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Title: A comparative study of native and heterologous enzyme production in Bacillus subtilis
Author: Cruz, Rita Alexandra Leal
ISNI:       0000 0004 6499 8412
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2017
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Biotechnology plays a central role in setting the course for a resource-efficient and sustainable Bioeconomy. In particular, the industrial production of enzymes using microorganisms offers an environmentally friendly alternative to many traditional chemical processes. Bacillus subtilis is a widely recognized microbial cell factory, able to secrete proteins in tens of grams per litre. However, this organism has developed a variety of quality control and stress response mechanisms designed to facilitate efficient growth and survival in a wide range of natural environments. These pose significant challenges for improving the production efficiency of an increasingly large variety of heterologous commercial proteins. We used two industrially relevant enzymes to study and compare, in parallel, the challenges associated with the production of native and heterologous enzyme by B. subtilis, focusing on the later stages of protein secretion, and the potential of a synthetic translocase for optimising production strains. The enzymes showed very distinct production profiles and secretion kinetics under the same growth and gene expression conditions. We investigated the effect of these enzymes on the regulation of the main genes involved in the secretion pathway. To this end, we analysed the impact of over expressing the model enzymes on secretion stress by fusing the GFP reporter gene to the htrA promoter and introducing this into a group of strains carrying precise deletions in ten genes encoding extracytoplasmic proteases. The results showed that increasing the expression of the Sec translocase proteins did not improve productivity, indicating that the number of Sec translocases it not a limiting factor for enzyme production. However, our data show that the complex regulatory architecture associated with the secretion stress and quality control mechanisms, indicate that post-translocational protein folding and proteolysis are not only inter-dependent but also influenced by the enzyme of interest, resulting in different levels of secretion stress induction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available