Objective Geraniol, a fragrance of great importance in the consumer goods industry, can be glucosylated by the UDP-glucose-dependent glucosyltransferase VvGT14a from Vitis vinifera , yielding more stable geranyl glucoside. Escherichia coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its intrinsic capability for UDP-glucose regeneration. The low water solubility and high cytotoxicity of geraniol can be overcome in a biphasic system where the non-aqueous phase functions as an in situ substrate reservoir. However, the effect of different process variables on the biphasic whole-cell biotransformation is unknown. Thus, the goal of this study was to identify potential bottlenecks during biotransformation with in situ geraniol supply via isopropyl myristate as second non-aqueous phase. Results First, insufficient UDP-glucose supply could be ruled out by measurement of intracellular UDP-glucose concentrations. Instead, oxygen supply was determined as a bottleneck. Moreover, the formation of the byproduct geranyl acetate by chloramphenicol acetyltransferase (CAT) was identified as a constraint for high product yields. The use of a CAT-deficient whole-cell biocatalyst prevented the formation of geranyl acetate, and geranyl glucoside could be obtained with 100% selectivity during a biotransformation on L-scale. Conclusion This study is the first to closely analyze the whole-cell biotransformation of geraniol with Escherichia coli expressing an UDP-glucose-dependent glucosyltransferase and can be used as an optimal starting point for the design of other glycosylation processes.

中文翻译：

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重组大肠杆菌全细胞生物转化无副产物香叶醇糖基化

目的香叶醇是消费品行业中非常重要的一种香料，它可以被葡萄中的UDP-葡萄糖依赖性葡萄糖基转移酶VvGT14a 糖基化，产生更稳定的香叶基糖苷。由于其固有的 UDP-葡萄糖再生能力，表达 VvGT14a 的大肠杆菌是这种生物转化的一种方便的全细胞生物催化剂。在双相系统中可以克服香叶醇的低水溶性和高细胞毒性，其中非水相充当原位底物储库。然而，不同过程变量对双相全细胞生物转化的影响尚不清楚。因此，本研究的目标是通过肉豆蔻酸异丙酯作为第二非水相原位供应香叶醇，确定生物转化过程中的潜在瓶颈。结果第一，UDP-葡萄糖供应不足可以通过测量细胞内UDP-葡萄糖浓度来排除。相反，氧气供应被确定为瓶颈。此外，氯霉素乙酰转移酶 (CAT) 形成的副产物乙酸香叶酯被确定为高产率的制约因素。使用缺乏 CAT 的全细胞生物催化剂阻止了乙酸香叶酯的形成，并且在 L 级生物转化过程中可以 100% 选择性地获得香叶基葡萄糖苷。结论 本研究首次密切分析香叶醇与表达 UDP 葡萄糖依赖性葡糖基转移酶的大肠杆菌的全细胞生物转化，并可用作设计其他糖基化过程的最佳起点。