Mining of Sucrose Synthases from Glycyrrhiza uralensis and Their Application in the Construction of an Efficient UDP-Recycling System

Abstract

Sucrose synthase (SUS) plays an important role in carbohydrate metabolism in plants. The SUS genes in licorice remain unknown. To reveal the sucrose metabolic pathway in licorice, all the 12 putative SUS genes of Glycyrrhiza uralensis were systematically identified by genome mining, and two novel SUSs (GuSUS1 and GuSUS2) were isolated and characterized for the first time. Furthermore, we found that the flexible N-terminus was responsible for the low stability of plant SUSs, and deletion of redundant N-terminus improved the stability of GuSUS1 and GuSUS2. The half-life of both GuSUS1 and GuSUS2 mutants was increased by 2-fold. Finally, the GuSUS1 mutant was coupled with UGT73C11 for the glycosylation of glycyrrhetinic acid (GA) with uridine 5′-diphosphate disodium salt hydrate (UDP) in situ recycling, and GA conversion was increased by 7-fold. Our study not only identified the SUS genes in licorice but also provided a stable SUS mutant for the construction of an efficient UDP-recycling system for GA glycosylation.

Mining of UDP‐glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids

“…The complementary DNA of G. uralensis was obtained as described before (Zhang et al, 2019). The target GuUGT genes were amplified by polymerase chain reaction (PCR) and the primers are listed in Table S1.…”

“…The crude enzymes of UGT73C33, UGT73F24, and UGT73F24m were prepared as described above, and GuSUS1m crude enzyme was prepared according to our previous report (Zhang et al, 2019). Twenty‐five milliliters substrate solution containing 700 μM GA (465 μM GA for UGT73C33), 1.3 μM UDP, 50 mM PBS (pH 6.5), 0.13 M sucrose was mixed with 75 ml UGTs crude enzyme, and 50 ml GuSUS1m crude enzyme.…”

“…Among various sugar types, glucosylation is a type of primary modification to natural products. Although several UGTs have been reported to recognize pentacyclic triterpenoids, most of them showed promiscuous activity; they can transfer glucose not only to the C‐3 OH site but also to the C‐29/30 COOH site (Dai et al, 2018; Zhang et al, 2019). This would result in an uncontrollable glycosylation process, forming a mixture of glucosylated derivatives and increasing the downstream separation and purification cost, which is not applicable for industry (Itkin et al, 2016; Olsson et al, 2016).…”

Biocatalytic Synthesis of Calycosin-7-O-β-D-Glucoside with Uridine Diphosphate–Glucose Regeneration System

“…In this respect, enzymatic glucosylation using regio-and stereoselective UGTs can alleviate these disadvantages [15,16]. Extensive studies indicate that sucrose synthase (SuSy) is a versatile biocatalyst that can synthesize costly uridine diphosphate glucose (UDPG) from abundant and cheap sucrose and catalytic amounts of uridine diphosphate (UDP) [17,18]. Furthermore, SuSy can be coupled with a UGT in a one-pot reaction that could recycle UDPG from UDP and sucrose, thereby providing a cost-effective approach for the glucosylation of natural products [19,20].…”

Microbial Cell Factory for Efficiently Synthesizing Plant Natural Products via Optimizing the Location and Adaptation of Pathway on Genome Scale

“…They have complex structures and various physiological as well as pharmacological activities. For instance, glycyrrhizin, betulinic acid, paclitaxel and resveratrol have antitumor activities, while lycopene, β-carotene, and astaxanthin possess antioxidant properties ( Lü et al, 2016 ; Zhao et al, 2017 ; Zhang L. et al, 2019 ; Chen et al, 2020 ; Sun et al, 2020 ). Obtaining natural products through extraction from cultivated plants faces the problem of low natural content, uncertain climate factors and destruction of the ecological environment ( Zhao and Li, 2018 ; Sun et al, 2019 ).…”