Journal
ACS SYNTHETIC BIOLOGY
Volume 7, Issue 5, Pages 1424-1435Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00055
Keywords
host-independent expression system; stable expression; single plasmid; T7 RNA polymerase; antisense RNA
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Funding
- National Natural Science Foundation of China [NSFC 21576153]
- Beijing Natural Science Foundation [5162019]
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Metabolic engineering and synthetic biology usually require universal expression systems for stable and efficient gene expression in various organisms. In this study, a host-independent and stable T7 expression system had been developed by integrating T7 RNA polymerase and its cognate transcriptional units in single plasmid. The expression of T7 RNA polymerase was restricted below its lethal threshold using a T7 RNA polymerase antisense gene cassette, which allowed long periods of cultivation and protein production. In addition, by designing ribosome binding sites, we further tuned the expression capacity of this novel T7 system within a wide range. This host-independent expression system efficiently expressed genes in five different Gram-negative strains and one Gram-positive strain and was also shown to be applicable in a real industrial D-p-hydroxyphenylglycine production system.
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