Abstract
Genes responsible for the anaerobic catabolism of benzoate in the thermophilic archaeon Ferroglobus placidus were expressed in the thermophilic lignocellulose-degrading bacterium Caldicellulosiruptor bescii, as a first step to engineering this bacterium to degrade this lignin metabolite. The benzoyl-CoA ligase gene was expressed individually, and in combination with benzoyl-CoA reductase and a putative benzoate transporter. This effort also assessed heterologous expression from a synthetically designed operon whereby each coding sequence was proceeded by a unique C. bescii ribosome binding site sequence. The F. placidicus benzoyl-CoA ligase gene was expressed in C. bescii to produce a full-length protein with catalytic activity. A synthetic 6-gene operon encoding three enzymes involved in benzoate degradation was also successfully expressed in C. bescii as determined by RNA analysis, though the protein products of only four of the genes were detected. The discord between the mRNA and protein measurements, especially considering the two genes lacking apparent protein abundance, suggests variable effectiveness of the ribosome binding site sequences utilized in this synthetic operon. The engineered strains did not degrade benzoate. Although the heterologously expressed gene encoding benzoyl-CoA ligase yielded a protein that was catalytically active in vitro, expression in C. bescii of six benzoate catabolism-related genes combined in a synthetic operon yielded mixed results. More effective expression and in vivo activity might be brought about by validating and using different ribosome binding sites and different promoters. Expressing additional pathway components may alleviate any pathway inhibition and enhance benzoyl-CoA reductase activity.
Highlights
Consolidated bioprocessing (CBP) is projected to be an inexpensive route to renewably produce bioethanol from lignocellulosic biomass (Lynd et al 2005, 2008)
The C. bescii expression platform used in this study has enabled C. bescii to produce ethanol (Chung et al 2014; Chung et al 2015a, b, c, d), detoxify furan aldehydes (Chung et al 2015a, b, c, d), and increased cellulolytic capability (Chung et al 2015a, b, c, d)
Pyrophosphate is a product in the biochemical reaction carried out by benzoyl-coenzyme A (CoA) ligase, and carryover of this chemical in lysates used in in vitro assays may be inhibiting the activity of the heterologous benzoyl-CoA ligase
Summary
Consolidated bioprocessing (CBP) is projected to be an inexpensive route to renewably produce bioethanol from lignocellulosic biomass (Lynd et al 2005, 2008). Caldicellulosiruptor bescii has emerged as a promising candidate microbial biocatalyst to achieve this (Chung et al 2014), and it is actively being engineered to produce a more effective consolidated bioprocessing host organism It is well suited for this, as it natively contains the ability to solubilize lignocellulosic biomass, using its array. Organisms that catabolize lignin monomers and other aromatic species initially modify these molecules using specialized pathways with narrow and non-overlapping specificity These intermediates are transformed, or ‘funneled’ to one of a few common metabolic intermediates, including benzoyl-Coenzyme A in anaerobic microorganisms (Harwood et al 1998). Dearomatization and subsequent degradation reactions are carried out on aromatic molecules activated with CoA moieties through a thioester bond This CoAthioester reduces the resonance in aromatic moieties by drawing electrons away from otherwise stable resonance structures. This reduces the energetic demand of aromatic ring reduction (Fuchs et al 2011)
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