWANSLFEDNAEFGLGFRLTV DQHRVRVLRLLDQFADKIPAELLTALKSDATPEVRREQVAALRQQLNDVAEAH ELLRDADALVEKSIWLIGGDGWAYDIGFGGLDHVLSLTENVNILVLDTQCYSNT GGQASKATPLGAVTKFGEHGKRKARKDLGVSMMMYGHVYVAQISLGAQLNQ TVKAIQEAEAYPGPSLIIAYSPCEEHGYDLALSHDQMRQLTATGFWPLYRFDP RRADEGKLPLALDSRPPSEAPEETLLHEQRFRRLNSQQPEVAEQLWKDAAAD LQKRYDFLAQMAGKAEKSNTD SEQ ID NO: 10 (Clostridium pasteurianum fdx; Cp.fdx) MAYKIADSCVSCGACASECPVNAISQGDSIFVIDADTCID CGNCANVCPVGAPVQE SEQ ID NO: 11 (E. coli PgpB) MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWL LKAAFWVTETVTQPWGVITHLILFGWFLWCLRFRIKAAFVLFAILAAAILVGQGV KSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLVKEQLAEEKNIPQY LRSHWQKETGFAFPSGHTMFAASWALLAVGLLWPRRRTLTIAILLVWATGVM GSRLLLGMHWPRDLVVATLISWALVATWLAQRICGPLTPPAEENREIAQRE QES SEQ ID NO: 12 (E. coli NudB) VINAA SEQ ID NO: 13 (E. coli Shine Dalgarno sequence) Petition: 870260009364, on January 30, 2026, page. 85 / 187 73 / 80 AGGAGG SEQ ID NO: 14 (Methanococcus maripaludis GAPOR) MNILIDGSRQNYEELEESEFPISFGINLHTKQETWKYDAF DEKNLFCFGKGILPIIGGHRLIFSFRSPLWDGFHFSAMGGAGYTFKDTGIQNVA ITGKCEVPTVIVLNGEEDKLKIEFMPFTEEITDIYEFNDKIIDLFKEKNYRAFLVGP ASKTTNMGGIYSQTIRNGKIVEGSEDWAARGGGGSVLYQAHNVLGVVFFGKK TPEKNLKEIVEEHYNKPYTKVVLEHTEKYRYSEEKKTGGTFGNNYHVTMELTP VFNWRMPFIDKNKRMKLHKKIIEYFVNRFDEEAIETKNWTNCGEPCPVVCKKY RKGLHVDYEPYEANGPCIGVFDIYAADKVVHTIDKLGFDAIEFGNLCSWTFELL DNGMLKPEEVGIEKPVFDISNFENDEDILKNSMHNAEQAVKLAEIIAFQTNEFG KICKSGTRRAGKILNEKYPDRIKDKKFEDFGVYDSFGERGQISPTMYWAIGNF MPYLIQGKYLTHYQCGVFLEPEELAELSVKNSIEEITLENLGICRFHRKWVTPIIE KLVKEMSDVNLNEESMELFKKIAKYDSNIGCPEMESERVKELIIAGAFEFENEK WSKEFENGNFDEYIKRVLEKYSELLEIDWKLKE SEQ ID NO: 15 (Heliobacterium modesticaldum, Hm.fdx1) MAYKITDACTACGACMDGCCVGAIVEGKKYSITSDCVD CGVCADKCPVDAIIPG SEQ ID NO: 16 (Pseudomonas aeruginosa, Pa.fdx) MSLKITDDCINCDVCEPECPNGAISQGEEIYVIDPNLCTE CVGHYDEPQCQQVCPVDCIPLDDANVESKDQLMEKYRKITGKA SEQ ID NO: 17 (Allochromatium vinosum, Cv.fdx) MALMITDECINCDVCEPECPNGAISQGDETYVIEPSLCTE CVGHYETSQCVEVCPVDCIIKDPSHEETEDELRAKYERITGEG SEQ ID NO: 18 (Cv.fdx C57A) MALMITDECINCDVCEPECPNGAISQGDETYVIEPSLCTE CVGHYETSQCVEVCPPVDAIIKDPSHEETEDELRAKYERITGEG SEQ ID NO: 19 (E. coli, Ec.yfhL) MALLITKKCINCDMCEPECPNEAISMGDHIYEINSDKCTE