Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers

• Published in: Applied and environmental microbiology Vol. 90; no. 1; p. e0195123
• Main Authors: Tanwee, Tania N N, Lipscomb, Gina L, Vailionis, Jason L, Zhang, Ke, Bing, Ryan G, O'Quinn, Hailey C, Poole, Farris L, Zhang, Ying, Kelly, Robert M, Adams, Michael W W
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 24.01.2024
• Subjects: 1,3-Butadiene; Acetoin; Alcohol dehydrogenase; Applied and Industrial Microbiology; Bacteria; Bacteria - metabolism; Base Composition; Biomass; Biosynthesis; Biotechnology; Butanediol; Butylene Glycols; Caldicellulosiruptor; Caldicellulosiruptor bescii; Cellulose; Cellulose - metabolism; Clostridiales - metabolism; Crystalline cellulose; Fuel production; Genomes; Hemicellulose; Lactates; Lignocellulose; Metabolic Engineering; Metabolism; Methyl ethyl ketone; Microorganisms; Panicum virgatum; Phylogeny; Plant biomass; Plants - metabolism; Product inhibition; Pyruvic acid; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Stereospecificity; Substrates; Sugars; Xylan; Xylans; Caldicellulosiruptor bescii; acetoin; metabolic flux; 2,3-butanediol; redox balance; thermophile; lignocellulose
• ISSN: 0099-2240; 1098-5336; 1098-5336
• DOI: 10.1128/aem.01951-23

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in , an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C and C sugars. In addition grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of .IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.