Mining of efficient microbial UDP-glycosyltransferases by motif evolution cross plant kingdom for application in biosynthesis of salidroside

Abstract

The plant kingdom provides a large resource of natural products and various related enzymes are analyzed. The high catalytic activity and easy genetically modification of microbial enzymes would be beneficial for synthesis of natural products. But the identification of functional genes of target enzymes is time consuming and hampered by many contingencies. The potential to mine microbe-derived glycosyltransferases (GTs) cross the plant kingdom was assessed based on alignment and evolution of the full sequences and key motifs of target enzymes, such as Rhodiola-derived UDP-glycosyltransferase (UGT73B6) using in salidroside synthesis. The GTs from Bacillus licheniformis ZSP01 with high PSPG motif similarity were speculated to catalyze the synthesis of salidroside. The UGTBL1, which had similarity (61.4%) PSPG motif to UGT73B6, displayed efficient activity and similar regioselectivity. Highly efficient glycosylation of tyrosol (1 g/L) was obtained by using engineered E. coli harboring UGTBL1 gene, which generated 1.04 g/L salidroside and 0.99 g/L icariside D2. All glycosides were secreted into the culture medium and beneficial for downstream purification. It was the first report on the genome mining of UGTs from microorganisms cross the plant kingdom. The mining approach may have broader applications in the selection of efficient candidate for making high-value natural products.

Multi‐modular engineering of Saccharomyces cerevisiae for high‐titre production of tyrosol and salidroside

“…Conversely, microbial biosynthesis provides a sustainable and economically feasible platform for producing natural products originally derived from plants (Lee et al ., 2012 ; Borodina and Nielsen, 2014 ; Paddon and Keasling, 2014 ). In recent years, E. coil was successfully used to produce tyrosol and salidroside (Satoh et al ., 2012 ; Bai et al ., 2014 ; Chung et al ., 2017 ; Fan et al ., 2017 ; Xue et al ., 2017 ; Liu et al ., 2018 ; Yang et al ., 2019 ). Through these studies, titres of tyrosol achieved using sugar (glucose, or xylose and glucose mixture) as the carbon source, have increased from 69.08 mg l −1 (0.5 mM) to 1.47 g l −1 (Satoh et al ., 2012 ; Liu et al ., 2018 ; Yang et al ., 2019 ).…”

“…1 ). Salidroside is produced via the glycosylation of tyrosol at the 8‐OH group, which is catalysed by a regio‐specific uridine 5’‐diphospho‐glucosyltransferase (UGT; Fan et al ., 2017 ). Previous studies increased tyrosol titres to 927.68 ± 25.26 mg l −1 in shake flasks and 8.37 g l −1 in a bioreactor in a haploid industrial S. cerevisiae strain HLF‐Dα (Jiang et al ., 2018 ; Torrens‐Spence et al ., 2018 ; Guo et al ., 2019 ; Guo et al ., 2020 ).…”

In Vivo Production of Five Crocins in the Engineered Escherichia coli

“…Two plant UGTs, GjUGT75L6 from G. jasminoides and CsUGT74AD2 from C. sativus , are reported to be involved in the biosynthesis of crocin III and crocin V. , Additionally, CsUGT2 from C. sativus displayed catalytic activity capable of producing products with more than nine glucose molecules attached to crocetin . 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).…”

“…32 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, 33,34 phlor- etin, 35 and salidroside. 36 Microbial UGTs have also been mined from Bacillus and have been shown to catalyze the synthesis of crocin III and crocin V. 15,24 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.…”

Current Status of Microbial Phenylethanoid Biosynthesis

“…This indicated that UGT73B6 is not specific for salidroside synthesis at least in E. coli. The recent result of tyrosol biotransformation into salidroside using E. coli expressing UGT from Bacillus licheniformis showed the production of both salidroside and icariside D2 [51]. The UGTs found so far attach glucose to both the phenolic hydroxyl group and alcoholic hydroxyl group.…”