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. 2022 May 14;21(1):85.
doi: 10.1186/s12934-022-01802-8.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

Affiliations

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

Ira Lauer et al. Microb Cell Fact. .

Abstract

Background: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging.

Results: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L-1 butanol and 133 mg L-1 hexanol from fructose in complex medium, and 174 mg L-1 butanol and 15 mg L-1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L-1) at the expense of butanol (158 mg L-1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L-1 butanol and 393 mg L-1 hexanol.

Conclusions: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Keywords: Acetogens; Biofuels; Butanol; Hexanol; Syngas fermentation; Wood-Ljungdahl pathway.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of fermentation products and CoA-ester intermediates in C. kluyveri. A GC–MS analysis of fermentation broth from a culture of C. kluyveri revealed the presence of butyrate, caproate, and traces of butanol and hexanol. Ethanol and acetate were derived from the medium and 1,3-propanediol served as the internal standard. B A volume of 50 mL of a logarithmically grown C. kluyveri culture (OD600nm = 0.9) was collected by centrifugation and re-suspended in 500 µL of a quenching solution. The cell debris was removed by centrifugation and sterile filtration. CoA-esters (acetyl-CoA, butyryl-CoA, 3-hydroxybutyryl-CoA, hexenoyl-CoA, hexanoyl-CoA and 3-hydroxylhexanoyl-CoA) were measured by LC–MS/MS with octanoyl-CoA as the internal standard
Fig. 2
Fig. 2
Designed pathway for butanol and hexanol biosynthesis and the corresponding plasmids. A Schematic representation of the heterologous butanol and hexanol biosynthesis pathway. ThlA1/2: thiolase A1/2; Hbd1/2: hydroxybutyryl-CoA-dehydrogenase 1/2; Crt1/2: crotonase 1/2; Bcd-EtfA/B complex = Bcd 1/2: butyryl-CoA-dehydrogenase 1/2; EtfA/B 1/2: electron transferring protein A/B 1/2 (all genes from C. kluyveri); AdhE2: bifunctional aldehyde-alcohol-dehydrogenase from C. acetobutylicum. Two molecules of acetyl-CoA are condensed by acetyl-CoA acetyltransferase (ThlA) to form acetoacetyl-CoA, which is then reduced to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (Hbd) with NADH as the electron donor. In the next step, 3-hydroxybutyryl-CoA is dehydrated by crotonase (Crt) to form crotonyl-CoA, which is then reduced with NADH and oxidized ferredoxin (Fd) by a Flavin-based electron bifurcating butyryl-CoA dehydrogenase (Bcd) to form butyryl-CoA. From here, butanol can be produced via two reduction steps with NADH as the electron donor, catalyzed by a bifunctional aldehyde dehydrogenase/alcohol dehydrogenase (AdhE2). Alternatively, for the generation of hexanol, a third acetyl group derived from acetyl-CoA is added to the butyryl-CoA to produce 3-ketohexanoyl-CoA. Analogous to the steps in which acetoacetyl-CoA is converted to butyryl-CoA, 3-ketohexanoyl-CoA is again reduced with NADH, dehydrated, and reduced again by a bifurcating butyryl-CoA dehydrogenase to form hexanoyl-CoA. A bifunctional aldehyde dehydrogenase/alcohol dehydrogenase then catalyzes the final reduction steps to form hexanol, with NADH as the electron donor. Butyrate and caproate are potential side products and can be derived from butyryl-CoA and hexanoyl-CoA, respectively, with butyryl phosphate and caproyl phosphate as the corresponding intermediates. After reduction of the acids butyrate and caproate to the respective aldehydes via the native AOR (aldehyde:ferredoxin oxidoreductase) the molecules can be further converted to the alcohols butanol and hexanol. These alternative pathways allow the conservation of energy in form of ATP production via substrate level phosphorylation and are shown in grey (adapted from [26]) B Schematic representation of pIM Hex#15. The integration cassette consisting of a butanol-hexanol biosynthesis cluster and the adjacent ermC sequence is flanked by the mycomar sites (ITR–inverted terminal repeats) which allow integration catalyzed by the xylose-inducible Himar1 transposase. C Schematic representation of the Ccar1 cassette with the butanol/ hexanol biosynthesis cluster of C. carboxidivorans (pIM Ccar1)
Fig. 3
Fig. 3
Comparison of plasmid-carrying and genomic integration strains. Maximum product concentrations and optical densities achieved by C. lju pIM Hex#15 (A, B) and C. lju Hex#15gInt (D, E) in serum bottle cultivations (A, D) and 2-L fermentations (B, E) with ATCC 1754 medium continuously supplied with 20% CO2 and 80% H2 as the carbon and energy sources. Data are means ± SD (n = 3 biological replicates, data in B: n = 1). Enzyme concentrations as determined by LC–MS/MS from C. lju pIM Hex#15 (C) and C. lju Hex#15gInt (F) in the early logarithmic (OD600 ~ 0.3–0.5), late logarithmic (OD600 ~ 0.5–1.0) and stationary phases (OD600 > 1.0) in a 2-L fermentation
Fig. 4
Fig. 4
Maximum product concentrations and optical densities achieved during the stationary phase with strains C. lju Hex#15gInt ΔermC and C. lju Hex#15gInt Ccar1gInt at 37 °C (A) and 30 °C (B) in 125-mL serum bottles containing 25 mL ATCC 1754 medium supplied with 20% CO2 and 80% H2 as the carbon and energy sources. Data are means ± SD (n = 3 biological replicates)
Fig. 5
Fig. 5
Fermentation of C. lju Hex#15gInt Ccar1gInt at 30 °C in ATCC 1754 medium at 2-L scale with a continuous supply of 20% CO2 and 80% H2 as the carbon and energy sources. AE Fermentation with pH control (pH 6.0). FJ Fermentation without initial pH control. A, F Fermentation course showing the formation of biomass and heterologous products over time. B, G The pH of the fermentation broth plotted over time. C, H Maximum product concentrations and optical densities. D, I Abundance of heterologous enzymes encoded by the Hex#15 construct. E, J Abundance of heterologous enzymes encoded by the Ccar1 construct

References

    1. Diender M, Stams AJ, Sousa DZ. Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas. Biotechnol Biofuels. 2016;9:82–92. doi: 10.1186/s13068-016-0495-0. - DOI - PMC - PubMed
    1. Dürre P. Biobutanol: an attractive biofuel. Biotechnol J. 2007;2:1525–1534. doi: 10.1002/biot.200700168. - DOI - PubMed
    1. Vojtisek-Lom M, Beránek V, Mikuška P, Křůmal K, Coufalík P, Sikorová J, Topinka J. Blends of butanol and hydrotreated vegetable oils as drop-in replacement for diesel engines: effects on combustion and emissions. Fuel. 2017;197:407–421. doi: 10.1016/j.fuel.2017.02.039. - DOI
    1. De Poures MV, Sathiyagnanam AP, Rana D, Rajesh Kumar B, Saravanan S. 1-Hexanol as a sustainable biofuel in DI diesel engines and its effect on combustion and emissions under the influence of injection timing and exhaust gas recirculation (EGR) Appl Therm Eng. 2017;113:1505–1513. doi: 10.1016/j.applthermaleng.2016.11.164. - DOI
    1. Breitkreuz K, Menne A, Kraft A. New process for sustainable fuels and chemicals from bio-based alcohols and acetone. Biofuels Bioprod Biorefin. 2014;8:504–515. doi: 10.1002/bbb.1484. - DOI

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