Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates

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Title: Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates
Author(s): Mordaka, PM
Heap, JT
Item Type: Journal Article
Abstract: Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus −35 and −10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthl was 79% or 58%, instead of 25% as before. Synthetic promoters from these “stringent” libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected “stringency” of promoter sequences in Clostridium, not reported previously.
Publication Date: 10-Jan-2018
Date of Acceptance: 10-Jan-2018
URI: http://hdl.handle.net/10044/1/55865
DOI: https://dx.doi.org/10.1021/acssynbio.7b00398
ISSN: 2161-5063
Publisher: American Chemical Society
Start Page: 672
End Page: 681
Journal / Book Title: ACS Synthetic Biology
Volume: 7
Issue: 2
Copyright Statement: © 2018 American Chemical Society
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Biotechnology and Biological Sciences Research Council
Funder's Grant Number: BB/M002454/1
BB/M002454/1
Keywords: Clostridium acetobutylicum
gene expression
metabolic engineering
promoter library
synthetic promoter
Publication Status: Published
Embargo Date: 2019-01-10
Appears in Collections:Faculty of Natural Sciences



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