Lebre, PHB; (2014) Evaluation of Staphylococcus carnosus as an Alternative Host Organism for Whole-Cell Biocatalysis. Doctoral thesis , UCL (University College London).

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Abstract

The aim of this project is to study the use of Staphylococcus carnosus as an alternative host in whole-cell biocatalysis, using the NADPH-dependent conversion of cyclic ketones by cyclohexanone monooxygenase (CHMO) as the model system. The use of whole cells in industrial biocatalysis has been actively researched due to their promise as biological factories that can perform complex reactions in environments where the use of isolated enzymes would not be feasible. However, the majority of whole-cell processes rely on a small range of organisms that, while been extensively characterized and easily engineering to express enzyme biocatalysts, have some inherent limitations that hinder their application to many industrial processes. Thus. There is a need to find novel microorganisms that can fill in the shortcoming of those conventional strains. In this project, a flexible shuttle vector was constructed to allow for the cloning and expression of CHMO in both E. coli and S. carnosus by using a dual promoter system in which one of the promoters could be replaced. Higher levels of CHMO expression were achieved in E. coli strains cloned with the shuttle vector compared to an expression vector used in previous studies. No biocatalyst expression was detected in S. carnosus. The shuttle vector was subsequently modified to allow for the quantitative characterization of different synthetic promoters based on the gemone of S. carnosus. A novel in-silico promoter selection methodology based on the codon bias was developped to allow for the rational selection of potential genomics promoters. Two synthetic promoters based on sequences upstream of ribosomal proteins rplK and rplJ were subsequently shown to allow for protein expression in both E. coli and S. carnosus. The stronger of these two promoter was inserted into the CHMO expression vector, but the resulting construct did not manage to express detectable levels of the biocatalyst in S. carnosus. It was thus concluded that S. carnosus was not a suitable host for biocatalysis with CHMO, and further studies with other enzymes would have to be conducted to access its suitability as a general biocatalytic host.

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