Metabolic engineering for microbial production of terpenoid products

Abstract

The invention relates to methods and bacterial strains for making terpene and terpenoid products, the bacterial strains having improved carbon pull through the MEP pathway and to a downstream recombinant synthesis pathway.

1 . A method for production of a terpene or terpenoid product, comprising:
 providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP pathway) and converts the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway;   wherein IspG and IspH are overexpressed in the bacterial strain such that IspG activity and IspH activity are balanced to provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, while preventing accumulation of HMBPP at an amount that feeds back and reduces MEP pathway flux and terpene or terpenoid productivity; the strain optionally comprising one or more genetic modifications that enhance supply and/or transfer of electrons through the MEP pathway and/or to terpene and terpenoid products, and culturing the bacterial strain to produce the terpene or terpenoid product.   
     
     
         2 . The method of  claim 1 , wherein the bacterial strain is a bacteria selected from  Escherichia  spp.,  Bacillus  spp.,  Corynebacterium Rhodobacter  spp.,  Zymomonas  spp.,  Vibrio  spp., and  Pseudomonas  spp. 
     
     
         3 . The method of  claim 2 , wherein 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.    
     
     
         4 . The method of  claim 3 , wherein the bacterial strain is  E. coli.    
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the bacterial strain expresses dxs, ispD, ispE, and idi as recombinant genes, which are optionally expressed as an operon. 
     
     
         6 . The method of  claim 5 , wherein the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which are optionally expressed as 1, 2, or 3 operons. 
     
     
         7 . The method of  claim 5  or  6 , wherein the recombinant genes of the MEP pathway are expressed from one or more plasmids or are integrated into the chromosome. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein IspG and IspH are overexpressed by introducing recombinant ispG and ispH genes into the bacterial strain. 
     
     
         9 . The method of  claim 8 , wherein the recombinant IspG and/or IspH enzymes comprises one or more beneficial mutations, or is an ortholog having improved properties or activity under conditions used for culturing. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the activity and/or expression of recombinant IspH is higher than the activity and/or expression of the recombinant IspG. 
     
     
         11 . The method of  claim 10 , wherein the expression of the recombinant IspH is higher than the expression of the recombinant IspG. 
     
     
         12 . The method of  claim 11 , wherein the recombinant IspH and IspG are expressed from an operon, with IspH gene positioned before the IspG gene in the operon. 
     
     
         13 . The method of  claim 12 , wherein the operon is expressed under control of a strong promoter. 
     
     
         14 . The method of any one of  claims 1  to  13 , wherein expression of the recombinant IspG and IspH enzymes are balanced by modifying the promoter strength, gene copy number, and/or ribosome binding site sequence of the ispG and/or ispH recombinant genes. 
     
     
         15 . The method of any one of  claims 1  to  14 , wherein HMBPP does not accumulate in cells substantially more than in a parent strain that does not comprise the recombinant ispG and ispH genes. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein the recombinant ispG and ispH genes are expressed from a plasmid or are integrated into the chromosome. 
     
     
         17 . The method of any one of  claims 1  to  16 , wherein the expression or activity of a recombinant idi gene is tuned to increase terpene or terpenoid production. 
     
     
         18 . The method of  claim 17 , wherein the expression of the recombinant idi gene is tuned by modifying the promoter strength, gene copy number, position in an operon, or ribosome binding site. 
     
     
         19 . The method of any one of  claims 1  to  18 , wherein HMBPP does not accumulate at greater than 10 mg/g of dry cell weight. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the bacterial strain comprises one or more genetic modifications that enhance supply and/or transfer of electrons through the MEP pathway and/or to terpene and terpenoid products, and which optionally oxidizes pyruvate and/or reduces ferredoxin. 
     
     
         21 . The method of  claim 20 , wherein the bacterial strain contains an overexpression of an oxidoreductase. 
     
     
         22 . The method of  claim 21 , wherein the oxidoreductase is a pyruvate:flavodoxin oxidoreductase (PFOR). 
     
     
         23 . The method of  claim 22 , wherein the PFOR is YdbK. 
     
     
         24 . The method of  claim 21 , wherein the bacterial strain comprises a recombinant YdbK gene. 
     
