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Biotechnology

Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

Michael T. Guarnieri, Yat-Chen Chou, Davinia Salvachúa, Ali Mohagheghi, Peter C. St. John, Darren J. Peterson, Yannick J. Bomble, Gregg T. Beckham
Haruyuki Atomi, Editor
Michael T. Guarnieri
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Yat-Chen Chou
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Davinia Salvachúa
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Ali Mohagheghi
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Peter C. St. John
Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Darren J. Peterson
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Yannick J. Bomble
Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Gregg T. Beckham
National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, USA
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Haruyuki Atomi
Kyoto University
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DOI: 10.1128/AEM.00996-17
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  • FIG 1
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    FIG 1

    A. succinogenes metabolism and genetic modifications. Gray arrows indicate native flux from pentose and hexose sugars. Green arrows indicate overexpression targets. Red arrows indicate genetic knockout targets. 13DPG, 3-phospho-d-glyceroyl phosphate; 2PG, d-glycerate 2-phosphate; 3PG, 3-phospho-d-glycerate; 6PGC, 6-phospho-d-gluconate; AC, acetate; ACALD, acetaldehyde; ACCOA, acetyl-CoA; ACTP, acetyl phosphate; DHAP, dihydroxyacetone phosphate; E4P, d-erythrose 4-phosphate; ETOH, ethanol; F6P, d-fructose 6-phosphate; FDP, d-fructose 1,6-bisphosphate; FOR, formate; FUM, fumarate; G3P, glyceraldehyde 3-phosphate; G6P, d-glucose 6-phosphate; GLC, d-glucose; MAL, l-malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; R5P, alpha-d-ribose 5-phosphate; RU5P, d-ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; SUCC, succinate; XU5P, d-xylulose 5-phosphate; XYL, d-xylose; XU, d-xylulose; LAC, d-lactate; pflB, pyruvate formate lyase; ackA, acetate kinase; PCK, phosphoenolpyruvate carboxykinase; MDH, malate dehydrogenase; FUM, fumarase.

  • FIG 2
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    FIG 2

    Example of a knockout cassette, ΔpflB (top) and plasmid vector for the overexpression of SA pathway genes (bottom). A.s. Ori frag, DNA fragment required for replication in Actinobacillus succinogenes.

  • FIG 3
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    FIG 3

    Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) compared to those of the knockout strains ΔackA, ΔpflB, and ΔpflB ΔackA. Scatter plots present bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production: SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H). The bar graphs show SA titers (I), yields and metabolic yields (J), and overall and maximum instantaneous productivities (K). SA “yield” is the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in all cases (since sugars were mostly consumed at that time). The maximum instantaneous productivity was observed at 12 h for the control and 48 h for the knockout strains.

  • FIG 4
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    FIG 4

    Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) and overexpression constructs such as the single-gene overexpression strains pFUM, pPCK, and pMDH and the triple-gene overexpression strain pPFM. The scatter plots show bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production, SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H), for the wild-type, pMDH, and pPFM strains. The scatter plots corresponding to pMDH and pPFM strains are shown in Fig. S1 in the supplemental material. The bar graphs show (I) SA titers, (J) yields and metabolic yields, and (K) overall and maximum instantaneous productivities for the five strains. SA “yield” is calculated as the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield but considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in wild type, pMDH, and pPFM and at 72 h (and marked with an asterisk) in pFUM and pPCK. The maximum instantaneous productivity was observed at 12 h for the control, 25 h for pMDH and pPFM, 21 h for pFUM, and 24 h for pPCK.

  • FIG 5
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    FIG 5

    Evaluation of different fermentation and metabolic parameters in wild-type A. succinogenes (130Z) compared to those of the ΔackA, ΔpflB, and ΔpflB ΔackA strains carrying pPMF. The scatter plots show bacterial growth (A), utilization of xylose (B), and utilization of glucose (C). (D to H) Acid production: SA (D), acetic acid (AA) (E), formic acid (FA) (F), pyruvic acid (PA) (G), and lactic acid (LA) (H). The bar graphs show SA titers (I), yields and metabolic yields (J), and overall and maximum instantaneous productivities (K). SA “yield” is calculated as the ratio of SA (g/liter) and the sugars consumed (g/liter) at the end of the fermentation. SA “metabolic yield” is calculated as the yield but considering the dilution factor at the end of the fermentation. The overall productivity was calculated at 96 h in the wild-type strain and at 144 h in the rest of the strains. The maximum instantaneous productivity was observed at 12 h for the control, ∼72 h for the ΔackA and ΔpflB strains carrying pPMF, and 98 h for the ΔpflB ΔackA strain carrying pPMF.

