Metabolic engineering Escherichia coli for efficient production of icariside D2

Abstract

BackgroundIcariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources.ResultsWe metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture.ConclusionsWe for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity

“…To gain UGTs with a high activity for glucosylation of phenolic aglycons, we compared eight UGTs from plants or bacteria, which were previously identified to have considerable substrate promiscuity and catalytic efficiency towards phenolic substrates. These UGTs include: UGT85A1 from A. thaliana and UGT72B14 from Rhodiola sachalinensis , applied to produce salidroside [ 15 , 26 ]; RrUGT3 from R. rosea , reported for efficient production of icariside D2 [ 18 ]; RrUGT33 and RrUGT17 from R. rosea , characterized as the key UGTs for biosynthesis of tyrosol glucosides in Rhodiola genus [ 24 ]; UGT73B6 from R. sachalinensis and its mutant UGT73B6F389S (UGT73B6FS), used in synthesis of many aromatic glucosides [ 27 ]; and YjiC from Bacillus licheniformis , having the capability to synthesize many types of glucosides [ 15 , 28 ].…”

“…In the era of synthetic biology, a growing number of biosynthetic systems for O -glucosides based on in vitro enzymatic cascades and in vivo metabolic engineering have been developed. For example, the microbial cell factories for salidroside, gastrodin, and icariside D2 have been constructed by integrating different uridine diphosphate (UDP) glucosyltransferases (UGT) and the fine-tuned aglycon supplying pathways [ 15 – 18 ]. Various biocatalytic systems for unnatural C -glucosides [ 19 , 20 ] and O -glucosides [ 21 – 23 ] have also been established by taking advantage of the substrate promiscuity of UGTs.…”

Metabolic Engineering of Escherichia coli for de Novo Production of Betaxanthins

“…We speculate that the poor growth of strain BTA6 may be caused by insufficient nitrogen nutrients of M9 medium. Yeast extract, as a general nitrogen source essential for coculture system, was designed to make a positive strain–strain interaction . When the M9 medium supplemented with 1 g/L of yeast extract (M9Y) is used, the growth of the coculture has an obvious improvement, and more glucose is consumed (Figure D).…”

“…Yeast extract, as a general nitrogen source essential for coculture system, was designed to make a positive strain− strain interaction. 30 When the M9 medium supplemented with 1 g/L of yeast extract (M9Y) is used, the growth of the coculture has an obvious improvement, and more glucose is consumed (Figure 6D). The percentage of BHS3 tends to be stable at approximately 20% after 12 h. It seems that the addition of yeast extract mainly improves the growth of strain BTA6 and balances the population of two strains in the coculture during the fermentation, leading to more histidinebetaxanthin production.…”

De novo biosynthesis of paracetamol from glucose by metabolically engineered Escherichia coli

“…With the developments of biotechnology, signi cant advances have been made on engineered microorganism for the biosynthesis of a variety of chemicals and drugs from renewable resources [11][12][13][14][15]. Paracetamol is one of the most commonly used drugs and is widely regarded as a safe and effective drug for the treatment of mild to moderate pain and fever.…”