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.

Keywords: Vibrio natriegens; fermentation optimization; pET expression system; recombinant protein; synthetic biology.

FIGURE 1

Evaluation of the soluble expression of 196 genes of interest (GOIs) in Escherichia coli and Vibrio natriegens. (A) Flowchart about shake-flask fermentation of 196 GOIs and soluble expression comparison between E. coli BL21(DE3) and V. natriegens VnDX. (B) Compare the soluble expression of the same GOI in E. coli and V. natriegens through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, 0.15 OD₆₀₀ (10-well gel) or 0.075 OD₆₀₀ (15-well gel) prepared crude protein samples were loaded to the corresponding protein gel wells. V, V. natriegens VnDX; E, E. coli BL21(DE3); Blue box, GOI possessing higher expression in VnDX than BL21(DE3); Orange box, GOI possessing the same expression in VnDX and BL21(DE3); Gray box, GOI possessing higher expression in BL21(DE3) than VnDX. 1#: Alanine racemase from Bacillus subtilis 168; 2#: N-acetyl amino acid racemase from Alcaligenes sp.; 3#: N-acetyl amino acid racemase from Deinococcus radiodurans NCHU1003; 4#: N-acetyl amino acid racemase from Amycolatopsis sp. TS-1-60; 5#: Maltooligosaccharide trehalose synthetase from Arthrobacter sp. Q36; 6#: Maltooligosaccharide trehalose synthetase from Arthrobacterium S34; 7#: D-carbamoylase (K34E) from Burkholderia pickettii. (C) Pie chart about the soluble expression results of 196 GOIs. VnDX > BL21(DE3): GOI possessing higher expression in VnDX than BL21(DE3); VnDX = BL21(DE3): GOI possessing the same expression in VnDX and BL21(DE3); VnDX < BL21(DE3): GOI possessing higher expression in BL21(DE3) than VnDX.

FIGURE 2

Analysis of the spectrum of highly expressed genes of interest (GOIs) in V. natriegens VnDX. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) comparison of 20 highly expressed GOIs. Here, 0.15 OD₆₀₀ (10-well gel) or 0.075 OD₆₀₀ (15-well gel) prepared crude protein samples were loaded to the corresponding protein gel wells. V, V. natriegens VnDX; E, E. coli BL21(DE3). Numbers above the SDS-PAGE lanes represent strain numbers of 20 highly expressed GOIs in both hosts. The detailed information of these GOIs was marked with blue asterisk in Supplementary Table 3. (B) Classification, sources, and protein length analysis of 20 GOIs with high expression in V. natriegens. Numbers of horizontal axis represents V. natriegens strain numbers of 20 highly expressed GOIs. The GOI indicated by the three triangles corresponds to three enzymes in (C). (C) Quantity comparison of three highly expressed GOIs with poor expression in E. coli. MxRDK: Ribonucleoside diphosphate kinase from M. xanthus, CIBT3888 and CIBT4194; AsNAR: N-acetyl amino acid racemase from Alcaligenes sp. CIBT3942 and CIBT4248; PaIS: Isoprene synthase (K308R, C533W) from Populus alba (codon Optimized for E. coli) CIBT4283 and CIBT4269. Error bars represent the SD of n = 3 technical replicates. Quantitative analysis was calculated by software Quantity One 1-D; GraphPad Prism software was used to analyze the significance by t-test, ****P < 0.0001.

FIGURE 3

Optimization of “what to try first” protocol for V. natriegens and testing the general applicability of the consensus protocol. (A) Growth curve of VnDX/pGDH in TBv2, BHIv2, and LBv2 medium and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of soluble glucose dehydrogenase (GDH) in 12 and 24 h. Here, 0.075 OD₆₀₀ crude protein samples were loaded for SDS-PAGE. (B) The curve of GDH catalytic efficiency per OD₆₀₀ [U/(ml⋅OD)] at 28 or 30°C. Induction occurred at 0 h. (C) Optimization of induction point and temperature of VnDX/pGDH. (D) Compare growth and catalytic efficiency of GDH between VnDX and BL21(DE3) under consensus “what to try first” fermentation conditions. (E) Five genes of interest (GOIs) were tested for the general applicability of the “what to try first” protocol. AbRED, carbonyl reductase from Acinetobacter baylyi CIBT3993; LkRED, carbonyl reductase from Lactobacillus kefiri CIBT3995; TvDAO, D-amino acid oxidase from Trigonopsis variabilis CIBT4021; NaRED, carbonyl reductase from Novosphingobium aromaticivorans DSM 12444 CIBT3990; GDH, glucose dehydrogenase from Bacillus subtilis. BL21(DE3), the optimal fermentation condition for E. coli BL21(DE3); VnDX before, V. natriegens fermentation condition before optimization; VnDX after, the fermentation condition using our improved “what to try first” protocol. Error bars represent the SD of n = 3 technical replicates.