Transcriptome-enabled discovery and functional characterization of enzymes related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum and their application for metabolic engineering

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.

Isolation and characterization of a multifunctional flavonoid glycosyltransferase from Ornithogalum caudatum with glycosidase activity

“…A transcriptome dataset of O. caudatum was reported previously [22][23][24] . The assembled sequences (104,180 unigenes) were thus aligned by Blast X to protein databases like nr, Swiss-Prot, KEGG and COG (e-value < 0.00001) with the aim of retrieving unigenes sharing the high sequence similarity with flavonoid GTs.…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…Of these software packages, citations have been found for JCat and OPTIMIZER for expression systems such as E. coli (Fahimi et al 2016;Guo et al 2016;Karkhah & Amani 2016;Zhao et al 2016), S. cerevisiae (Ask et al 2013;Guadalupe-Medina et al 2013;Li et al 2015;Milne et al 2015;Solis-Escalante et al 2013), N. benthamiana (Binder et al 2014), HEK293 cells (Shah et al 2015), B. subtilis (Reilman et al 2014), C. crescentus (Ko et al 2013), P. putida (Dammeyer et al 2013;Dammeyer et al 2011), S.…”

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

“…The procedure of transcriptome sequencing of O. saundersiae was the same as that of O. caudatum [28][29][30][31] . In brief, total RNA was extracted from the frozen bulbs of O. saundersiae using TRIzol reagent (CWBIO Co., Ltd., Beijing, China) according to the manufacturer's instructions.…”

“…To verify the authenticity of the candidate unigene, cDNA isolation was performed using gene-specific primers by a nested PCR assay as previously described (Supplementary Information Table S1) [28][29][30][31] . The obtained amplicon was inserted into pEASY TM -Blunt plasmid (TransGen Co., Ltd., Beijing, China) and then transformed into E. coli Trans1-T1 competent cells for recombinant plasmid selection ( Supplementary Information Table S2).…”

“…Also, to improve heterologous expression of OsSGT1, pET28a-OsSGT1 was cotransformed into E. coli BL21(DE3) strain with a chaperone plasmid pGro7 (Takara Biotechnology Co., Ltd., Dalian, China) as introduced by Yin et al 27 The expression of OsSGT1 was induced by IPTG at a final concentration of 0.3 mmol/L. The expressed OsSGT1 was checked by SDS-PAGE and Western-blot analyses as described by Guo et al 31 Next, the BL21(DE3) [pET28a-OsSGT1þpGro7] suspension cells were disrupted in a high-pressure homogeniser (APV-2000, Albertslund, Denmark) operated at 800 bar. Disrupted cells were centrifuged at 12,000 rpm for 30 min to discard the pellet.…”