     
         25 . The method of  claim 24 , wherein the recombinant YdbK gene is integrated into the chromosome or expressed from a plasmid, which is optionally under the control of a weak or intermediate strength promoter. 
     
     
         26 . The method of any one of  claims 21  to  25 , wherein the strain comprises one or more P450 enzymes for the production of a terpenoid compound. 
     
     
         27 . The method of any one of  claims 21  to  26 , further comprising an overexpression of one or more of a flavodoxin, flavodoxin reductase, ferredoxin, and ferredoxin reductase. 
     
     
         28 . The method of any one of  claims 1  to  27 , wherein the expression or activity of IspG and IspH is balanced with respect to the expression or activity of Dxr, Dxs, IspD, IspE and IspF to reduce MEcPP metabolite in the culture. 
     
     
         29 . The method of any one of  claims 1  to  27 , wherein the bacterial strain comprises an overexpression of PgpB and/or NudB. 
     
     
         30 . The method of  claim 29 , wherein the bacterial strain expresses at least one recombinant pgpB and/or nudB gene. 
     
     
         31 . The method of  claim 29 , wherein the expression of the recombinant PgpB and/or NudB is tuned to provide higher terpene or terpenoid product titer, optionally by varying promoter strength, gene copy number, position in an operon, and/or ribosome binding site. 
     
     
         32 . The method of  claim 31 , wherein the recombinant PgpB and/or NudB is expressed under control of a weak or intermediate strength promoter. 
     
     
         33 . The method of any one of  claims 30  to  32 , wherein the recombinant pgpB or nudB is integrated into the chromosome or expressed from a plasmid. 
     
     
         34 . The method of any one of  claims 1  to  33 , wherein the bacterial strain has one or more additional modifications to increase co-factor availability or turnover, the one or more additional modifications optionally selected from recombinant expression of a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); overexpression of ydhV and/or ydhY; downregulation, inactivation, or deletion of gshA; and recombinant expression of CHAC1 and/or CHAC2. 
     
     
         35 . A method for draining excess carbon flux from the MEP pathway in a bacterial cell, comprising:
 providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an upstream methylerythritol phosphate pathway (MEP pathway) and optionally converts the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway;   wherein the bacterial strain overexpresses one or more MEP pathway genes and overexpresses PgpB and/or NudB,   culturing the bacterial strain to produce MEP carbon.   
     
     
         36 . The method of  claim 35 , wherein the bacterial strain is a bacteria selected from  Escherichia  spp.,  Bacillus  spp.,  Corynebacterium  spp.,  Rhodobacter  spp.,  Zymomonas  spp.,  Vibrio  spp., and  Pseudomonas  spp. 
     
     
         37 . The method of  claim 35 , wherein the bacterial strain is a species selected from  Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymornonas mobilis, Vibrio natriegens,  or  Pseuclomonas putida.    
     
     
         38 . The method of  claim 37 , wherein the bacterial strain is  E. coli.    
     
     
         39 . The method of any one of  claims 35  to  38 , wherein he bacterial strain expresses at least one recombinant PgpB and/or NudB gene. 
     
     
         40 . The method of  claim 39 , wherein the expression of the recombinant PgpB and/or NudB is tuned to provide higher terpene or terpenoid product titer, optionally by varying promoter strength, gene copy number, position in an operon, and/or ribosome binding site. 
     
     
         41 . The method of  claim 40 , wherein the recombinant PgpB and/or NudB is expressed under control of a weak or intermediate strength promoter. 
     
     
         42 . The method of any one of  claims 39  to  41 , wherein the recombinant PgpB or NudB is integrated into the chromosome or expressed from a plasmid. 
     
     
         43 . The method of any one of  claims 35  to  42 , wherein the bacterial strain overexpresses IspG and IspH such that IspG activity and IspH activity are balanced to provide increased carbon flux to HMBPP intermediate, while preventing accumulation of HMBPP at an amount that substantially reduces cell growth, viability, MEP flux, or product titer. 
     
     
         44 . The method of  claim 43 , wherein HMBPP does not accumulate at greater than 10 mg/g of dry cell weight. 
     
     
         45 . The method of any one of  claims 35  to  44 , wherein the bacterial strain expresses dxs, ispD, ispF, and idi as recombinant genes, which are optionally expressed as an operon. 
     
