High-level production of Rhodiola rosea characteristic component rosavin from D-glucose and L-arabinose in engineered Escherichia coli.

Rosavin is the characteristic component of Rhodiola rosea L., an important medicinal plant used widely in the world that has been reported to possess multiple biological activities. However, the endangered status of wild Rhodiola has limited the supply of rosavin. In this work, we successfully engineered an Escherichia coli strain to efficiently produce rosavin as an alternative production method. Firstly, cinnamate: CoA ligase from Hypericum calycinum, cinnamoyl-CoA reductase from Lolium perenne, and uridine diphosphate (UDP)-glycosyltransferase (UGT) from Bacillus subtilis (Bs-YjiC) were selected to improve the titer of rosin in E. coli. Subsequently, four UGTs from the UGT91R subfamily were identified to catalyze the formation of rosavin from rosin, with SlUGT91R1 from Solanum lycopersicum showing the highest activity level. Secondly, production of rosavin was achieved for the first time in E. coli by incorporating the SlUGT91R1 and UDP-arabinose pathway, including UDP-glucose dehydrogenase, UDP-xylose synthase, and UDP-xylose 4-epimerase, into the rosin-producing stain, and the titer reached 430.5 ± 91.4 mg/L. Thirdly, a two-step pathway derived from L-arabinose, composed of L-arabinokinase and UDP-sugar pyrophosphorylase, was developed in E. coli to further optimize the supply of the precursor UDP-arabinose. Furthermore, 1203.7 ± 32.1 mg/L of rosavin was produced from D-glucose and L-arabinose using shake-flask fermentation. Finally, the production of rosavin reached 7539.1 ± 228.7 mg/L by fed-batch fermentation in a 5-L bioreactor. Thus, the microbe-based production of rosavin shows great potential for commercialization. This work provides an effective strategy for the biosynthesis of other valuable natural products with arabinose-containing units from D-glucose and L-arabinose.

[Advances in microbial synthesis of plant natural products].

Plant natural products are one of the main sources of small molecule drugs, nutraceuticals, cosmetics and fragrances, and play an important role in economy development. At present, the way of obtaining plant natural products mainly depends on direct extraction from plants, which is farm land occupying and time consuming. The contents of active ingredients in plants are usually low, and thus the production cost is high. By elucidating the biosynthetic pathways and reconstructing the pathways in microbial cells, plant natural products can be produced by fermentation using renewable raw materials. Microbial biosynthesis provides a new route for the supply of plant natural products. This review summarizes the research progress of microbial synthesis of terpenoids, flavonoids, phenylpropanoids and other important natural products of plants in Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Current research challenges and future prospects are also briefly discussed.

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction.

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6'-O-ß-xylopyranosyl)-O-ß-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.

Producing Gram-Scale Unnatural Rosavin Analogues from Glucose by Engineered Escherichia coli.

Cinnamyl alcohol glycosides (CAGs) are key active ingredients of the precious medicinal plant Rhodiola rosea L., which has diverse pharmacological activities. The quality of R. rosea extracts is standardized to the contents of rosavin, a cinnamyl alcohol disaccharide, along with salidroside. The supply of rosavin and analogues is limited by both the inefficiency of chemical synthesis methods and the shortage of natural resources. Herein, we achieved de novo synthesis of a series of rosavin analogues by engineered Escherichia coli strains. First, cinnamyl alcohol was synthesized by expression of phenylalanine ammonia-lyase (PAL), hydroxycinnamate:CoA ligase, and cinnamyl-CoA reductase in a phenylalanine high-producing strain. UGT73C5 from Arabidopsis thaliana and a sugar chain elongating glycosyltransferase from Catharanthus roseus, CaUGT3 sequentially catalyzed the formation of an unnatural cinnamyl alcohol diglucoside, named rosavin B. Then, these biosynthetic enzymes were transformed into a tyrosine high-producing strain, except that PAL was replaced by a tyrosine ammonia-lyase, and synthesis of mono- and diglucosides of p-coumaryl alcohol with sugars attached to aliphatic or phenolic hydroxyl position was achieved. Finally, fed-batch fermentation was conducted for the strain producing rosavin B, and the titer reached 4.7 g/L. Tri- and tetraglucosides of cinnamyl alcohol were also produced by fed-batch fermentation. In summary, seven rosavin analogues including six unnatural compounds were produced from glucose by microorganisms. This work expanded the structural diversity of CAGs, which holds promise to discover new analogues with improved pharmaceutical properties. The study also paves the way for producing CAGs in a sustainable and cheap way.

Production of Cinnamyl Alcohol Glucoside from Glucose in Escherichia coli.

Rosin, a cinnamyl alcohol glucoside, is one of the important ingredients in Rhodiola rosea, which is a valuable medicinal herb used for centuries. Rosin displayed multiple biological activities. The traditional method for producing rosin and derivatives is direct extraction from R. rosea, which suffers from limited availability of natural resources and complicated purification procedure. This work achieved de novo biosynthesis of rosin in Escherichia coli. First, a biosynthetic pathway of aglycon cinnamyl alcohol from phenylalanine was constructed. Subsequently, the UGT genes from Rhodiola sachalinensis (UGT73B6) or Arabidopsis thaliana (UGT73C5) were introduced into the above recombinant E. coli strain to produce rosin. Then the phenylalanine metabolic pathway of E. coli was optimized by genetic manipulation, and the production of rosin by the engineered E. coli reached 258.5 ± 8.8 mg/L. This study lays a significant foundation for microbial production of rosin and its derivatives using glucose as the renewable carbon source.

Heterologous biosynthesis of costunolide in Escherichia coli and yield improvement.

Costunolide, the main bioactive compound of the medicinal plant, Radix Aucklandiae, is a sesquiterpene lactone (SL) and has a broad range of biological activities. It is also a precursor of many biologically-active SLs and is a branching point in the biosynthesis of SLs. Here we have reconstituted the costunolide biosynthetic pathway in Escherichia coli by co-expression of three genes (GAS, GAO, LsCOS) involved in costunolide biosynthesis and eight genes involved in converting acetyl-CoA into farnesyl diphosphate from mevalonate pathway. Costunolide production was then detected. By screening and optimization of cultured medium and inducing temperature, costunolide yield was up to 100 mg l(-1) in E. coli.

Production of salidroside in metabolically engineered Escherichia coli.

Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.