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

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.

Leveraging substrate flexibility and product selectivity of acetogens in two‐stage systems for chemical production

“…Most of H 2 ‐based CO 2 fermentations are single stage processes. Even though the number of added‐value compounds achievable by H 2 ‐based CO 2 fermentation is on the rise (Lauer et al, 2022 ; Mook et al, 2022 ; Weitz et al, 2021 ) using metabolically engineered acetogens (Bourgade et al, 2021 ; Lee et al, 2022 ; Song et al, 2022 ), high‐profile demonstrations of process scalability is restricted to acetate and ethanol (Fackler et al, 2021 ). A possible strategy to broaden the product spectrum of CO 2 gas fermentation consists of considering the gas fermentation mediated by acetogens in the context of two‐stage processes.…”

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO

“…Although butanol was supplied exogenously in this work, certain acetogens such as C. carboxidivorans can produce naturally butanol and hexanol from CO [39,40]. Also, engineered acetogens including C. autoethanogenum have been described to autotrophically produce butanol [37,[39][40][41][42][43]. Besides longer-chain alcohols such as butanol and hexanol, C. carboxidivorans can naturally produce longer-chain acyl-CoAs such as butyryl-CoA which could serve as basis for further expansion of autotrophic ester biosynthesis.…”

Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production

“…[473][474][475][476] Lauer et al expressed 13 genes from C. kluyveri and C. acetobutylicum in C. ljungdahlii that could convert acetyl-CoA produced by the Wood-Ljungdahl pathway into butyryl-CoA and hexanoyl-CoA, eventually forming butanol (174 mg L À1 ) and hexanol alcohols (15 mg L À1 ). 477 Leang et al redirected carbon flow by mutating adhE1 and adhE2 (putative bifunctional aldehyde/alcohol dehydrogenases) in C. ljungdahlii to enhance acetate synthesis. 478 In another example, to increase carbon flux toward 3-hydroxybutyrate (3HB) synthesis, Woolston et al downregulated the expression level of phosphotransacetylase (pta) and aldehyde:ferredoxin oxidoreductase (aor2) in C. ljungdahlii by the inducible CRISPRi system, resulting in a significant decrease in acetate production and a concomitant increase in 3HB production in heterotrophic conditions.…”