CVGHYETPTCQKVCPIPNTIVKDPAHVETEEQLWDKFVLMHHADKI SEQ ID NO: 20 (Clostridium acetobutylicum, Ca.fdx) Petition 870260009364, of 30 / 01 / 2026, p. 86 / 187 74 / 80 MAYKITDACVSCGSCASECPVSAISQGDTQFVIDADTCI ECGNCANVCPVGAPVQE SEQ ID NO: 21 (E. coli, Ec.fdx) MPKIVILPHQDLCPDGAVLEANSGETILDAALRNGIEIEHA CEXCACTTCHCIVREGFDSLPESSEQEDDMLDKAWGLEPESRLSCQARVTD EDLVVEIPRYTINHAREH SEQ ID NO: 22 (Ectothiorhodospira shaposhnikovii, Ev2.fdx) MERLSEDDPAAQALEYRHDASSVQHPAYEEGQTCLNC LLYTDASAQDWGPCSVFPGKLVSANGWCTAWVAR SEQ ID NO: 23 (Pseudomonas putida, Pp1.fdx) MSLIITDDCINCDVCEPECPNAAISQGEEIYVIDPNLCTQC VGHYDEPQCQQVCPVDCIPLDEAHPETHDELMEKYKRITGKA SEQ ID NO: 24 (Pseudomonas putida, Pp2.fdx) MSLIITDDCINCDVCEPECPNEAISQGEEIYVIDPNLCTQC VGHYDEPQCQQVCPVDCIPLDEAHPETEEELMAKYRRIT SEQ ID NO: 25 (Azotobacter vinelandii fldA2; Av.fldA2) MAKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDALNVNR VSAEDFAQYQFLILGTPTLGEGELPGLSSDCENESWEEFLPKIEGLDFSGKTV ALFGLGDQVGYPENYLDALGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVD GKFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL SEQ ID NO: 26 (Azotobacter chroococcum fldA2; Ac.fldA2) MAKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDAVNVNR VSAEDFAQYQFLILGTPTLGEGELPGLSSDCENESWEEFLPKIEGLDFSGKTV ALFGLGDQVGYPENFLDAMGELHSFFTERGAKVVGAWSTDGYEFEGSTAVV DGKFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL SEQ ID NO: 27 (E. coli, Ec.fldA) MAITGIFFGSDTGNTENIAKMIQKQLGKDVADVHDIAKSS KEDLEAYDILLLGIPTWYYGEAQCDWDDFFPTLEEIDFNGKLVALFGCGDQED Petition: 870260009364, on January 30, 2026, page. 87 / 187 75 / 80 YAEYFCDALGTIRDIIEPRGATIVGHWPTAGYHFEASKGLADDDHFVGLAIDED RQPELTAERVEKWVKQISEELHLDEILNA SEQ ID NO: 28 (B. subtilis, Bs.fldA) MAKALITYASMSGNTEDIAFIIKDTLQEYELDIDCVEINDM DASCLTSYDYVLIGTYTWGDGDLPYEAEDFFEEVKQIQLNGLKTACFGSGDYS YPKFCEAVNLFNVMLQEAGAAVYQETLKIELAPETDEDVESCRAFARGFLAWA DYMNKEKIHVS SEQ ID NO: 29 (Synechocystis sp., Scy.pfor) MSLPTYATLDGNEAVARVAYLLSEVIAIYPITPSSPMGEW SDAWAAEHRPNLWGTVPLVVEMQSEGGAAGTVHGALQSGALTTTFTASQGL MLMLPNMHKIAGELTAMVLHVAARSLAAQGLSIFGDHSDVMAARNTGFAMLS SNSVQEAHDFALIATATSFATRIPGLHFFDGFRTSHEEQKIELLPQEVLRGLIKD EDVLAHRGRALTPDRPKLRGTAQNPDVYFQARETVNPFYASYPNVLEQVME