Design and Reconstruction of Regulatory Parts for Fast-Growing Vibrio natriegens Synthetic Biology

Abstract

The fast-growing Vibrio natriegens is an attractive robust chassis for diverse synthetic biology applications. However, V. natriegens lacks the suitable constitutive regulatory parts for precisely tuning the gene expression and, thus, recapitulating physiologically relevant changes in gene expression levels. In this study, we designed, constructed, and screened the synthetic regulatory parts by varying the promoter region and ribosome binding site element for V. natriegens with different transcriptional or translational strengths, respectively. The fluorescence intensities of the cells with different synthetic regulatory parts could distribute evenly over a wide range of 5 orders of magnitude. The selected synthetic regulatory parts had good stability in both nutrient-rich and minimal media. The precise combinatorial modulation of galP (GalP = galactose permease) and glk (Glk = glucokinase) from Escherichia coli by using three synthetic regulatory parts with different strengths was confirmed in a phosphoenolpyruvate:carbohydrate phosphotransferase system with inactive V. natriegens strain to alter the glucose transport. This work provides the simple, efficient, and standardized constitutive regulatory parts for V. natriegens synthetic biology.

Metabolic engineering of Vibrio natriegens

“…To drive adjusted gene expression in V. natriegens, synthetic promoter libraries have been constructed and characterized [28,31]. Permutation of promoter elements (−35, −10, and UP element), spacer sequences, ribosomal-binding sites, and terminator sequences yielded a library which varies up to five orders of magnitude in gfp expression.…”

“…Examples include several reporter constructs (e.g. GFP, sfGFP, mCherry, mSCFP3, and LacZ, worked nicely, whereas functionality of RFP, YFP, and eBFP2 was poor) as well as proteins, that are largely insoluble, typically yielded at low concentrations in E. coli, isotopically labeled proteins or even multimeric membrane protein complexes [6,7,20,28,31,[36][37][38].…”

Biosensor-based isolation of amino acid-producing Vibrio natriegens strains

“…Interestingly, the relative maximum output did not necessarily correspond to the promoter strength measured in previous studies ( Stukenberg et al, 2021 ; Tschirhart et al, 2019 ), but this could be due to the different reporter or media used. Cultivation media were shown to have an influence on promoter functioning in V. natriegens ( Wu et al, 2020 ). Another explanation could be the lack of promoter insulation, since the Anderson promoter sequences are relatively short (35bp), and only a short RBS was added (21bp).…”

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.…”