Recent progress in engineering Clostridium autoethanogenum to synthesize the biochemicals and biocommodities

Abstract

Excessive mining and utilization fossil fuels has led to drastic environmental consequences, which will contribute to global warming and cause further climate change with severe consequences for the human population. The magnitude of these challenges requires several approaches to develop sustainable alternatives for chemicals and fuels production. In this context, biological processes, mainly microbial fermentation, have gained particular interest. For example, autotrophic gas-fermenting acetogenic bacteria are capable of converting CO, CO₂ and H₂ into biomass and multiple metabolites through Wood-Ljungdahl pathway, which can be exploited for large-scale fermentation processes to sustainably produce bulk biochemicals and biofuels (e.g. acetate and ethanol) from syngas. Clostridium autoethanogenum is one representative of these chemoautotrophic bacteria and considered as the model for the gas fermentation. Recently, the development of synthetic biology toolbox for this strain has enabled us to study and genetically improve their metabolic capability in gas fermentation. In this review, we will summarize the recent progress involved in the understanding of physiological mechanism and strain engineering for C. autoethanogenum, and provide our perspectives on the future development about the basic biology and engineering biology of this strain.

Keywords: Biofuel; Carbon fixation; Clostridium autoethanogenum; Gas fermentation; Genetic engineering.

Fig. 1

Detailed mechanism of WLP and energy conservation mechanism in C. autoethanogenum. NAD⁺: nicotinamide adenine dinucleotide; NADP⁺: nicotinamide adenine dinucleotide phosphate. Fdh, formate dehydrogenase; THF, tetrahydrofolate; CoFeSP, corrinoid iron-sulfur protein; Acetyl-CoA, acetyl coenzyme A; CODH: carbon monoxide dehydrogenase; ADP, adenosine diphosphate; Pi, phosphate; ATP, adenosine triphosphate; Etf: electron transfer flavoprotein; The electron-bifurcating enzyme Nfn is responsible for the interconvesion of Fd, NADH and NADPH. C. autoethanogenum oxidizes hydrogen an enzyme complex of hydrogenase (HytA-E) and formate dehydrogenase (Fdh), achieving the reduction of ferredoxin (Fd) and NAD⁺ or NADP⁺. The membrane-bound Rnf complex and ATPase are proposed to couple the electron transfer from reduced ferredoxin (Fd²⁻) to NAD⁺ with the generation of ATP.

Fig. 2

The genetic tools available for the C. autoethanogenum. ClosTron method is most widely used tools to disrupt gene expression in C. autoethanogenum. The CRISPR-Cas genome editing system has been efficiently used in C. autoethanogenum. The homologous recombination system for gene deletion is based on double crossover. CRISPR: clusted regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; Chr: chromosome; DSB: double-stranded break; HR: homologous recombination; LHA: left homology arm; RHA: right homology arm.

Fig. 3

The main products of wild-type or engineered C. autoethanogenum. NAD⁺: nicotinamide adenine dinucleotide; NADP⁺: nicotinamide adenine dinucleotide phosphate. ThlA, thiolase; CoA-SH: Coenzyme A; Acetyl-CoA: Acetyl Coenzyme A; Hbd: 3-hydroxybutyryl-CoA dehydrogenase; Crt: crotonase; Bcd: butyryl-CoA dehydrogenase; Fd²⁻: reduced ferredoxin; Fd: ferredoxin; Ptb: phosphotransbutyrylase; Pi:phosphate; BuK: butyrate kinase; ATP: adenosine triphosphate; ADP: adenosine diphosphate; AdhE: aldehyde/alcohol dehydrogenase; CtfA/B: electron-transferring flavoprotein A and electron-transferring flavoprotein B; 2,3-Bdh: 2,3-butanediol dehydrogenase; Adc:acetoacetate decarboxylase; sAdh: secondary alcohol dehydrogenase; Pfor: pyruvate:ferredoxin oxidoreductase; Als: acetolactate synthase; BudA: acetoin decarboxylase; Ldh: lactate dehydrogenase; Pta: phosphotransacetylase; AOR: aldehyde:ferredoxin oxidoreductase; Ack: acetate kinase; Cit/D/E/F: citrate lyase; AcnB: aconitase; AceA: isocitrate lyase; GhrA: glyoxylate reductase; AldA: Glycolaldehyde dehydrogenase; fucO: lactaldehyde reductase. r-Box: reverse ꞵ-oxidation.