QFGQLTGRHYRPYEYCGHPEAERVIVLMGSGAETAQETVDFLTAQGEKVGLL KVRLYRPFAGDRLVNALPKTVQKIAVLDRCKEPGSIGEPLYQDVLTAFFEAGM MPKIIGGRYGLSSKEFTPAMVKGVLDHLNQTNPKNHFTVGINDDLSHTSIDYDP SFSTEADSVVRAIFYGLGSDGTVGANKNSIKIIGEDTDNYAQGYFVYDSKKSGS VTVSHLRFGPNPILSTYLISQANFVACHQWEFLEQFEVLEPAVDGGVFLVNSP YGPEEIWREFPRKVQQEIIDKNLKVYTINANDVARDAGMGRRTNTVMQTCFFA LAGVLPREEAIAKIKQSVQKTYGKKGQEIVEMNIKAVDSTLAHLYEVSVPETVS DDAPAMRPVVPDNAPVFVREVLGKIMARQGDDLPVSALPCDGTYPTATTQW EKRNVGHEIPVWDPDVCVQCGKCVIVCPHAVIRGKVYEEAELANAPVSFKFTN AKDHDWQGSKFTIQVAPEDCTGCGICVDVCPAKNKSQPRLRAINMAPQLPLR EQERENWDFFLDLPNPDRLSLNLNKISHQQMQEPLFEFSGACAGCGETPYLK LVSQLFGDRMLVANATGCSSIYGGNLPTTPWAQNAEGRGPAWSNSLFEDNA EFGLGFRVAIDKQTEFAGELLKTFAGELGDSLVSEILNNAQTTEADIFEQRQLVEQVKQRLQNLETPQAQMFLSVADYLVKKSVWIIGGDGWAYDIGYGGLDHVLA SGRNVNILVMDTEVYSNTGGQASKATPRAAVAKFAAGGKPSPKDLGLMAMT YGNVYVASIAMGAKNEQSIKAFMEAEAYPGVSLIIAMSHMGHQMHQQ KELVDSGRWLLYRYNPLADEGKNPLQLDMGSPKVAIDKTVYSENRFAMLTR Petition 870260009364, of 30 / 01 / 2026, p. 88 / 187 76 / 80 SQPEEAKRLMKLAQGDVNTRWAMYEYLAKRSLGGEINGNNHGVSPSPPEVIAK SV SEQ ID NO: 30 (Kluyvera intermedia, Ki.pfor) MSGKMKTMDGNAAAAWISYAFTDVAAIYPITPSTPMAEN VDEWTAQGKKNLFGQPVRLMEMQSEAGAAGAVHGALQAGALTTTYTASQGL LLMIPNLYKIAGELLPGVFHVSARALATNSLNIFGDHQDVMAVRQTGCAMLAE NNVQQVMDLSAVAHLSAIKGRVPFINFFDGFRTSHEIQKIEVLEHEALAPLLDQ EALNLFRRNALNPDHPVIRGTAQNPDIYFQEREASNRFYQALPDIVEGYMAEIY RITGREYHLFDYYGSPDAEQIIIAMGSVCDTIQEVVDAMIDSGEKVGLVSVHLF RPFSLAHFMAKIPASVKRIAVLDRTKEPGAQAEPLCLDVKNAFYHHDNPPLIVG GRYALGGKDVLPGHIVSVFENLKKPLPMDGFTVGIFDDVTHTSLPVPAYDIHVS REGITACKFWGLGSDGTVSANKNAIKIIGDNTSMFAQAYFAYDSKKSGGITMS HLRFGKRPITSPYLIHNADFIACSQQSYVDKYDLLDGINPGGIFLLNCTWFGEE VERHLPNKMKRIIARQGVRFYTLNAVDIARKLGLGGRFNMLMQAAFFKLTDIID AKTASEHLKKAVAKSYGSKGQNVVDMNNAAIDLGMDALQEIIVPDHWAYVEE EANNDGKLMPDFIRNILEPMNRQNGDKLPVSAFLGMEDGTFPPGTAAWEKRG IAMQVPVWQPEGCTQCNQCAFICPHAAIRPALLSSEEREAAPVALLSKVAQGA KHYEYHLAVSPLDCSGCGNCVDICPSKGKALAMKPLDSQRHMVPVWDHALA LAPKENPFSKATVKGCQFEPPLLEFSGACAGCGETPYARLITQLFGDRMMIAN ATGCSSIWGASAPSIPWTTNHKGQGPAWANSLFEDNAEFGLGMMLGGRAIR EQLASDAASVLERPLHPDLQQALRDWLEHKDLGEGTRARAEKLSALLAAEKGDDDLLNRLYQNQDYFTKRSQWIFGGDGWAYDIGFGGLDHVLASGEDVNILVF DTEVYSNTGGQSSKSTPVAAIAKFAAEGKRTRCKDLGMMAVSYGNVYVAQVA