Efficient Synthesis of Crocins from Crocetin by a Microbial Glycosyltransferase from Bacillus subtilis 168

Abstract

Crocins are the most important active ingredient found in Crocus sativus, a well-known "plant gold". The glycosyltransferase-catalyzed glycosylation of crocetin is the last step of biosynthesizing crocins and contributes to their structural diversity. Crocin biosynthesis is now hampered by the lack of efficient glycosyltransferases with activity toward crocetin. In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of the PSPG motif and the N-terminal motif of the target plant-derived UGT75L6 and Cs-GT2. Bs-GT from Bacillus subtilis 168, an enzyme with a higher activity of glycosylation toward crocetin than that of Bc-GTA, was characterized. The efficient synthesis of crocins from crocetin catalyzed by microbial GT (Bs-GT) was first reported with a high molecular conversion rate of 81.9%, resulting in the production of 476.8 mg/L of crocins. The glycosylation of crocetin on its carboxyl groups by Bs-GT specifically produced crocin-5 and crocin-3, the important rare crocins.

In Vivo Production of Five Crocins in the Engineered Escherichia coli

“…Glycosylation is one of the most common and important reactions in biological systems, because it can improve the hydrophilicity of lipophilic compounds and determine their pharmacokinetics. − The pharmacological actions of crocin I and crocin II are well-studied, and these two types of crocins are the most abundant forms in crocin-producing plants . The in vivo production of crocin V and crocin III in E. coli has been reported, , but the biosynthesis of other crocins and high productivity of crocin biosynthesis remains challenging. Here, we produce five types of crocins from crocetin using plant-derived glycosyltransferases in E. coli for the first time.…”

“…Microbial UGTs have provided great advantages for functional catalysis and the highly efficient bioproduction of natural compounds in microbe synthetic biology, and they have been found to accept many small molecules as substrates to produce glycosylated derivatives with high promiscuity, as exemplified by the biosynthesis of the natural products ginsenoside, , phloretin, and salidroside . Microbial UGTs have also been mined from Bacillus and have been shown to catalyze the synthesis of crocin III and crocin V. , In G. jasminoides , the expression pattern of GjUGT74F8 and GjUGT94E13 is highly consistent with the accumulation of crocins (PCT: CN2018/114419). Compared to the reported UGTs, the expression of GjUGT74F8 and GjUGT94E13 in E. coli could produce five types of crocins in vivo .…”

“…Crocetin and crocin V have been successfully produced in engineered Escherichia coli and Saccharomyces cerevisiae , ,, but these strains had low productivity. In addition, two microbial glycosyltransferases (Bs-GT from Bacillus subtilis 168 and Bc-GTA from Bacillus cereus WQ9–2) were shown to catalyze the glycosylation of crocetin to produce crocin III and crocin V . Furthermore, three enzymes (YdhE, YjiC, and Yojk) from B. subtilis might be involved in crocin biosynthesis; however, the combination of these three enzymes only produced low levels of crocin V .…”

Research progress on biosynthesis and synthetic biology of crocin

“…参与西红花苷生物 合成的微生物糖基转移酶研究也备受关注. 基于已报 道的西红花苷生物合成糖基转移酶, Ding等人 [94] . 近几年, 大宗常用中药 如人参 [99] 、三七 [100,101] 、丹参 [102] 、灵芝 [103] 、红景 [32,43,49] .…”

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside

“…To date, substantial efforts have been made to reveal glucosylation enzymes and successfully excavated more and more uridine diphosphate (UDP)-dependent glucosyltransferases (UGTs) [11][12][13][14]. The UGTs with high regioselectivity and substrate specificity have attracted remarkable attention due to their great potential application in biotechnology [15][16][17].…”