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Lookup NU author(s): Christopher Sauer, Rita Cruz, Dr Leendert Hamoen, Professor Colin Harwood
This is the authors' accepted manuscript of an article that has been published in its final definitive form by American Chemical Society, 2018.
For re-use rights please refer to the publisher's terms and conditions.
Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a “brute-force” approach by designing and synthesizing a large combinatorial library comprising ∼12,000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned five orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5’-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gap and xynA constructs were due to differences in 5’-end mRNA folding and consequential differences in the of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.
Author(s): Sauer C, van Themaat EVL, Boender LGM, Groothuis D, Cruz R, Hamoen LW, Harwood CR, van Rij T
Publication type: Article
Publication status: Published
Journal: ACS Synthetic Biology
Year: 2018
Volume: 7
Issue: 7
Pages: 1773-1784
Print publication date: 20/07/2018
Online publication date: 25/06/2018
Acceptance date: 25/06/2018
Date deposited: 27/06/2018
ISSN (electronic): 2161-5063
Publisher: American Chemical Society
URL: https://doi.org/10.1021/acssynbio.8b00110
DOI: 10.1021/acssynbio.8b00110
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