MGADKAQTLRAIAEAEAWPGPSLVIAACINHCGLKAGMSIKRAGRAGRAGRAG YWHLWRYNPQLLAKGKNPFILDSEEPEESFRDFLMGEVRYASLGRTSPEVAD SLFAQTEQDAKDRYAQYRRLAGE SEQ ID NO: 31 (Desulfovibrio africanus, Da.pfor) MGKKMMTTDGNTATAHVAYAMSEVAAIYPITPSS™GE EADDWAAQGRKNIFGQTLTIREMQSEAGAAGAVHGALAAGALTTTFTASQGL LLMIPNMYKISGELLPGVFHVTARAIAAHALSIFGDHQDIYAARQTGFAMLASSS Petition 870260009364, of 30 / 01 / 2026, p. 89 / 187 77 / 80 VQEAHDMALVAHLAAIESNVPFMHFFDGFRTSHEIQKIEVLDYADMASLVNQK ALAEFRAKSMNPEHPHVRGTAQNPDIYFQGREAANPYYLKVPGIVAEYMQKV AALTGRSYKLFDYVGAPDAERVIVSMGSSCETIEEVINHLAAKGDKIGLIKVRLY RPFVSEAFFAALPASAKVITVLDRTKEPGAPGDPLYLDVCSAFVERGEAMPKIL AGRYGLGSKEFPAMVKSVYDNMSGAKKNHFTVGIEDDVTGTSLPVDNAFAD TTPKGTIQCQFWGLGADGTVGANKQAIKIIGDNTDLFAQGYFSYDSKKSGGITI SHLRFGEKPIQSTYLVNRADYVACHNPAYVGIYDILEGIKDGGTFVLNSPWSSL EDMDKHLPSGIKRTIANKKLKFYNIDAVKIATDVGLGGRINMIMQTAFFKLAGVL PFEKAVDLLKKSIHKAYGKKGEKIVKMNTDAVDQAVTSLQEFKYPASWKDAPA ETKAEPKTNEFFKNVVKPILTQQGDKLPVSAFEADGRFPLGTSQFEKRGVAIN VPQWVPENCIQCNQCAFVCPHSAILPVLAKEEELVGAPANFTALEAKGKELKG YFRIQINTLDCMGCGNCADICPPKEKALVMQPLDTQRDAQVPNLEYAARIPV KSEVLPRDSLKGSQFQEPLMEFSGACSGCGETPYVRVITQLFGERMFIANAT GCSSIWGASAPSMPYKTNSLGQGPAWGNSLFEDAAEYGFGMNMSMFARRT HLADLAAKALESDASGDVKEALQGWLAGKNDPIKSKEYGDKLKKLLAGQKDG LLGQIAAMSDLYTKKSVWIFGGDGWAYDIGYGGLDHVLASGEDVNVFVMDTE VYSNTGGQSSKATPTGAVAKFAAAGKRTGKKDLARMVMTYGYVYVATVSMG YSKQQFLKVLKEAESFPGPSLVIAYATCINQGLRKMGKSQDVMNTAVKSGYWPLFRYDPRLAAQGKNPFQLDSKAPDGSVEEFLMAQNRFAVLDRSFPEDAK RLRAQVAHELDVRFKELERMAATNIFESFAPAGGKADGSVDFGEGAEFCTRD DTPMMARPDSGEACDQNRAGTSEQQGDLSKRTKK SEQ ID NO: 32 (Nostoc sp., Ns.pfor) MSQTFATIDGNEAVARVAYKLNEVIAIYPITPSSAMGEWA DAWMAEGRPNLWGTVPSVVQMQSEGGAAGAVHGALQTGSLSTTFTASQGL LLMIPNLYKIGGELTSMVVHVAARSLATHALSIFGDHSDVMAARGTGFAMLCS ASVQESHDFALIAHAATLDTRVSFLHFFDGFRTSHEVQKVELLADDDDVRSLINE DKIFAHRARALTPDSPLLRGTAQNPDVFFQAREGANPYYNACPAIVQGIMDKF GERTGRYYQIYEYHGASDADRLIIIMGSGCETVHETVDYLNARGEKVGVLKVR LFRPWDVERFVQALPHSVQAIAVLDRTKEPGSAGEPLYQDVVTAIHEGWVNK NNSPVPSPQSPVPKIIGGRYGLSSKEFTPAMVKAVFDNLAQATPKNHFTIGIND Petition 870260009364, of 30 / 01 / 2026, p. 90 / 187 78 / 80 DVTHTSLEYDPSFSTEPDNVVRAMFYGLGSDGTVGANKNSIKIIGEGTDNYAQ GYFVYDSKKSGSMTVSHLRFGSQPIRSTYLIDQANFIGCHHWGFLERIEVLNA