Production of salidroside in metabolically engineered Escherichia coli

Abstract

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.

Figure 1. Metabolic pathways for tyrosol (3), salidroside (1), and icariside D2 (4) biosynthesis in recombinant E. coli strains from glucose.

(a) The artificial synthetic pathway for salidroside (1) production from 2 consisting of enzymes, yeast pyruvate decarboxylase ARO10, endogenous ADH of E. coli, and UGT73B6 from Rhodiola. (b) Metabolic flux enhancement of precursor supply of 2. Single arrows represent one-step conversions, while single dashed arrows represent multiple steps. Bold arrows represent gene overexpression. The dashed lines indicate feedback inhibitions. The fork arrows represent gene deletion. Abbreviations: G6P, 6-phosphate-D-glucose; PYR, pyruvate; G3P, glyceraldehyde-3-phosphate; F6P, fructose-6-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; UDP-glucose, Uridine 5′-diphosphoglucose; DAHP, 3-deoxy-arabino-heptulonate7-phosphate; DHQ, 3-dehydroquinic acid; CHA, chorismic acid; DHS, dehydroshikimate; SHIK, shikimate; S3P, shikimate 3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PP, phenylpyruvate; PHE, phenylalanine; 4HPP, 4-hydroxyphenylpyruvate; TYR, tyrosine; AroG*, feedback resistant mutant of AroG; TyrA*, feedback resistant mutant of TyrA; FeaB, phenylacetaldehyde dehydrogenase; ADH, alcohol dehydrogenase.

Figure 2. Plasmids construction.

Plasmids pBb1, pBb2 and pBb3 containing L-tyrosine pathway genes were used to stepwise enhance metabolic flux toward 4-hydroxyphenylpyruvate (2); plasmids pTrc1 and pTrc2 harbored genes encoding for enzymes involved in biosynthesis of tyrosol and salidroside.

Figure 3. Production of tyrosol (3) in recombinant strains.

(a) HPLC analysis of tyrosol (3) production in the fermentation supernatant of recombinant strains. 1. BMGF1, E. coli MG1655 (ΔfeaB) harboring plasmid pTrc1 (P_Trc-ARO10); 2. BMGF0, E. coli MG1655 (ΔfeaB) harboring empty vector pTrcHisB (control); 3. Standard tyrosol (3). (b) Mass spectrum of tyrosol (3) from fermentation supernatant of strain BMGF1. (c) The influence of gene deletions on the tyrosol production. All the strains derived from BMGF1. The genes pykA, tyrR, pykF and pheA were sequentially deleted to enhance the metabolic flux toward 4-hydroxyphenylpyruvate (2), resulting strains BMGP1, BMGT1, BMGK1 and BMGA1. (d) The influence of genes overexpression on the tyrosol production. BMGA2, BMGA3, BMGA4 and BMGA5 were derived from BMGA1 harboring empty vector pBb0 (as control), pBb1 (P_LtetO−1-tyrA*^syn-aroG*^syn-ppsA), pBb2 (P_LtetO−1-tyrA*^syn-aroG*^syn-ppsA and P_lacUV5-tktA-aroE) and pBb3 (P_LtetO−1-tyrA*^syn-aroG*^syn-ppsA, P_lacUV5-tktA-aroE and P_lacUV5-aroD-aroB^op), respectively. Three replicates were performed, and the error bars represented standard deviation.

Figure 4. Salidroside (1) and icariside D2 (4) were produced by metabolically engineered strain carrying Rhodiola-derived glycosyltransferase.

(a) HPLC analysis of tyrosol (3) and glycosylated derivatives in fermented supernatant sample of strains. 1. BMGA6 derived from BMGA5 by introduction of glycosyltransferase UGT73B6; 2. BMGA5 was a tyrosol over-producing strain; 3. Tyrosol (3) and salidroside (1) standard. (b) HR-ESI-MS of salidroside (3) from fermented supernatant of BMGA6. (c) Gastrodin (7), a main bioactive component from Tianma and its homolog icariside D2 (4). (d) HR-ESI-MS of icariside D2 (4) from fermented supernatant of strain BMGA6.