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Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

ABSTRACT: Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

ORGANISM(S): Clostridium autoethanogenum

PROVIDER: GSE115837 | GEO | 2019/02/03

REPOSITORIES: GEO

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Project description:Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model (GEM). CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a four-fold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by five-fold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin-reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

Project description:A hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on a variety of CO-containing feed stocks such as by-product gas generated from steel-mill process. In this study we applied a long-term adaptive evolution to enhance H2 productivity. Through serial transfer of cell cultures with carbon monoxide (CO) as an energy source, we observed physiological changes in cell density, CO consumption rate and H2 production rate. To understand the underlying mechanism for the changes, we performed systems analysis of genomic, transcriptomic and epigenomic data. Genomic analysis of an evolved strain, designated as 156T, revealed that single or multiple bases were substituted, deleted or inserted in the sequence of the parental strain. A single point mutation in a putative transcriptional regulator (TON_1525) seemed to play a pivotal role in changing cellular phenotypes by increasing the expression level of genes of a CO dehydrogenase-hydrogenase gene cluster. Additionally, a mutation in an aromatic amino acid permease (TON_0820) contributed to increasing cell growth. Transcriptomic analysis revealed that genes belonging to the categories of transcription, translation and energy metabolism in archaeal Clusters of Orthologous Genes (arCOGs) were significantly changed. In particular, genes involved in energy conservation via CO oxidation were highly upregulated. Epigenomic analysis suggested that methylation change might be a way of gene regulation in a hyperthermophilic archaeon. The evolved 156T strain showed highly enhanced hydrogen productivity with CO at high flow rates of 800 ml min-1 and above, indicating the adaptation rendered the strain less sensitive to high CO. The 156T strain was demonstrated to be appropriate for H2 production by using synthetic gas obtained by coal gasification. This study is the first example to show that evolutionary engineering is very effective in enhancing H2 productivity of a hyperthermophilic archaeon on CO

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Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

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