AAHGATILLNSPYNAATVWENLPLKVRLQILDKQLKLYVINANQVARDSGMGG RINTIMQVCFFALAGVLPEVQAIAKIKQAIEKTYGKKGVEVVRMNLQAVDQTLE NLHEVKIPIEEKGKWIDEEALLSNQSPFSTSAPKFVRDVLGKIMVWQGDDLPV STLPPDGTFPTGTAKWEKRNVAQEIPVWDTDICVQCSKCVMVCPHAAIRAKV YQPSELENAPPTFKSVDAKDRDFANQKFTIQVAPEDCTGCAICVNVCPAKNKS EPSLKAINMANQLPLREQERDNWDFFLNLPNPDRRNLKLNQIRQQQLQEPLF EFSGACAGCGETPYVKLLTQLFGDRSVIANATGCSSIYGGNLPTTPWTKNND GRGPAWSNSLFEDNAEFGFGYRLSLDKQAEFAAELLQQFSTEVGDNLVDSIL KAPQKTEADIWEQRQRIELLKQQLDKIPTFDPNLKSKIQNLKSLADYLVKKSVW IIGGDGWAYDIDFGGIDHVIASGRNVNILVMDTEVYSNTGGQSSKATPKAAVAK FAASGKPAQKKDMGLMAMNYGNVYVASVALGAKDDQTLKAFLEAEAFDGPSI IIAYSHCIAHGINMTTGMNQQKALVESGRWLLYRYNPLLQEQGKNPLQLDMRS PTQSVEQSMYQENRFKMLTKSKPEVAKQLLEQAQAEVDARWQMYQYLASR SEQ ID NO: 33 (E. coli, Ec.ydhV) MANGWTGNILRVNLTTGNITLEDSSKFKSVGGMGFGY KIMYDEVPPGTKPFDEANKLVFATGPLTGSGAPCSSRVNITSLSLTFTKGNLVV DAHMGGGFFAAQMKFAGYDVIIIEGKAKSPVWLKIKDDKVSLEKADFLWGKGTR ATTEEICRLTSPETCVAAIGQAGENLVPLSGMLNSRNHSGGAGTGAIMGSKNL KAIAVEGTKGADRQEMKRLNDYMTELIGANNNHVVPSTPQSWAEYSDP KSRWTARKGLFWGAAEGGPIETGEIPPGNQNTVGFRTYKSVFDLGPAAEKYT VKMSGCHSCPIRCMTQMNIPRVKEFGVPSTGGNTCVANFVHTTIFPNGPKDF EDKDDGRVIGNLVGLNLFDDYGLWCNYGQLHRDFTYCYSKGVFKRVLPAEEY AEIRWDQLEAGDVNFIKDFYYLRVGELSHLADGSYAIAERWNLGEEYWG YAKNKLWSPFGYPVHHANEASAQVGSIVNCMFNRDCMTHTHINFIGSGLPLKL QREVAKELFGSEDAYDETKNYTPINDAKIKYAKWSLLRVCLHNAVTLCNWVW PMTVSPLKSRNYRGDLALEAKFFKAITGEEMTQEKDLLAAERIFTLHRAYTVKL MQTKDMRNEHDLICSWVFDKDPQIPVFTEGTDKMDRDDMHASLTMFYKEMG WDPQLGCPTRETLQRLGLEDIAADLAAHNLLPA Petition 870260009364, de 30 / 01 / 2026, pág. 91 / 187 79 / 80 SEQ ID NO: 34 (E. coli, Ec.ydhY) MNPVDRPLLDIGLTRLEFLRISGKGLAGLTIAPALLSLLGC KQEDIDSGTVGLINTPKGVLVTQRARCTGCHRCEISCTNFNDGSVGTFFSRIKI HRNYFFGDNGVGSGGGLYGDLNYTADTCRQCKEPQCMNVCPIGAITWQQKE GCITVDHKRCIGCSACTTACPWMMATVNTESKKSSKCVLCGECANACPTGAL KIIEWKDITV SEQ ID NO: 35 (Gilliamella apicola, Ga.pfor) MIISDANSAVSSVAYRANEVIAIYPITPSSSMAEQASTWA EFDKPNVFGDIPRVVEMQSEAGAIATVHGALMTGALATSFTSSQGLLLMIPSLY KIAGELTPFVLHVAARTVATHALSIFGDHSDVMSVRQTGFAMLCSSSVQEAQD LALISQIASFKSRIPFVHFFDGFRTSHEVNKIYPLSDEDIHDLLPHEAIKAYRSRA LTPDKPMIRGTSANPDTYFQCREAINSYYDNAYQHVVDAMTDFEKQTGRKYQ PFEYYGASDAERIIVIMGSGASTSKEVIDYLLKENQKVGVVIVRLFRPFSAQHLL AVIPDSVKKIAVLDRTKEPGAQAEPLYLDIMTAFAESLSRGERNTIPQIVGGRY