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Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824

Willson, Benjamin J.; Kovács, Katalin; Wilding-Steele, Tom; Markus, Robert; Winzer, Klaus; Minton, Nigel P.

Authors

Benjamin J. Willson

Tom Wilding-Steele

Robert Markus

NIGEL MINTON nigel.minton@nottingham.ac.uk
Professor of Appliedmolecular Microbiology



Abstract

Background: The use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy.
Results: The genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays.
Conclusions: We have been able to demonstrate the synthesis and in vivo assembly of a four component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant strains display an improved growth phenotype on xylan and an increase in released reducing sugar from several substrates including untreated powdered wheat straw. This constitutes an important milestone towards the development of a truly cellulolytic strain suitable for CBP.

Citation

Willson, B. J., Kovács, K., Wilding-Steele, T., Markus, R., Winzer, K., & Minton, N. P. (2016). Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824. Biotechnology for Biofuels, 9(1), https://doi.org/10.1186/s13068-016-0526-x

Journal Article Type Article
Acceptance Date May 10, 2016
Online Publication Date May 23, 2016
Publication Date 2016-12
Deposit Date Sep 12, 2016
Publicly Available Date Sep 12, 2016
Journal Biotechnology for Biofuels
Electronic ISSN 1754-6834
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 9
Issue 1
Article Number 109
DOI https://doi.org/10.1186/s13068-016-0526-x
Keywords Cellulosome, Scaffoldin, Sortase anchoring, Consolidated bioprocessing, Clostridium acetobutylicum
Public URL http://eprints.nottingham.ac.uk/id/eprint/36481
Publisher URL https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0526-x
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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