Department of Mechanical Engineering, Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany.
Bioprocess Technology, Evonik Operations GmbH, Rodenbacher Chaussee 4, 63457, Hanau-Wolfgang, Germany.
Biotechnol Lett. 2021 Jan;43(1):247-259. doi: 10.1007/s10529-020-02993-z. Epub 2020 Aug 28.
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.
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.
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.
香叶醇是消费品行业中非常重要的香料，可以被葡萄(Vitis vinifera)中的 UDP-葡萄糖依赖型葡糖基转移酶 VvGT14a 葡基化，生成更稳定的香叶基葡糖苷。由于大肠杆菌(Escherichia coli)具有内在的 UDP-葡萄糖再生能力，因此其可作为该生物转化的方便的全细胞生物催化剂。香叶醇的低水溶性和高细胞毒性可以在两相体系中克服，其中非水相充当原位底物库。然而，不同过程变量对两相全细胞生物转化的影响尚不清楚。因此，本研究的目的是通过以异丙基肉豆蔻酸作为第二非水相来原位提供香叶醇，确定生物转化过程中的潜在瓶颈。
首先，通过测量细胞内 UDP-葡萄糖浓度，可以排除 UDP-葡萄糖供应不足的情况。相反，氧供应被确定为一个瓶颈。此外，氯霉素乙酰转移酶（CAT）的副产物香叶基乙酸的形成被确定为高产物收率的限制因素。使用缺乏 CAT 的全细胞生物催化剂可防止香叶基乙酸的形成，并且在 L 规模的生物转化过程中，可以 100%选择性地获得香叶基葡糖苷。
本研究首次密切分析了表达 UDP-葡萄糖依赖型葡糖基转移酶的大肠杆菌对香叶醇的全细胞生物转化，可为其他糖基化过程的设计提供最佳起点。