GLSSKEFDPRSVLGIFNELSLEKPRPRFTVGIYDDITGLSLPLPDKTIPQKSALE ALFYGLGSDGTVSATKNNIKIIGDSSPFYVQGYFVYDSKKAGGLTTSHLRVNLD PIDSPYLITSAHFIGCHQDQFIDKYQIVDKLKNDGIFLLNTPYNKDEIWHRLPKEV QVQLIKKRAHFYIINAAKIARECNLGARINTVMQAAFFHLSDIFKNDFSISQLKEV IAKSYSSKGQELVENNWKALDLAITSLEQIPLNCVDQSSPSMPPIVPNNAPDFV KTVTATMLAGLGDSLPVSAFPPDGAWPTGTTKWEKRNIAEEIPIWKSELCTQC NHCAVACPHAAIRAKVVEPDAMLNAPDTLESLEVKARDMKGQRYVLQVAPED CTGCNLCVEVCPSRDRNNFDIKAINMQPRIDNLDTQRVNFEFFSALPDRDIKSL DRIDIRTSQLITPLFEYSGACAGCGETPYIKLLTQLYGDHLAIANATGCSSIYGG NLPSTPYTTDRSGRGPAWANSLFEDNAEFALGYRITYNQHRKRALRLLDHLA GEISPEIVITLQSSDATIAEKRTQVDLLREQLKHIDSAEAKELLEDTNYLIDKSVWAIGGDGWAYDIGFGGLDHVMSLTDNVNILVLDTQCYSNTGGQQSKATPMGAV SKFADLGKHKARKDLGVSIMMYGHVYVAQVALGSQLNQTLKALQEAEAYDGP SLVIAYSPCEEHGYDLAKSHEQMKDLVKSGGFWPLEGWRPGYDRKLDVLDVLPG SKSPNSEALSSILLKEQRFRRLETLEPTVANILHERSTKMVESKYRFLQMLSSY SDIETPPDS Petition 870260009364, of 30 / 01 / 2026, p. 92 / 187 80 / 80 SEQ ID NO: 36 (Nostoc sp., Ns.fpr) MSNQGAFDGAANVESGSRVFVYEVVGMRQNEETDQT NYPIRCSGSVFIRVPYNRMNQEMQRITRLGGKIVTIQTVSALQQLNGRTTIATV INCLUDING KHADVPVNLYRPNAPFIGKVISNEPLVKEGGIGIVQHIKFDLTGGNLKYIEGQSI GIIPPGVDKNGKPEKLLYSIASTRHGDDVDDKTISLCVRQLEYKHPESGETVY GVCSTYLTHIEPGSEVKITGPVGKEMLLPDDPEANVIMLATGGTYRTGIRW MFKDAERAANPEYQFKGFSWLVFGVPTTPNILYKEELEEIQQKYPDNFRLTYAI SREQKNPQGGRMYIQDRVAEHADELWQLIKNQTTHYICGLRGMEEGIDAAL SAAAAKEGVTWSDYQKDLKKAGRWHVETY SEQ ID NO: 37 (Synechococcus sp., Sco.fpr) MYGITSTANSTGNQSYANRLFIYEVVGLGGDGRNENSL VRKSGTTFITVPYARMNQEMQRITKLGGKIVSIRPAEDAAQIVSEGQSSAQASA QSPMASSTKIVHPKTTDTSVPVNIYRPKTPFLGKCIENYELVDEGGSGTVRHVT FDISEGDLRYLEGQSIGIIPPGEDKNGKPHKLRLYSIASTRHGDMEDNKTVSLC VRQLEYQDPESGETVYGVCSTYLCNLPVGTDDVKITGPVGKEMLLPDDEDAT VVMLATGTGIAPFRAFLWRMFKEQHEDYKFKGKAWLIFGVPYTANILYKDDFE KMAAENPDNFRLTYAISREQKTADGGKVYVQSRVSEYADELFEMIQKPNTHV YMCGLKGMQPPIDETFTAEAEKRGLNWEEMRRSMKKEHRWHVEVY SEQ ID NO: 38 (E. coli, Ec.fpr) MADWVTGKVTKVQNWTDALFSLTVHAPVLPFTAGQFTK LGLEIDGERVQRAYSYVNSPDNPDLEFYLVTPDGKLSPRLAALKPGDEVQVV SEAAGFFVLDEVPHCETLWMLATGTAIGPYLSILQLGKDLDRFKNLVLVHAARY AADLSYLPLMQELEKRYEGKLRIQTVVSRETAAGSLTGRIPALIESGELESTIGL PMNKETSHVMLCGNPQMVRDTQQLLKETRQMTKHLRRRPGHMTAEHYW Petition 870260009364, de 30 / 01 / 2026, pág. 93 / 187

