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.

Keywords: AraC; Induction; Membrane-bound dehydrogenase; PBAD promoter; mNeonGreen; β-D-Glucuronidase UidA.

Fig. 1

Comparisons of the G. oxydans strains 621H and BP.6. a Growth (OD₆₀₀) in shake flasks in complex medium with 4% (w/v) d-mannitol or 1% (w/v) l-arabinose as well as both 4% (w/v) d-mannitol plus 1% (w/v) l-arabinose. b Arabinose concentrations in complex medium with 4% (w/v) d-mannitol plus 1% (w/v) l-arabinose in shake flasks. Data represent mean ± SD from three biological replicates

Fig. 2

Comparisons of the G. oxydans strains 621H and BP.6. a Growth and UidA activity in Miller units (MU) in strains 621H and BP.6 carrying plasmid pBBR1MCS-5-araC-P_BAD-uidA in l-arabinose-induced and non-induced condition in shake flasks. b Growth and specific mNeonGreen (mNG) fluorescence in strains 621H and BP.6 carrying plasmid pBBR1MCS-5-araC-P_BAD-mNG in l-arabinose-induced and non-induced condition in shake flasks. The mNG fluorescence was measured in a Tecan reader. The specific fluorescence was calculated from absolute fluorescence per OD₆₀₀. c Growth according to backscatter and specific mNG fluorescence in strains 621H and BP.6 carrying plasmid pBBR1MCS-5-araC-P_BAD-mNG in l-arabinose-induced and non-induced condition in microscale BioLector cultivations. The specific fluorescence was calculated from absolute fluorescence per backscatter. For induction, always 1% (w/v) l-arabinose was added to the d-mannitol medium. For all experiments, data represent mean ± SD from three biological replicates

Fig. 3

l-Arabinose-dependent modulation of expression in the G. oxydans strains 621H (a) and BP.6 (b) carrying plasmid pBBR1MCS-5-araC-P_BAD-mNG in microscale BioLector cultivations. Reporter gene mNG expression was induced with increasing concentrations of l-arabinose from 0.03 to 2% (w/v) as indicated. Data represent mean ± SD from three biological replicates

Fig. 4

FACS analysis of the G. oxydans strains 621H (a) and BP.6 (b) carrying plasmid pBBR1MCS-5-araC-P_BAD-mNG. Cells were grown in shake flasks with d-mannitol medium and induced with 1% (w/v) l-arabinose. FACS analysis was performed 8 and 26 h after induction. After 26 h, cells were transferred into fresh l-arabinose- and d-mannitol-free medium adjusted to pH 6 followed by incubation on a rotary shaker for 2 h. As a control, 621H or BP.6 cells without plasmids also grown in d-mannitol medium with 1% (w/v) l-arabinose were used. Total counts per sample represent 100,000 events and only appears to be different due to the logarithmic scale on the x-axis

Fig. 5

l-Arabinose-inducible reporter gene expression in DASbox fermentations in pH-controlled conditions (pH 6). Both, mNG and UidA activity remained high in G. oxydans 621H carrying plasmid pBBR1MCS-5-araC-P_BAD-mNG (a) or pBBR1MCS-5-araC-P_BAD-uidA (b) 24 h after induction with 1% (w/v) l-arabinose when pH 6 was maintained

Fig. 6

Scheme of the pBBR1MCS-5-based araC-P_BAD plasmid with a multiple cloning site (a) and sequence information details (b). The ribosome-binding site AGGAGA (GOX_RBS) is included and usable when the insert cloning is carried out on the 5′-end by NdeI; otherwise, another RBS needs to be included in the insert upstream of the gene of interest. The iGEM terminator sequence of BBa_B1002 is located downstream of the multiple cloning site (MCS)