     
         46 . The method of  claim 45 , wherein the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which are optionally expressed as 1, 2, or 3 operons. 
     
     
         47 . The method of  claim 45  or  46 , wherein the recombinant genes of the MEP pathway are expressed from one or more plasmids or are integrated into the chromosome. 
     
     
         48 . The method of any one of  claims 43  to  47 , wherein IspG and IspH are overexpressed by introducing at least one recombinant IspG and IspH gene into the bacterial strain. 
     
     
         49 . The method of  claim 48 , wherein the activity and/or expression of recombinant IspH is higher than the activity and/or expression of the recombinant ispG. 
     
     
         50 . The method of  claim 49 , wherein the recombinant IspH and ispG are expressed as an operon, with IspH positioned before IspG in the operon, wherein the operon is optionally expressed under control of a strong promoter. 
     
     
         51 . The method of any one of  claims 43  to  50 , wherein the recombinant ispG and IspH genes are expressed from a plasmid or are integrated into the chromosome. 
     
     
         52 . The method of any one of  claims 35  to  51 , wherein the expression or activity of a recombinant Idi gene is tuned to increase terpene or terpenoid production. 
     
     
         53 . The method of any one of  claims 42  to  52 , wherein the bacterial strain contains an overexpression of an oxidoreductase. 
     
     
         54 . The method of  claim 53 , wherein the oxidoreductase is a pyruvate:flavodoxin oxidoreductase (PFOR), 
     
     
         55 . The method of  claim 54 , wherein the PFOR is YdbK. 
     
     
         56 . The method of  claim 55 , wherein a recombinant YdbK gene is integrated into the chromosome or expressed from a plasmid. 
     
     
         57 . The method of any one of  claims 53  to  56 , wherein the strain comprises one or more P450 enzymes for the production of a terpenoid compound. 
     
     
         58 . The method of any one of  claims 53  to  57 , further comprising an overexpression of one or more of a flavodoxin, flavodoxin reductase, ferredoxin, and ferredoxin reductase. 
     
     
         59 . The method of any one of  claims 35  to  58 , wherein the bacterial strain has one or more additional modifications to increase co-factor availability or turnover, the one or more additional modifications optionally selected from recombinant expression of a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); overexpression of ydhV and/or ydhY; downregulation, inactivation, or deletion of gshA; and recombinant expression of CHAC1 and/or CHAC2. 
     
     
         60 . The method of any one of  claims 1  to  59 , wherein the bacterial strain has reduced or eliminated PDH mediated conversion of pyruvate to acetyl-CoA. 
     
     
         61 . The method of  claim 60 , wherein the bacterial strain expresses a mutant aceE, wherein the mutation is G267C. 
     
     
         62 . The method of  claim 60 , wherein the bacterial strain has a deletion or inactivation of aceE. 
     
     
         63 . The method of any one of  claims 1  to  62 , wherein the bacterial strain over expresses one or more non-native fdx and/or fldA homologs. 
     
     
         64 . The method of  claim 63 , wherein the fdx homolog is selected from Hm.fdx1 ( Heliobacterium modesticaldum ), Pa.fdx ( Pseudomonas aeruginosa ), Cv.fdx ( Allochromatium vinosum ), Ca.fdx ( Clostridium acetobulylieum ), Cp.fdx ( Clostridium pasteurianum ), Ev2. fdx ( Ectothiorhodospira shaposhnikovii ), Pp1.fdx ( Pseudomonas putida ) and Pp2.fdx ( Pseudomonas putida ), or a derivative of any of the foregoing. 
     
     
         65 . The method of  claim 63 , wherein the fidA homolog is selected from Ac.fldA2 ( Azotobacter chroococcum ), Av.fldA2 ( Azotobacter vinelandii ), and Bs.fldA ( B. subtilis ), or a derivative thereof. 
     
     
         66 . The method of any one of  claims 1  to  65 , wherein the terpene or terpenoid product comprises at least one compound selected from: Farnesene, Amorphadiene, 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. 
     