Claims

1 / 5 CLAIMS 1. A method for producing a terpene or terpenoid product, characterized by comprising: providing an Escherichia coli strain expressing a terpenoid biosynthesis pathway, the terpenoid biosynthesis pathway comprising an upstream methylerythritol phosphate (MEP) pathway and a recombinant downstream synthesis pathway, wherein the bacterial strain overexpresses IspG and IspH relative to the wild-type bacterial strain and one or more recombinant oxidoreductase enzymes enhance the supply and / or transfer of electrons to IspG and IspH, and culturing the E. coli strain to produce the terpene or terpenoid product, wherein said one or more recombinant oxidoreductases are pyruvate:flavodoxin oxidoreductases (PFOR).

2. Method according to claim 1, characterized in that IspG and IspH genes are overexpressed when recombinant ispG and ispH genes are introduced into the E. coli strain.

3. A method according to claim 1 or 2, characterized in that the IspG and IspH enzymes are overexpressed in the E. coli strain by complementation with recombinant ispG and ispH genes, and / or in that the activity and / or expression of recombinant IspH is higher than the activity and / or expression of recombinant IspG.

4. A method, according to any one of claims 1 to 3, characterized in that the expression of recombinant IspH is higher than the expression of recombinant IspG.

5. Method according to claim 4, characterized in that recombinant IspH and IspG genes are expressed from an operon, with the IspH gene positioned before the IspG gene in the operon.

6. Method, according to any one of claims 1 to 5, characterized in that the expression of recombinant IspG and IspH enzymes is such that the expression of recombinant IspH is superior to the expression of recombinant IspG by means of modification of the promoter strength, gene copy number and / or ribosome binding site sequence of recombinant ispG and / or ispH genes.

7. A method according to any one of claims 1 to 6, characterized in that HMBPP does not accumulate in cells more than in a source strain that does not comprise the recombinant ispG and ispH genes.

8. A method, according to any one of claims 1 to 7, characterized by the expression or activity of a recombinant gene encoding Idi being adjusted to increase terpene or terpenoid production.

9. Method, according to any one of claims 1 to 8, characterized by PFOR comprising an amino acid sequence selected from Ec. YdbK (SEQ ID NO: 9) and Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35).

10. Method, according to any one of claims 1 to 9, characterized by PFOR comprising an amino acid sequence of SEQ ID NO:

9.

11. A method, according to any one of claims 1 to 10, characterized in that a polynucleotide encoding PFOR is integrated into a chromosome or expressed from a plasmid.

12. A method, according to any one of claims 1 to 11, characterized by the E. coli strain expressing two or more recombinant electron carriers.

13. Method according to claim 12, characterized by a recombinant electron carrier having a redox potential in the range of 400 to 550 mV.

14. Method according to claim 12, characterized by a recombinant electron carrier having a redox potential in the range of 400 to 500 mV.

