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.

中文翻译：

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用于乙酸细菌氧化葡糖杆菌的可调的1-阿拉伯糖诱导型表达质粒

摘要

乙酸细菌（AAB）氧化葡糖杆菌不完全氧化多种碳水化合物，因此在工业上用于氧化生物转化。对于氧化单孢菌，没有可用的系统允许基于质粒的可调节表达。我们发现，升-arabinose诱导P _BAD启动子和转录调节阿糖胞苷大肠杆菌MC4100中表现非常出色G.杆菌。分别基于pBBR1的质粒显示报告基因β-葡萄糖醛酸苷酶和mNeonGreen的基础表达非常低，用1％的1-阿拉伯糖诱导多达480倍，从0.1到1％的1-阿拉伯糖具有可调性。在G. oxydans 621H，1-阿拉伯糖被膜结合的葡萄糖脱氢酶氧化，这在多缺失菌株BP.6中是不存在的。然而，AraC-P _BAD在两个菌株的指数期表现相似，这表明在野生型氧化假单胞菌菌株中应用AraC-P _BAD不需要基因敲除。然而，氧化产物阿拉伯糖酸在固定相期间强烈促进了621H培养物中生长培养基的酸化，这导致621H（pH 3.3）中的报道基因活性急剧下降，而在BP.6培养物（pH 4.4）中则没有明显降低。这些活动可以强烈仅通过培养在固定式电池迅速增加d将无甘露醇的培养基的pH值调整为6，这表明报道分子几乎不会降解，而是变得无活性。在pH控制的生物反应器中，这些报告基因活性在固定相（pH 6）中保持较高水平。最后，我们基于pBBR1MCS-5用araC -P _BAD创建了一个多克隆载体。在一起，我们展示了在氧化单孢菌中AraC-P _BAD系统的卓越功能和良好的可调谐性，该系统也可能在其他AAB中使用。

关键点

•我们发现，来自大肠杆菌MC4100的AraC-P _BAD 系统在氧化单孢菌中是很好的可调性。

•在没有阿糖胞苷或 升-arabinose从P表达_BAD 极低。

• 该araC-P _BAD 系统在其他乙酸细菌中也可以完全发挥作用。