| Summary: | Carbon monoxide is a greenhouse gas mainly produced by anthropogenic sources, but the electrons liberated for CO oxidation toCO2 have a very low potential of -520 mV. Interestingly, CO can be produced in the syngas by the gasification of renewable sources. Hence, to exploit these low potential electrons and carbon from renewable sources, the general objective of this project was to engineer Escherichia coli to use carbon monoxide as the sole energy and carbon source. One of the engineering points was the heterologous expression in E. coli of the glyceraldehyde-3-phosphate oxidoreductase (GAPOR) to function in the gluconeogenic direction to fix the low potential electrons from the carbon monoxide.
The already characterised GAPOR from the mesophilic Methanococcus maripaludis has been produced in a mutant E. coli strain optimised for crude extract enzyme assay. Three genes were deleted from the E. coli Rosetta-gami 2(DE3): iscR to improve the availability of the [4Fe-4S] cofactor in the cell; selA and hypF to diminish the benzyl viologen reduction background activity observed in the preliminary test. The glyceraldehyde-3-phosphate (G3P) oxidation activity of GAPOR in cell crude extract and affinity purified has been tested, but no activity was observed. In parallel, the enzymes from the M. maripaludis molybdenum cofactor biosynthesis pathway were co-expressed with GAPOR to improve the G3P-dependent activity. However, no enzymatic activity has been detected. A new LC-MS method of molybdopterin cofactor identification has been used to detect the cofactor, but so far, no cofactor was observed in the protein extract. The expression of the newly discovered bacterial homologous genes of GAPOR: GOR-SL from the mesophilic bacterium Geosporobacter ferrireducens, was tentatively expressed in E. coli, but no soluble enzyme has been detected. The expression of both proteins was tested in aldehyde ferredoxin oxidoreductase expressing bacteria Clostridium autoethanogenum and Clostridium acetobutylicum under the control of different inducible and constitutive promoters. GAPOR and GOR expression has been observed in the soluble fraction of C. acetobutylicum. NoG3P-dependent activity has been detected during the enzyme assay on the purified enzyme and no impact on the cell solvent production in batch culture was either seen.
This study did not allow the production of active enzymes; nevertheless, it opened a new path of research in the archaeal molybdenum cofactor biosynthesis pathway and the pursuit of the investigation in GAPOR and GOR-SL-like protein in C. acetobutylicum.
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