Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw

Bashir, Zeenat; Sheng, Lili; Anil, Annamma; Lali, Arvind; Minton, Nigel P.; Zhang, Ying

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 abile 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 fuscii, whereasstrain 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 2-fold 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 hours 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 to desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels.

Citation

Bashir, Z., Sheng, L., Anil, A., Lali, A., Minton, N. P., & Zhang, Y. (2019). Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw. Biotechnology for Biofuels, 12(1), Article 199. https://doi.org/10.1186/s13068-019-1540-6

• Journal Article Type: Article
• Acceptance Date: Aug 5, 2019
• Online Publication Date: Aug 20, 2019
• Publication Date: 2019-12
• Deposit Date: Aug 12, 2019
• Publicly Available Date: Aug 21, 2019
• Journal: Biotechnology for Biofuels
• Electronic ISSN: 1754-6834
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 12
• Issue: 1
• Article Number: 199
• DOI: https://doi.org/10.1186/s13068-019-1540-6
• Public URL: https://nottingham-repository.worktribe.com/output/2418518
• Publisher URL: https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1540-6
• Additional Information: Received: 14 January 2019; Accepted: 6 August 2019; First Online: 20 August 2019; : Not applicable.; : All authors consented to the publication of this work.; : The authors declare that they have no competing interests.
• Contract Date: Aug 21, 2019