     
         67 . The method of any one of  claims 1  to  66 , wherein the strain comprises a terpene synthase enzyme that has one or more modifications to improve one or more of enzyme kinetics, terpene or terpenoid product profile, stability, and temperature tolerance. 
     
     
         68 . The method of any one of  claims 1  to  67 , wherein the strain is cultured with a C1, C2, C3, C4, C5, or C6 carbon source. 
     
     
         69 . The method of  claim 68 , wherein the carbon source is glucose, sucrose, or glycerol. 
     
     
         70 . The method of  claim 68  or  69 , wherein the bacterial strain is cultured at a temperature between 22° C. and 37° C. 
     
     
         71 . The method of any one of  claims 1  to  70 , wherein culturing step is a fed-batch process comprising a first phase where bacterial biomass is created, followed by a terpene or terpenoid production phase. 
     
     
         72 . The method of  claim 71 , wherein the culture is at least about 100 L. 
     
     
         73 . The method of  claim 72 , wherein the culture is from about 300 L to about 1,000,000 L. 
     
     
         74 . The method of any one of  claims 68  to  73 , wherein the culture is maintained under aerobic conditions. 
     
     
         75 . The method of  claim 74 , wherein the culture is maintained under micro-aerobic conditions. 
     
     
         76 . The method of  claim 74  or  75 , wherein the biomass production phase occurs under aerobic conditions, followed by reducing the oxygen levels after from about 10 to about 20 hours. 
     
     
         77 . The method of any one of  claims 68  to  76 , wherein the production phase includes feeding a nitrogen source, which optionally comprises ammonium hydroxide. 
     
     
         78 . The method of any one of  claims 68  to  77 , wherein the production phase includes feeding a carbon source, which optionally comprises glucose, sucrose, and/or glycerol. 
     
     
         79 . The method of any one of  claims 76  to  78 , wherein the nitrogen and carbon feeding is initiated when a predetermined amount of batch media is consumed. 
     
     
         80 . The method of  claim 78  or  79 , wherein the nitrogen feed rate is from about 8 L per hour to about 20 L per hour. 
     
     
         81 . The method of any one of  claims 68  to  80 , further comprising, monitoring or testing the accumulation of indole in the culture. 
     
     
         82 . The method of any one of  claims 68  to  81 , comprising monitoring or testing the accumulation of MEcPP in the culture. 
     
     
         83 . The method of any one of  claims 68  to  82 , further comprising, monitoring or testing the level of HMBPP in the cells. 
     
     
         84 . The method of any one of  claims 1  to  83 , further comprising, recovering the terpene or terpenoid product. 
     
     
         85 . The method of  claim 84 , wherein the product is a volatile terpene or terpenoid product. 
     
     
         86 . The method of  claim 85 , wherein the terpene or terpenoid product is recovered from an organic or hydrophobic phase that is mechanically separated from the culture. 
     
     
         87 . The method of  claim 86 , wherein the terpene or terpenoid product is harvested from the liquid and/or solid phase. 
     
     
         88 . The method of any one of  claims 85  to  87 , wherein the product is purified by distillation, which is optionally simple distillation, steam distillation, fractional distillation, wipe-film distillation, or continuous distillation. 
     
     
         89 . The method of any one of  claims 85  to  88 , wherein the product is purified by sequential extraction and purification. 
     
     
         90 . The method of any one of  claims 85  to  89 , wherein the product is purified by chromatography-based separation and recovery, which is optionally supercritical fluid chromatography. 
     
     
         91 . The method of  claim 84 , wherein the product is a non-volatile terpene or terpenoid product. 
     
     
         92 . The method of  claim 91 , wherein the product is an extracellular product recovered from the culture medium. 
     
     
         93 . The method of  claim 91 , wherein the product is an intracellular product recovered from harvested cell material. 
     
     
         94 . The method of any one of  claims 91  to  93 , wherein the product is poorly soluble and is recovered by filtration, optionally with solvent extraction. 
     
     
         95 . The method of  claim 94 , wherein the solvent is ethanol. 
     
     
         96 . The method of any one of  claims 91  to  95 , wherein the product is recovered by chromatography-based separation, which is optionally liquid chromatography. 
     
     
         97 . The method of  claim 96 , wherein the product is recovered by filtration. 
     
