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 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.

Identification of Gradient Promoters of Gluconobacter oxydans and Their Applications in the Biosynthesis of 2-Keto-L-Gulonic Acid

“…Three broad-spectrum promoters (P bs1 , P bs2 , and P bs3 ) with different strengths, were generated by random mutation and characterized. In a recent study, a newly tunable L-arabinose-inducible P BAD promoter was discovered to be useful in G. oxydans 621H, and the activity of this promoter was affected by the pH of the medium (Fricke et al, 2020). In summary, the identification of gradient promoters in this study expanded the toolbox of available promoters, and these promoters revealed promising prospects in metabolic engineering of G. oxydans for high-value products.…”

On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

“…Until now, an L-arabinose-inducible araC-P araBAD system on the pBBR1MCS-5 backbone is the only regulatable system available for Gluconobacter (Fricke et al 2020). The mechanism of transcriptional gene regulation by AraC is different compared to those of the pure repressor proteins TetR and LacI, which just dissociate from their operator DNA when their respective inducer is bound to the protein.…”

“…The L-arabinose concentrations required for highest induction in G. oxydans were much higher than the ones typically used for E. coli. Attempts to increase the arabinose sensitivity of the araC-P araBAD system by expression of the L-arabinose transporter gene araE in tandem with araC from the promoter of araC had a strong negative effect on the growth of G. oxydans and the expression performance (Fricke et al 2020). With a pBBR1MCS-5 derivative carrying araE separately under control of the weak constitutive G. oxydans promoter P GOX0384 no G. oxydans 621H transformants could be obtained yet, suggesting a severe growth defect upon araE expression in G. oxydans, even in the absence of L-arabinose (our unpublished results).…”

“…In follow-up studies on cellulose-forming (Cel + ) and cellulose-negative (Cel -) mutants of K. xylinus the broad-host-range cloning vector pVK100 and cosmid vector pRK311 were used to prepare gene libraries of K. xylinus to search for genes expressed from their native promoters and complementing the Celphenotype (Fjaervik et al 1991;Valla et al 1989). The screenings revealed a uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase gene designated celA involved in cellulose biosynthesis for which UDPG is the precursor, and a gene encoding phosphoglucomutase activity, which (Fricke et al 2020). As further analyzed and discussed in the study, the decrease of the mNG fluorescence in strain 621H at stronger induction is pH-dependent due to enzymatic oxidation of L-arabinose forming L-arabinonic acid and can be avoided by either using the multi-deletion strain BP.6 or pH-controlled condition catalyzes the second step of UDPG synthesis from glucose by isomerization of glucose-6-phosphate to glucose-1phosphate.…”

“…In AAB, for a long time, only constitutive target gene expression and in a very few studies only weakly regulatable target gene expression was achieved since, for example, the transfer of heterologous regulatable systems such as the well-known classical examples of the TetR-, AraC-, and LacI-dependent systems and their application was not really successful yet in AAB due to high (leaky) expression already in the absence of the respective inducer (Florea et al 2016a;Teh et al 2019). Only two regulatable expression systems, an N-acyl homoserine lactone-inducible luxR-P lux system for Komagataeibacter rhaeticus and an Larabinose-inducible araC-P araBAD system for Gluconobacter oxydans with up to 480-fold induction have recently been reported for the two AAB species (Florea et al 2016a;Fricke et al 2020). To provide a comprehensive overview of expression systems, their latest developments and the use cases in AAB we have searched and evaluated the literature for relevant AAB studies.…”