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Whole cells of Escherichia coli overexpressing a glucosyltransferase from Vitis vinifera were used for the glucosylation of geraniol to geranyl glucoside. A high cell density cultivation process for the production of whole-cell biocatalysts was developed, gaining a dry cell mass concentration of up to 67.6 ± 1.2 g L(-1) and a glucosyltransferase concentration of up to 2.7 ± 0.1 g protein L(-1) within a process time of 48 h. Whole-cell batch biotransformations in milliliter-scale stirred-tank bioreactors showed highest conversion of geraniol at pH 7.0 although the pH optimum of the purified glucosyltransferase was at pH 8.5. The biocatalytic batch process performance was improved significantly by the addition of a water-immiscible ionic liquid (N-hexylpyridinium bis(trifluoromethylsulfonyl)imid) for in situ substrate supply. The so far highest final geranyl glucoside concentration (291 ± 9 mg L(-1)) and conversion (71 ± 2 %) reported for whole-cell biotransformations of geraniol were achieved with 5 % (v/v) of the ionic liquid.

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BACKGROUND Flavor and fragrance (F&F) compounds are increasingly produced by biotechnological instead of chemical means, as this allows them to be labeled natural additives. However, since most F&F compounds exhibit cytotoxicity towards common microbiological production hosts, in situ product removal strategies using two‐phase partitioning bioreactors are desirable, making the rational selection of effective non‐aqueous phases a crucial step. Here, thermodynamic first‐principles methods and the experimental determination of partition coefficients were used to differentiate between sequestering phases with high and low thermodynamic affinity towards 17 important F&F compounds. RESULTS The approach was highly successful, enabling identification of outstanding extractants for several F&F compounds. Moreover, it was shown that certain solvent classes (e.g. long‐chained alcohols and a variety of esters) function as efficient sequestering phases across all classes of target F&F compounds, whereas other solvent types (such as alkanes), and the liquid polymer silicone oil exhibit poor partitioning behavior. Finally, the tested absorptive solid polymers generally did not constitute effective sequestering phases for the target compounds, due to high fractions of hard/crystalline segments, however, this suggests that designing polymers with a higher proportion of soft segment could lead to enhanced sorptive capacity. CONCLUSIONS This study is the first in the current literature to systematically analyze sequestering phase choices for important F&F compounds and can serve as a guide for researchers working on biphasic microbial F&F production. © 2017 Society of Chemical Industry

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 UDPglucose 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 UDPglucose 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.