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

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

PROVIDER: PRJNA476240 | ENA |

REPOSITORIES: ENA

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

Project description: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.

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Metabolic and transcriptional response to the induction of a heterologous poly-3-hydroxybutyrate pathway in Phaeodactylum tricornutum

Project description:The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host and how this influences trans- and native-gene expression. In this study we used episomal vectors to express the heterologous poly-3-hydroxybutyrate (PHB) pathway in diatoms, which produces precursors for polymeric bioplastics from acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N-limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferentially shunted into the endogenous metabolism over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA.

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