Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw

Abstract

Background A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are able to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP. Results In this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (β-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fusca ; whereas, strain BZ9 contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii . All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately twofold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 h of growth. Conclusion We engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass into desired products represents a new paradigm for C...

Strategies towards Reduction of Cellulases Consumption: Debottlenecking the Economics of Lignocellulosics Valorization Processes

“…The ethanologenic bacteria Zymomonas mobilis, which is extensively studied for second generation ethanol, was engineered to secrete hydrolytic enzymes and was able to produce 43 g/L and 32 g/L of ethanol from CMC and NaOH-pretreated sugar cane bagasse, respectively [119]. In a combination of both strategies used for the development of CBP microorganisms, the bacteria Escherichia coli and Geobacillus thermoglucosidasius were heavily engineered in order to simultaneously enable them with cellulase-secreting abilities and improved capacity to produce ethanol [120,121]. These resulted in 0.19 g/L of ethanol from nitric acid/ammonia pretreated wheat straw using the modified G. thermoglucosidasius strain and 7.6 g/L of ethanol from acid pretreated Arundo donax cane with the E. coli strain.…”

A systematic review of the genera Geobacillus and Parageobacillus: their evolution, current taxonomic status and major applications

“…thermoglucosidasius strains BZ243 and BZ244, together with the engineered ethanologenic G. thermoglucosidasius strain LS242 (∆ ldh , pdh up , ∆ pfl ) as a control, microaerobic fermentation was demonstrated [98]. Culture supernatants from these strains were used for measurement of ethanol production by HPLC at growth time points of 24, 48 and 72 h. The results showed 2-fold (4.2 mM ethanol) and 1.6-fold (3.7 mM ethanol) increase in ethanol production in the recombinant G.…”

“…In a recent study, Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to utilize lignocellulosic biomass. By assimilation of the cglT (β-1, 4-glucosidase) gene from Thermoanaerobacter brockii into the genome, two different strains, BZ9 and BZ10, were created and genes encoding different cellulolytic enzymes were localized on autonomous plasmids [98]. To investigate the production of ethanol from pretreated wheat straw using the recombinant G.…”

Digestibility of Wheat and Cattail Biomass Using a Co-culture of Thermophilic Anaerobes for Consolidated Bioprocessing

“…Although T. fusca does not naturally produce any ethanol, it has been engineered to produce propanol, indicating its potential use for biofuel production [105]. While the oligosaccharide-utilizing Geobacillus thermoglucosidasius is relatively well-known for its CBP potential [47,106], natively-cellulolytic, ethanolgenic species from the Geobacillus genus have also been discovered [107,108]. While these nativelycellulolytic Geobacillus species have naturally low ethanol yields [108], the engineering of highethanol phenotypes in closely-related species [51] highlights their potential.…”