Abstract
The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the l-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters β-glucuronidase and mNeonGreen, up to 480-fold induction with 1% l-arabinose, and tunability from 0.1 to 1% l-arabinose. In G. oxydans 621H, l-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in d-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB.Key points• We found the AraC-PBADsystem from E. coli MC4100 was well tunable in G. oxydans.• In the absence of AraC orl-arabinose, expression from PBADwas extremely low.• This araC-PBADsystem could also be fully functional in other acetic acid bacteria.
Highlights
Controlled expression of target genes to produce proteins for various purposes in a bacterial cell culture at a defined time point is often beneficial or even required in basic research and in biotechnological applications
We found that the AraC-PBAD system derived from E. coli K12 MC4100 performed very well in G. oxydans with the pBBR1 plasmid backbone
The regulatable expression plasmids tested in acetic acid bacteria (AAB) species and reported so far suffered from very leaky expression in the absence of the respective inducer, resulting in very low induction ratios for the promoters PBAD, PTet or PLux, while the overall expression was strong according to the reporter activities (Florea et al 2016; Teh et al 2019)
Summary
Controlled expression of target genes to produce proteins for various purposes in a bacterial cell culture at a defined time point is often beneficial or even required in basic research and in biotechnological applications. For acetic acid bacteria (AAB), including the most frequently used and studied genera Acetobacter, Gluconobacter, Gluconacetobacter, Komagataeibacter, and Acidiphilium, a low-cost, tight, and strongly inducible expression system has not been reported yet in the literature to the best of our knowledge. In Komagataeibacter rhaeticus iGEM, the TetR-dependent system from transposon Tn10 with the inducible promoter Ptet exhibited approximately only 1.5-fold induction due to high leakiness in the absence of the inducer anhydrotetracycline (Florea et al 2016). The native Larabinose-inducible AraC-dependent PBAD system from E. coli exhibited approximately only 5- to 12-fold induction in Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 53582, and Komagataeibacter rhaeticus iGEM due to high basal expression and required a high concentration (4%, w/v) of the inducer L-arabinose (Teh et al 2019). The performance of IPTGinducible LacI-dependent expression has not been reported yet for AAB according to the literature
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