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Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase.

ABSTRACT: Glycyrrhetinic acid (GA) is a principal bioactive pentacyclic triterpenoid from Glycyrrhiza uralensis. Uridine diphosphate-dependent glycosyltransferases (UGTs) have been widely used to catalyze glycosylation of diverse nature products for the development of potential therapeutic compounds. In this study, we have characterized a UGT109A3 from Bacillus subtilis, which can glycosylate both the free C3 hydroxyl and C30 carboxyl groups of GA to yield a unique 3, 30-O-β-D-diglucoside-GA. By coupling the microbial UGT109A3 to plant sucrose synthase (SUS), GA-diglucoside could be biosynthesized in an efficient and economical way. With a fed-batch glycosylation, a large scale of GA-diglucoside (6.26 mM, 4.98 g/L in 8 h) could be enzymatically transformed from GA. The obtained GA-diglucoside showed a significant water solubility improvement of around 3.4 × 10³ fold compared with that of the parent GA (29 μM). Moreover, it also exhibited dose-dependent cytotoxicity toward human colon carcinoma Caco-2 cell line according to MTT assay, having an IC₅₀ at 160 μM. This study not only establishes efficient platform for producing GA-glucosides, but is also valuable for developing further the biosynthesis of other complex glycosylated natural products.

SUBMITTER: Ali MY

PROVIDER: S-EPMC8218813 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase.

Ali Mohamed Yassin MY Chang Qing Q Yan Quande Q Qian Zheng Z Guo Xiang X Thow Kieran K Wu Jinhong J Zhang Yong Y Feng Yan Y

Frontiers in bioengineering and biotechnology 20210608

Glycyrrhetinic acid (GA) is a principal bioactive pentacyclic triterpenoid from <i>Glycyrrhiza uralensis</i>. Uridine diphosphate-dependent glycosyltransferases (UGTs) have been widely used to catalyze glycosylation of diverse nature products for the development of potential therapeutic compounds. In this study, we have characterized a UGT109A3 from <i>Bacillus subtilis</i>, which can glycosylate both the free C3 hydroxyl and C30 carboxyl groups of GA to yield a unique 3, 30-O-β-D-diglucoside-GA ...[more]

PMID: 34169062

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Project description:Backgroundβ-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids.ResultsA new gene of β-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L β-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing β-caryophyllene production to 100.3 mg/L. The highest production of β-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of β-caryophyllene by 20% to 5142 mg/L.ConclusionA new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce β-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of β-caryophyllene than the previous studies. During the fermentation process, we observed that β-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.

Dataset Information

Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase.

Publications

Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glucosides by Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase.

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