A tunable l-arabinose-inducible expression plasmid for the acetic acid bacterium Gluconobacter oxydans

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 P(BAD) 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-P(BAD) performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-P(BAD) 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-P(BAD) based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-P(BAD) system in G. oxydans that could possibly also be used in other AAB. KEY POINTS: • We found the AraC-P(BAD) system from E. coli MC4100 was well tunable in G. oxydans. • In the absence of AraC or l-arabinose, expression from P(BAD) was extremely low. • This araC-P(BAD) system could also be fully functional in other acetic acid bacteria. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-020-10905-4) contains supplementary material, which is available to authorized users.