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  • TABLE 1

    Bacterial strains and plasmids

    Strain or plasmidDescriptionReference or source
    Strains
        Escherichia coli
            DH5-αF− Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK− mK−) phoA supE44 λ− thi-1 gyrA96 relA1Invitrogen
        Actinobacillus succinogenes
            130ZWild type
            ΔpflB::bla130Z derivative; contains loxP-bla-lox loxP integration replacing pflB; AmprThis study
            ΔpflBΔpflB::bla derivative; contains one loxP siteThis study
            ΔackA130Z derivative; contains ackA knockoutThis study
            ΔpflBΔackA130Z derivative; contains pflB and ackA knockoutsThis study
    Plasmids
        pLGZ920E. coli-A. succinogenes shuttle vector; Ampr; contains PpckA22
        pL920CmpLGZ920 derivative; Cmr; Cmr gene under the control of PblaThis study
        pPCKpLGZ920 derivative; Ampr; pckA under the control of PpckAThis study
        pMDHpLGZ920 derivative; Ampr; mdh under the control of PmdhThis study
        pFUMpLGZ920 derivative; Ampr; fum under the control of PpckAThis study
        pPMFpLGZ920 derivative; Cmr; pckA under the control of PpckA; mdh under the control of Pmdh; fum under the control of PfumThis study
        pLCreCmpLGZ920 derivative; Cmr; cre under the control of PpckAThis study
  • TABLE 2

    Primers used in this study

    PrimerSequence (5′ to 3′)Usage
    YC-1CGTTAACCGTGGGAATCAGTTTGTTAGGAATGpflB up fragment; Gibson Assembly
    YC-2CGAAGTTATTGTAATACTTCCTTTTGCTAGTATTGATAATGAAATCCTGTAAGpflB up fragment; Gibson Assembly
    YC-3CCATAACTTCGTATAATGTATGCTATACGAAGTTATTTGGGGTAACGTAATAAAAATGpflB down fragment; Gibson Assembly
    YC-4TCTCTCCTTCGCGGAATAAAATATCCACTTCpflB down fragment; Gibson Assembly
    YC-5CTAGCAAAAGGAAGTATTACAATAACTTCGTATAATGTATGCTATACGAAGTTATAATTCTTGAAGACGAAAGGGCCTCGTGbla-loxP fragment; Gibson Assembly
    YC-6CGTATAGCATACATTATACGAAGTTATGGGGTCTGACGCTCAGTGGAACGAAAACTCbla-loxP fragment; Gibson Assembly
    YC-7GCAGCAATAGAGGAAACACGGTTTGpckA fragment
    YC-8GGATTTGGTACCGTGCCGGCGGCCTAATAACCTGpckA fragment, KpnI site underlined
    YC-9CGAACCGAAGCGTTCCTGCGCGAGTAACGCmdh fragment; blunt-end ligation
    YC-10GCTTCCCATTAATCAAACGGCGGmdh fragment; blunt-end ligation
    YC-11CTCAAACAAACCGTGTTTCCTCTATTGCTGCpLGZ920 to linearize for mdh cloning
    YC-12AATTATCAATGAGGTGAAGTATGACATTTCGTATTGAAAAAGACACfum CDS fragment; Gibson Assembly
    YC-13GAATTCGAGCTCGGTACCCGGGGATCCCTGACCGTCTTCGGTGAATACTGATATAGfum CDS fragment; Gibson Assembly
    YC-14ACTTCACCTCATTGATAATTTAAAATTAAAAATCCpLGZ920 to linearize for fum cloning
    YC-15GGATCCCCGGGTACCGAGCTCGAATTCACTGpLGZ920 to linearize for mdh or fum cloning
    YC-16CGGTACCGAGCTCGAATTCACTGGCCGTCGpPCK to linearize for mdh and fum cloning
    YC-17GTCATCTTAACAGGTTATTAGGCCGCCGGCApPCK to linearize for mdh and fum cloning
    YC-18CCTTCGGCCGGCCCTGCCGTTTCGGAAAACTCACGCTTTACCCGfum fragment; FseI site underlined
    YC-19CCTTCGGCCGGCCCATAAAGAATCCAAGATAAACGAATTGGCfum fragment; FseI site underlined

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Evaluation of different fermentation and metabolic parameters (Fig. S1 and S2), t test data (Table S1), details of the carbon balance calculations (Table S2), and details of the degree of reduction balance (Table S3).

      PDF, 204K

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Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis
Michael T. Guarnieri, Yat-Chen Chou, Davinia Salvachúa, Ali Mohagheghi, Peter C. St. John, Darren J. Peterson, Yannick J. Bomble, Gregg T. Beckham
Appl. Environ. Microbiol. Aug 2017, 83 (17) e00996-17; DOI: 10.1128/AEM.00996-17

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Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis
Michael T. Guarnieri, Yat-Chen Chou, Davinia Salvachúa, Ali Mohagheghi, Peter C. St. John, Darren J. Peterson, Yannick J. Bomble, Gregg T. Beckham
Appl. Environ. Microbiol. Aug 2017, 83 (17) e00996-17; DOI: 10.1128/AEM.00996-17
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    • ABSTRACT
    • INTRODUCTION
    • RESULTS
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KEYWORDS

Actinobacillus succinogenes
biochemical
biorefinery
fermentation
metabolic engineering
succinic acid

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