Byproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant Escherichia coli.

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.

Rational selection of biphasic reaction systems for geranyl glucoside production by Escherichia coli whole-cell biocatalysts.

Geranyl glucoside, the glucosylated, high-value derivative of the monoterpenoid geraniol, has various applications in the flavor and fragrance industry and can be produced through whole-cell biotransformation of geraniol with Escherichia coli whole-cell biocatalysts expressing the glucosyltransferase VvGT14a. However, the low water solubility and high cytotoxicity of geraniol require the design of a proper biphasic system where the second, non-aqueous phase functions as an in-situ substrate reservoir. In this work, a rational selection strategy was applied for choosing suitable sequestering phases for geranyl glucoside production by whole-cell biotransformation of geraniol. Hansen solubility parameters and octanol/water distribution coefficients were used as first principle methods in combination with extensive database research to preselect 12 liquid and 6 solid sequestering phases. Subsequently, experimental approaches were applied to determine physicochemical characteristics and the distribution of geraniol and geranyl glucoside between the phases. Moreover, the effects of the sequestering phases on the whole-cell biocatalysts and on the produced geranyl glucoside concentration were measured during parallel biotransformations in milliliter-scale stirred-tank bioreactors. The fatty acid ester isopropyl myristate emerged as the best choice due to its low viscosity, very poor water solubility, low price and compatibility with the whole-cell biocatalyst. The biphasic system containing 20% (v/v) of this solvent boosted geranyl glucoside production (4.2-fold increase of geranyl glucoside concentration in comparison to aqueous system) and exhibits advantageous partitioning of geraniol into the organic phase (logD of 2.42±0.03) and of geranyl glucoside into the water phase (logD of -2.08±0.05). The systematic selection of a suitable biphasic system constitutes basic groundwork for the development of new bioprocesses involving geraniol. Moreover, this study can serve as a guideline for selecting sequestering phases for other whole-cell biotransformation processes.

Lipid production with Trichosporon oleaginosus in a membrane bioreactor using microalgae hydrolysate.

Lipid production by Trichosporon oleaginosus was first studied in fed-batch operated stirred-tank bioreactors on a milliliter- and liter-scale making use of typical sugar monomers and a sugar mixture that may be derived from microalgae biomass hydrolysis after the extraction of lipids. 20.3gL-1 lipids (58% of dry cell mass) were produced with T. oleaginosus in a defined medium at nitrogen starvation in the fed-batch process with a typical microalgae derived carbohydrate mixture (60% glucose, 20% mannose, 20% galactose). Real microalgae hydrolysate resulted in superior growth of T. oleaginosus but no enhanced lipid formation was possible due to nitrogen and phosphorus excess in the hydrolysate. Phosphate precipitation and the application of a continuously operated membrane bioreactor with total cell retention due to the low sugar concentrations (∼40gL-1) in the microalgae hydrolysate resulted in the production of 30gL-1 lipids (53% of dry cell mass) with T. oleaginosus at high space-time-yields of 0.33g lipids L-1h-1. A high apparent lipid yield of 0.43g lipids g-1 sugars consumed (130% of the theoretical maximum) was achieved with the microalgae hydrolysate most likely due to the additional conversion of other carbon sources (e.g. uronic acids, peptides) in the hydrolysate.

Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with Escherichia coli overexpressing a glucosyltransferase.

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.