15. Method according to claim 12, characterized in that a recombinant electron carrier has a redox potential in the range of 400 to 450 mV.

16. Method according to claim 12, characterized in that a recombinant electron carrier is a recombinant ferredoxin (fdx) or recombinant flavodoxin (Fld).

17. Method according to claim 1, characterized in that PFOR is YdbK.

18. Method according to claim 17, characterized in that the E. coli strain comprises a recombinant YdbK gene, or in that the recombinant YdbK gene is integrated into the chromosome or expressed from a plasmid.

19. Method according to claim 1, characterized by the E. coli strain expressing recombinant genes encoding dxs, ispD, ispF and idi.

20. Method according to claim 19, characterized by the E. coli strain expressing recombinant genes encoding dxs, dxr, ispD, ispE, ispF and idi.

21. A method, according to any one of claims 1 to 20, characterized by recombinant IspH expression or activity superior to recombinant IspG expression or activity, for reducing the MEcPP metabolite in culture.

22. A method, according to any one of claims 1 to 21, characterized in that the E. coli strain has reduced or eliminated the PDH-mediated conversion of pyruvate to acetyl-CoA.

23. Method according to claim 22, characterized by the E. coli strain expressing a mutant aceE, wherein the mutation is G267C.

24. Method according to claim 23, Petition 870260063250, dated 06 / 26 / 2026, p. 10 / 17 4 / 5 characterized by the E. coli strain having a deletion or inactivation of aceE.

25. Method, according to any one of claims 1 to 24, characterized by the bacterial strain overexpressing one or more non-native homologs of fdx and / or fldA.

26. Method, according to any one of claims 1 to 25, characterized in that the homolog of fdx is selected from Hm.fdxl (Heliobacterium modesticaldum) (SEQ ID NO: 15), Pa.fdx (Pseudomonas aeruginosa) (SEQ ID NO: 16), Cv.fdx (Allochromatium vinosum) (SEQ ID NO: 17), Ca.fdx (Clostridium acetobutylicum) (SEQ ID NO: 20), Cp.fdx (Clostridium pasteurianum) (SEQ ID NO: 10), Ev2.fdx (Ectothiorhodospira shaposhnikovii) (SEQ ID NO: 22), Pp1.fdx (Pseudomonas putida) (SEQ ID NO: 23) and Pp2.fdx (Pseudomonas putida) (SEQ ID NO: 24).

27. Method, according to any one of claims 1 to 26, characterized in that the homologue of fldA is selected from Ac.fldA2 (Azotobacter chroococcum) (SEQ ID NO: 26), Av.fldA2 (Azotobacter vinelandii) (SEQ ID NO: 25) and Bs.fldA (B. subtilis) (SEQ ID NO: 27).

28. Method, according to any one of claims 1 to 27, characterized by the terpene or terpenoid product comprising a compound selected from: Farnesene, Amorfadiene, Artemisinic acid, Artemisinin, Bisabolol, Bisabolene, alpha-Sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Rose Oxide, Sabinene, Steviol, Steviol Glycoside (including Rebaudioside D or Rebaudioside M), Taxadiene, Thymol and Valencene.

29. Method, according to any one of claims 1 to 28, characterized in that the culture step is a fed-batch process comprising a first phase in which an E. coli biomass is created, followed by a terpene or terpenoid production phase.

30. Method according to claim 29, characterized in that the culture can be 100 L.

31. Method, according to claim 29, characterized in that the culture volume can be 10,000 L.

32. Method, according to claim 29, characterized in that the culture volume can be 100,000 L.

33. Method, according to any one of claims 29 to 32, characterized in that the culture is maintained under microaerobic or aerobic conditions.

34. Method according to claim 33, characterized in that the biomass production phase occurs under aerobic conditions followed by a reduction in oxygen levels after 10 to 20 hours.

35. Method according to claim 29, characterized in that the production phase includes feeding with a nitrogen source.

36. Method according to claim 29, characterized in that the production phase includes feeding in a carbon source.

37. A method according to any one of claims 1 to 36, characterized by further comprising recovering the terpene or terpenoid product.

38. Method, according to any one of claims 1 to 37, characterized by the E. coli strain or cell overexpressing one or more of geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS). Petition 870260063250, dated 06 / 26 / 2026, p. 12 / 17

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