Abstract
Background(2S)-Pinocembrin is a chiral flavanone with versatile pharmacological and biological activities. Its health-promoting effects have spurred on research effects on the microbial production of (2S)-pinocembrin. However, an often-overlooked salient feature in the analysis of microbial (2S)-pinocembrin is its chirality.ResultsHere, we presented a full characterization of absolute configuration of microbial (2S)-pinocembrin from engineered Escherichia coli. Specifically, a transcriptome-wide search for genes related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum, a plant rich in flavonoids, was first performed in the present study. A total of 104,180 unigenes were finally generated with an average length of 520 bp. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping assigned 26 unigenes, representing three enzyme families of 4-coumarate:coenzyme A ligase (4CL), chalcone synthase (CHS) and chalcone isomerase(CHI), onto (2S)-pinocembrin biosynthetic pathway. A total of seven, three and one full-length candidates encoding 4CL, CHS and CHI were then verified by reverse transcription polymerase chain reaction, respectively. These candidates were screened by functional expression in E. coli individual or coupled multienzyme reaction systems based on metabolic engineering processes. Oc4CL1, OcCHS2 and OcCHI were identified to be bona fide genes encoding respective pathway enzymes of (2S)-pinocembrin biosynthesis. Then Oc4CL1, OcCHS2 and MsCHI from Medicago sativa, assembled as artificial gene clusters in different organizations, were used for fermentation production of (2S)-pinocembrin in E. coli. The absolute configuration of the resulting microbial pinocembrin at C-2 was assigned to be 2S-configured by combination of retention time, UV spectrum, LC–MS, NMR, optical rotation and circular dichroism spectroscopy. Improvement of (2S)-pinocembrin titres was then achieved by optimization of gene organizations, using of codon-optimized pathway enzymes and addition of cerulenin for increasing intracellular malonyl CoA pools. Overall, the optimized strain can produce (2S)-pinocembrin of 36.92 ± 4.1 mg/L.ConclusionsHigh titre of (2S)-pinocembrin can be obtained from engineered E. coli by an efficient method. The fermentative production of microbial (2S)-pinocembrin in E. coli paved the way for yield improvement and further pharmacological testing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0424-8) contains supplementary material, which is available to authorized users.
Highlights
Pinocembrin (1, Fig. 1), named 5, 7-dihydroxyflavanone or dihydrochrysin, is a kind of chiral flavonoid made up of two enantiomer forms, (2S)-pinocembrin (2, Fig. 1) and (2R)-pinocembrin (3, Fig. 1)
Results showed varied unigenes were assigned to every step of (2S)-pinocembrin (2) biosynthesis (Additional file 1: Table S1)
19, 3 and 4 unigenes showing high similarity with 4-coumarate:coenzyme A ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) were retrieved from transcriptome sequence, HO
Summary
Pinocembrin (1, Fig. 1), named 5, 7-dihydroxyflavanone or dihydrochrysin, is a kind of chiral flavonoid made up of two enantiomer forms, (2S)-pinocembrin (2, Fig. 1) and (2R)-pinocembrin (3, Fig. 1). In response to the poor yield of extraction from natural sources and poor chemical synthesis efficiency, research groups have directed their attention to the microbial production of (2S)-pinocembrin (2) [18,19,20]. This approach expresses the biosynthetic pathway of (2S)-pinocembrin (2) in many more amenable heterologous hosts to improve pinocembrin yields with a more economical and environment-friendly manner
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