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Metabolic Engineering
2020

The heterologous production of terpenes by the thermophile Parageobacillus thermoglucosidasius in a consolidated bioprocess using waste bread

Matthew Q.Styles, Edward A.Nesbitt, Timothy D.Hoffmann, Junichi Queen, Maria V.Ortenzi, David J.Leak

Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.

Abstract

Parageobacillus thermoglucosidasius is a genetically tractable thermophile that grows rapidly at elevated temperatures, with a doubling time at 65°C comparable to the shortest doubling times of Escherichia coli. It is capable of using a wide variety of substrates, including carbohydrate oligomers, and has been developed for the industrial production of ethanol. In this study, P. thermoglucosidasius NCIMB11955 has been engineered to produce the sesquiterpene τ-muurolol by introduction of a heterologous mevalonate pathway constructed using genes from several thermophilic archaea together with a recently characterised thermostable τ-muurolol synthase. P. thermoglucosidasius naturally uses the methylerythritol phosphate pathway for production of the terpene precursor, isopentenyl pyrophosphate, while archaea use a version of the mevalonate pathway. By introducing the orthogonal archaeal pathway it was possible to increase the flux through to sesquiterpene biosynthesis. Construction of such a large metabolic pathway created problems with genetic vector introduction and stability, so recombinant plasmids were introduced by conjugation, and a thermostable serine integrase system was developed for integration of large pathways onto the chromosome. Finally, by making the heterologous pathway maltose-inducible we demonstrate that the new strain is capable of using waste bread directly as an autoinduction carbon source for the production of terpenes in a consolidated bioprocess.

Keywords: Consolidated bioprocess, Thermophile, Terpenes, Parageobacillus thermoglucosidasius.

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