Glycosyltransferase cascades are promising tools of biocatalysis for natural product glycosylation, but their suitability for actual production remains to be shown. Here, we demonstrate at a scale of 100 g isolated product the integrated biocatalytic production of nothofagin, the natural 3'-C-β-D-glucoside of the polyphenol phloretin. A parallel reaction cascade involving coupled C-glucosyltransferase and sucrose synthase was optimized for the one-pot glucosylation of phloretin from sucrose via an UDP/UDP-glucose shuttle. Inclusion complexation with the highly water soluble 2-hydroxypropyl-β-cyclodextrin pushed the phloretin solubility to its upper practical limit (∼120 mM) and so removed the main bottleneck on an efficient synthesis of nothofagin. The biotransformation thus intensified had excellent performance metrics of 97% yield and ∼50 g /L at a space-time yield of 3 g/L/hr. The UDP-glucose was regenerated up to ∼220 times. A scalable downstream process for efficient recovery of nothofagin (≥95% purity; ≥65% yield) was developed. A tailored anion-exchange chromatography at pH 8.5 was used for capture and initial purification of the product. Recycling of the 2-hydroxypropyl-β-cyclodextrin would also be possible at this step. Product precipitation at a lowered pH of 6.0 and re-dissolution in acetone effectively replaced desalting by size exclusion chromatography in the final step of nothofagin purification. This study therefore, reveals the potential for process intensification in the glycosylation of polyphenol acceptors by glycosyltransferase cascades. It demonstrates that, with up- and downstream processing carefully optimized and suitably interconnected, a powerful biocatalytic technology becomes available for the production of an important class of glycosides difficult to prepare otherwise.

Nucleotide sugar‐dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine‐5′‐diphosphate glucose (UDP‐glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole‐cell biotransformation. The UDP‐glc production had excellent performance metrics of ∼100 gproduct/L, 86% yield (based on UDP), and a total turnover number of 103 gUDP‐glc/gcell dry weight at a space‐time yield of 10 g/L/h. Using efficient chromatography‐free DSP, the UDP‐glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. © 2016 Wiley Periodicals, Inc.

Sucrose synthase (SuSy) catalyzes in the presence of a pyrimidine or purine nucleoside diphosphate (NDP) the conversion of sucrose to the corresponding nucleotide‐activated derivative of glucose (NDP‐glucose). To realize the potential of SuSy for NDP‐glucose synthesis fully, a nucleoside monophosphate (NMP) should be employed in the reaction, for it is a much more cost‐effective substrate than NDP. Therefore we explored in this study the use of polyphosphate kinases (PPK) from class II and III of family 2 which catalyze in the presence of polyphosphate (polyP) the conversion of NMP into NDP. A biocatalytic cascade of PPK (from Meiothermus ruber) and SuSy (from Acidithiobacillus caldus) was established for NDP‐glucose production. The synthetic efficiency of the cascade reflected the NMP substrate specificity of the PPK, following the order of nucleoside monophosphate: adenosine (AMP)>guanosine (GMP)>cytidine (CMP)>uridine (UMP)>deoxy‐thymidine (dTMP). The efficiency was also influenced by the concentrations of magnesium (Mg2+) and polyphosphate (polyP) as well as by the pH. An optimized synthesis at 45 °C and pH 5.5 gave 81 mM (48 g L−1) ADP‐glucose from 100 mM AMP and 132 mM polyP in the presence of an excess of sucrose (1 M) and 25 mM Mg2+. The productivity was 2.0 g L−1 h−1 despite using an enzyme concentration of only 150 μg mL−1. Isolation of ADP‐glucose (∼99% purity) by anion‐exchange chromatography required prior removal of the polyP, which was achieved by fractional precipitation with ethanol. The herein developed coupling with PPK, to form the NDP substrate from NMP in situ, could be generally useful to advance NDP‐sugar synthesis by Leloir glycosyltransferases.magnified image

Sugar nucleotide‐dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology‐based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l‐arabinose‐inducible expression of the T7 phage‐derived Gp2 protein from a genome‐integrated site. The glycosyltransferase genes are encoded on conventional pET‐based expression plasmids that allow T7 RNA polymerase‐driven inducible expression by isopropyl‐β‐ d‐galactoside. Laboratory batch and scaled‐up (20 L) fed‐batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12‐fold as a result of production under growth‐decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed‐batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3‐fold) and quality (2.3‐fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth‐arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

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