     
         98 . The method of any one of  claims 89  to  96 , wherein the product is recovered by sequential extraction and purification. 
     
     
         99 . The method of  claim 92  or  93 , wherein the product is crystallized out of solution. 
     
     
         100 . A method for making an industrial or consumer product, comprising, incorporating the terpene or terpenoid made according to the method of any one of  claims 1  to  100  into said industrial or consumer product. 
     
     
         101 . The method of  claim 100 , wherein the industrial or consumer product is a flavor product, a fragrance product, a sweetener, a cosmetic, a cleaning product, a detergent or soap, or a pest control product. 
     
     
         102 . The method of  claim 100 , wherein the industrial or consumer product is a food, beverage, texturant, pharmaceutical, tobacco product, nutraceutical, oral hygiene product, or cosmetic product. 
     
     
         103 . The method of any one of  claims 1  to  102 , wherein the bacterial strain or cell overexpresses one or more of geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS). 
     
     
         104 . A bacterial strain having one or more genetic modifications that increase production of products from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) precursors; the bacterial strain producing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through an MEP pathway, and the MEP pathway comprising one or more overexpressed genes; the strain converting the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis pathway; wherein said genetic modifications comprise:
 (a) recombinant or modified IspG and IspH enzymes, the IspG and IspH enzymes having balanced expression or activity to prevent accumulation of HMBPP intermediate,   (b) a recombinant or modified gene encoding an enzyme that enhances supply and/or transfer of electrons through 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,   (c) an inactivation or deletion, or reduced expression or activity, of aceE or aceE enzyme complex, and optionally   (d) a recombinant or modified idi gene to tune activity for higher terpene or terpenoid production.   
     
     
         105 . The bacterial strain of  claim 104 , wherein the bacterial strain is a bacteria selected from  Escherichia  spp.,  Bacillus  spp.,  Corynebacterium  spp.,  Rhodobacter  spp.,  Zymomonas  spp.,  Vibrio  spp., and  Pseudomonas  spp. 
     
     
         106 . The bacterial strain of  claim 105 , wherein the bacterial strain is a species selected from  Escherichia coli, Bacillus subtillus, corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens,  or  Pseudomonas putida.    
     
     
         107 . The bacterial strain of  claim 106 , wherein the bacterial strain is  E. coli.    
     
     
         108 . The bacterial strain of any one of  claims 104  to  107 , wherein the bacterial strain expresses dxs, ispD, ispF, and idi as recombinant genes, which are optionally expressed as an operon. 
     
     
         109 . The bacterial strain of  claim 108 , wherein the bacterial strain expresses dxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which are optionally expressed as 1, 2, or 3 operons. 
     
     
         110 . The bacterial strain of  claim 108  or  109 , wherein the recombinant genes of the MEP pathway are expressed from one or more plasmids or are integrated into the chromosome. 
     
     
         111 . The bacterial strain of any one of  claims 104  to  110 , wherein HMBPP does not accumulate at greater than 10 mg/g of dry cell weight. 
     
     
         112 . The bacterial strain of any one of  claims 104  to  111 , wherein the recombinant ispG and ispH genes comprise one or more beneficial mutations, or encode an IspG or IspH ortholog having improved properties or activity. 
     
     
         113 . The bacterial strain of any one of  claims 104  to  112 , wherein the expression of recombinant IspH is higher than the expression of the recombinant IspG. 
     
     
         114 . The bacterial strain of  claim 113 , wherein the recombinant ispH and ispG are expressed as an operon, with ispH positioned before ispG in the operon. 
     
     
         115 . The bacterial strain of  claim 114 , wherein the operon is expressed under control of a strong promoter. 
     
     
         116 . The bacterial strain of any one of  claims 112  to  115 , wherein the recombinant ispG and ispH genes are expressed from a plasmid or are integrated into the chromosome. 
     
     
         117 . The bacterial strain of any one of  claims 104  to  116 , wherein the expression or activity of a recombinant idi gene is tuned to increase terpene or terpenoid production, optionally by modifying the promoter strength, gene copy number, position in an operon, or ribosome binding site. 
     
     
         118 . The bacterial strain of any one of  claims 104  to  117 , wherein the bacterial strain contains an overexpression of an oxidoreductase. 
     
