title: 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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  High 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.
authors: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. guoleiglly@163.com.
authors: Lei
authors: L
authors: Guo
authors: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. cx-pharmacy@126.com.|School of Medicine of Wuhan University, Wuhan, China. cx-pharmacy@126.com.
authors: Xi
authors: X
authors: Chen
authors: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. lena@imm.ac.cn.
authors: Li-Na
authors: LN
authors: Li
authors: School of Medicine of Wuhan University, Wuhan, China. tangweidoctor@163.com.
authors: Wei
authors: W
authors: Tang
authors: School of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China. panyiting@bipt.edu.cn.
authors: Yi-Ting
authors: YT
authors: Pan
authors: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. jianqiangk@imm.ac.cn.
authors: Jian-Qiang
authors: JQ
authors: Kong
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics*; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005285:Fermentation; D044950:Flavanones / Q000737:chemistry / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D060847:Metabolic Engineering / Q000379:methods*; D005810:Multigene Family; D031442:Ornithogalum / Q000201:enzymology* / Q000235:genetics*; D066244:Proton Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D044950:Flavanones; D011994:Recombinant Proteins; C016063:pinocembrin