Efficient Biosynthesis of Salidroside via Artificial in Vivo enhanced UDP-Glucose System Using Cheap Sucrose as Substrate

Abstract

Salidroside, a valuable phenylethanoid glycoside, is obtained from plants belonging to the Rhodiola genus, known for its diverse biological properties. At present, salidroside is still far from large-scale industrial production due to its lower titer and higher process cost. In this study, we have for the first time increased salidroside production by enhancing UDP-glucose supply in situ. We constructed an in vivo UDP-glucose regeneration system that works in conjunction with UDP-glucose transferase from Rhodiola innovatively to improve UDP-glucose availability. And a coculture was formed in order to enable de novo salidroside synthesis. Confronted with the influence of tyrosol on strain growth, an adaptive laboratory evolution strategy was implemented to enhance the strain's tolerance. Similarly, salidroside production was optimized through refinement of the fermentation medium, the inoculation ratio of the two microbes, and the inoculation size. The final salidroside titer reached 3.8 g/L. This was the highest titer achieved at the shake flask level in the existing reports. And this marked the first successful synthesis of salidroside in an in situ enhanced UDP-glucose system using sucrose. The cost was reduced by 93% due to the use of inexpensive substrates. This accomplishment laid a robust foundation for further investigations into the synthesis of other notable glycosides and natural compounds.

Figure 1

Diagram showcasing the synthetic coculture of S. cerevisiae and E. coli designed for the production of salidroside using a blend of glucose and sucrose. The AAS enzyme was introduced from the high tyrosine-producing pathway to form E. coli QH04 for tyrosyl synthesis. Sucrose synthase GmSUS from Glycine max and glycosyltransferase RrUGT33 from Rhodiola were introduced into endogenous UDP-glucose-producing S. cerevisiae to form strain QH03, which could achieve in situ UDP-glucose circulation and salidroside synthesis. QH03 and QH04 strains ingested sucrose and glucose, respectively, and cocultured stably. PcAAS: acetaldehyde synthases enzymes, 4-HPAA: 4-hydroxyphenylacetaldehyde, ADH: alcohol dehydrogenase, UDP: uridine diphosphate, UDP-glucose: uridine 5, 9-diphosphoglucose, RrUGT33: UDP-glucosyltransferase from Rhodiola, GmSUS: sucrose synthase from Glycine max, Agt1: Alpha-Glucoside Transporter.

Figure 2

Strain QH02 and QH03 used tyrosol as a substrate to produce salidroside. (A) The HPLC of fermentation broth after 120 h culture. Recombinant QH02 (expressing RrUGT33 alone in S. cerevisiae) and QH03(coexpressing GmSUS and RrUGT33 in S. cerevisiae) were incubated with tyrosol and sucrose, respectively. (B) Plot of the titer variation of strains QH02 and QH03 using tyrosol as substrate to produce salidroside by fermentation with sucrose and glucose. (C) The final titer of salidroside and molar conversion of tyrosol produced by QH02 and QH03 with different carbon sources. (D) Variation curves of the concentration and conversion rate of ethanol. Purple represents the alcohol and alcohol conversion produced by fermentation of the QH02 strain with glucose. Orange represents the alcohol and alcohol conversion produced by fermentation of the QH03 strain with sucrose. (E) Variation curves of OD600 nm. Purple circles represent the growth of strain QH02 in glucose, and orange squares represent the growth of QH03 in sucrose. The trials were conducted three times, with the error bars indicating the standard deviations.

Figure 3

Coculture engineering of the QH03 strain with the QH04 strain. (A) QH03 and QH04 strains were fermented in a sucrose and glucose mixture, sugar consumption comparison, and salidroside production. (B) Comparison of sugar consumption and growth of strain QH03-hxt-null in single glucose and sucrose, respectively. (C) Salidroside titer of QH03-hxt-null on the sole sucrose and mixture of glucose and sucrose. (D) Production of tyrosol and salidroside produced by coculture before and after modification of QH03 strain. The trials were conducted three times, with the error bars indicating the standard deviations.

Figure 4

Salidroside production through metabolic balancing of QH03-hxt-null and QH04 strains in a synergistic coculture. (A) Enhancing salidroside production by adjusting the sucrose-to-glucose ratio, starting with an initial S/G ratio of 1/1. (B) Improving salidroside production by adjusting the inoculation ratio of the QH03-hxt-null strain and QH04 strain, using an S/G ratio of 3/1. (C) Optimization of salidroside production by altering the content of maltose, inorganic salt mixture, and yeast extract with the S/G ratio of 3/1 and initial Q3/Q4 ratio of 2/1. (D) Optimization of salidroside production by altering the induction time of IPTG. The trials were conducted three times, with the error bars indicating the standard deviations.

Figure 5

Test of cytotoxicity of tyrosol to S. cerevisiae. (A) Effect of tyrosol at different concentrations on the growth of QH03-htx-null. (B) Tolerance of QH03-T5 strain to tyrosol after domestication. (C) Comparison of salidroside titer of QH03 strain and QH03-T5 strain. The trials were conducted three times, with the error bars indicating the standard deviations.