     
         119 . The bacterial strain of  claim 118 , wherein the oxidoreductase is a pyruvate:flavodoxin oxidoreductase (PFOR). 
     
     
         120 . The bacterial strain of  claim 119 , wherein the PFOR is YdbK. 
     
     
         121 . The bacterial strain of any one of  claims 104  to  120 , wherein the strain contains an enzyme that oxidizes pyruvate and/or reduces ferredoxin, which is optionally a recombinant YdbK gene, and which is optionally integrated into the chromosome or expressed from a plasmid. 
     
     
         122 . The bacterial strain of  claim 121 , wherein the strain comprises one or more P450 enzymes for the production of a terpenoid compound. 
     
     
         123 . The bacterial strain of  claim 121  or  122 , further comprising an overexpression of one or more of a flavodoxin, flavodoxin reductase, ferredoxin, and ferredoxin reductase. 
     
     
         124 . The bacterial strain of any one of  claims 104  to  123 , wherein the expression of the recombinant pgpB and/or nudB is tuned to provide higher terpene or terpenoid product titer, optionally by varying the promoter strength, gene copy number, position in an operon, and/or ribosome binding site. 
     
     
         125 . The bacterial strain of any one of  claims 104  to  124 , wherein the strain has one or more additional modifications selected from: recombinant expression of a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); overexpression of ydhV and/or ydhY; downregulation, inactivation, or deletion of gshA; and recombinant expression of CHAC1 and/or CHAC2. 
     
     
         126 . The bacterial strain of  claim 125 , wherein the recombinant pgpB and/or nudB is expressed under control of a weak or intermediate strength promoter. 
     
     
         127 . The bacterial strain of  claim 126 , wherein the recombinant pgpB or nudB is integrated into the chromosome or expressed from a plasmid. 
     
     
         128 . The bacterial strain of any one of  claims 104  to  127 , wherein the bacterial strain has reduced or eliminated PDH-mediated conversion of pyruvate to acetyl-CoA. 
     
     
         129 . The bacterial strain of  claim 128 , wherein the bacterial strain expresses a mutant aceE, wherein the mutation is G267C. 
     
     
         130 . The bacterial strain of  claim 129 , wherein the bacterial strain has a deletion or inactivation of aceE. 
     
     
         131 . The bacterial strain of any one of  claims 104  to  130 , wherein the bacterial strain overexpresses one or more non-native fdx and/or fldA homolog. 
     
     
         132 . The bacterial strain of  claim 131 , wherein the non-native fdx homolog is selected from Hm.fdx1 ( Heliobacterium modesticaldum ), Pa.fdx ( Pseudomonas aeruginosa ), Cv.fdx ( Allochromatium vinosum ), Ca.fdx ( Clostridium acetobutylicum ), Cp.fdx ( Clostridium pasteurianum ), and Ev2.fdx ( Ectothiorhodospira shaposhnikovii ), Ppl.fdx ( Pseudomonas putida ) and Pp2.fdx ( Pseudomonas putida ), or derivative of any of the foregoing. 
     
     
         133 . The bacterial strain of  claim 132 , wherein the fldA homolog is selected from Ac.fldA2 ( Azotobacter chroococcum ), Av.fldA2 ( Azotobacter vinelandii ), and Bs.fldA. ( B. subtilis ), or a derivative thereof. 
     
     
         134 . The bacterial strain of any one of  claims 104  to  133 , wherein the terpene or terpenoid product comprises at least one compound selected from: Farnesene, Amorphadiene, Artemisinic acid, Artemisinin, Bisabolol, Bisabolene, alpha-Sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal, Cubebol, Geraniol, Linionene, Menthol, Menthane, Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Rose oxide, Sabinene, Steviol, Steviol glycoside (including Rebaudioside D or Rebaudioside M), Taxadiene, Thymol, and Valencene. 
     
     
         135 . The bacterial strain of  claim 134 , wherein the strain comprises a terpene synthase enzyme that has one or more modifications to improve one or more of enzyme kinetics, terpene or terpenoid product profile, stability, and temperature tolerance. 
     
     
         136 . The bacterial strain of any one of  claims 104 - 135 , wherein the bacterial strain overexpresses one or more of geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS).