Vibrio natriegens as a pET-Compatible Expression Host Complementary to Escherichia coli

Abstract

Efficient and novel recombinant protein expression systems can further reduce the production cost of enzymes. Vibrio natriegens is the fastest growing free-living bacterium with a doubling time of less than 10 min, which makes it highly attractive as a protein expression host. Here, 196 pET plasmids with different genes of interest (GOIs) were electroporated into the V. natriegens strain VnDX, which carries an integrated T7 RNA polymerase expression cassette. As a result, 65 and 75% of the tested GOIs obtained soluble expression in V. natriegens and Escherichia coli, respectively, 20 GOIs of which showed better expression in the former. Furthermore, we have adapted a consensus “what to try first” protocol for V. natriegens based on Terrific Broth medium. Six sampled GOIs encoding biocatalysts enzymes thus achieved 50–128% higher catalytic efficiency under the optimized expression conditions. Our study demonstrated V. natriegens as a pET-compatible expression host with a spectrum of highly expressed GOIs distinct from E. coli and an easy-to-use consensus protocol, solving the problem that some GOIs cannot be expressed well in E. coli.

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags

“…While up to date E. coli still remains the first-to-try protein expression host, the direct transfer of plasmids into V. natriegens is time and cost saving and allows the implementation of a flexible toolbox of plasmids that can be used for both production hosts. According to a recent study by Xu et al ( 2021a ), the chance to obtain higher protein yields in V. natriegens from direct transfer of pET-plasmids is at least 10%. The additional codon-optimization of plasmids for V. natriegens will result in even higher yields.…”

“…Their core carbon metabolism was found to be similar during aerobic growth on glucose minimal medium and both use the same pathways to synthesize canonical amino acids (Long et al 2017 ). The genetic proximity to E. coli allows in many cases a direct transfer of E. coli optimized pET-plasmids into the engineered V. natriegens strain, Vmax™ Express (Synthetic Genomics 2017 ), that was equipped with a T7 RNA polymerase expression cassette (Xu et al 2021a ). Among others, Vmax is compatible with lacUV5 and ara BAD promoters, frequently used antibiotic resistance cassettes including the ones for chloramphenicol, kanamycin, ampicillin and tetracycline, as well as commonly used origins of replication like pMB1, pUC, p15A and derivatives (Thoma and Blombach 2021 ; Hoff et al 2020 ).…”

Intelligent host engineering for metabolic flux optimisation in biotechnology

“…Consequently, although not usually a focus of biotechnology, it remains the case that the more time cells spend in a fermentor non-productively the less good the process. This has led to the consideration of hosts such as Vibrio natriegens [50,[531][532][533][534][535][536][537][538][539][540][541], whose optimal doubling time can be as little as 7 min, some threefold quicker than the widely quoted 20 min for E. coli in rich media. Whether or not organisms such as V. natriegens turn out to be valuable production hosts, there is no doubt that understanding how to make cell growth quicker might help enhance the rates of recombinant protein production.…”

Rapid production of l‐DOPA by Vibrio natriegens, an emerging next‐generation whole‐cell catalysis chassis

“…Moreover, accurate control of induction timing is unnecessary because the protein expression level is impervious to biomass concentration of the induction timing, which is effortless and time‐saving (Becker et al ., 2019 ). V. natriegens may also highly express some genes, acting as an expression host superior to E. coli in some aspects (Xu et al ., 2021 ). Based on the above findings, it is reasonable to speculate that V. natriegens is a promising novel whole‐cell catalysis chassis for l ‐DOPA production.…”