Cloning And Characterization Of Genes In The Biosynthetic Pathway Of Pinocembrin And Construction Of Engineered Escherichia Coli For (2S)-Pinocembrin

With over 9000 substituted members and growing, flavonoids are the most numerous and widespread plant secondary metabolites found in fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, teas and red wines. Plant flavonoids have recently attracted increasing research interest due to their diverse biological functions, including antioxidant, antiviral,antibacterial, antiobesity, and anticancer. They are subdivided into flavonols, flavones, flavonones, isoflavones, catechins, and anthocyanins according to variability in molecular structure.Pinocembrin, belonging to flavanons, was isolated from various plants.Pinocembrin has a variety of pharmacological activities, such as neuroprotective effect, antimicrobial activity, and antioxidant efficacy. Pinocembrin was approved as class I drugs to its phase Ⅱ clinical trial by CFDA in 2009, mainly used for the treatment of ischemic stroke. As a promising compound, the manufacturing technologies of pinocembrin, including chemical synthesis, extraction from plant and synthetic biology technology, have attracted many attentions. Compared with the first two technologies, synthetic biology technology has many advantages, such as environment-friendly and low-cost. Microbial production offers an alternate way to produce these compounds on an industrial scale in a more economical and environment-friendly manner.Pinocembrin can be synthesized biologically using microorganisms such as Escherichia coli, which has been used to synthesize diverse natural compounds. It is synthesized from phenylalanine by the action of three enzymes:4-coumarate:CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). Because of the stereo-selectivity of CHI, pinocembrin, obtained by synthetic biology technology, is 2S configuration which plays an important role in new drugs application.Farnesyl diphosphate synthase (FPPS) catalyzes the sequential head-to-tail coupling of DMAPP with two molecules of IPP, producing farnesyl diphosphate(FPP), which lies at the branch point of the pathway of isoprenoid synthesis. FPP is the precursor of a structurally diverse class of sesquiterpenes that are widely distributed in plant kingdom including a diversity of functionally important compounds, such as artemisinin, a very famous anti-malaria drug. The study of FPPS from O.caudatum can provide gene element for construction of erpenoids biosynthetic pathway in vitro.In this study, we mainly focus on (25)-pinocembrin produced by synthetic biology technology. The genes of Osa4CL,OsaCHS and OsaCHI, which play key role in the biosynthesis pathway of pinocembrin, were cloned and induced to express. Then, we study on the characterization of three key enzymes and constructed pinocembrin engineering strain. At last, several strategies were applied to improve the titer of pinocembrin.1. Cloning and expression of the key enzymes, Osa4CL, OsaCHS and OsaCHITotal RNA was extracted from sterile bulb tissue of O.caudatum, and been used as a template for reverse transcription to obtain the cDNA after transcriptome sequencing and analysis. And then, according to the method of In-Fusion, primers were designed for target genes. In this study, seven genes coding for 4CL, designated as OsaCLl,OsaCL2, OsaCL3, OsaCL4, OsaCLS, OsaCL6 and OsaCL7, a gene coding for CHS, designated as OsaCHS1, OsaCHS2 and OsaCHS3, one gene coding for CHI, designated as OsaCHI, were firstly isolated from O.caundersiae and Osa4CL1, Osa4CL2, Osa4CL3, Osa4CL4, Osa4CL5, Osa4CL6, Osa4CL7, OsaCHS1, OsaCHS2, OsaCHS3 and OsaCHI were expressed successfully by gene expression analysis.2. Characterization of the key enzymes and the construction of (2S)-pinocembrin strainIf the strain of pinocembrin, constructed by three enzymes with only one OsaCHS2 whose function is unknown in the biosynthetic pathway, can produce pinocembrin, we can conclude that OsaCHS2 can catalyze cinnamoyl CoA to form pinocembrin chalcone. In this study, we made functional analysis of OsaCHS and OsaCHI by constructing strain. The product of strain, (2S)-pinocembrin, was unambiguously determined by HPLC, ESI-MS,NMR and CD.3. The optimization for (2S)-pinocembrin strainIn order to improve the yield, lots of modular pathway engineering strategies were applied, such as the selection of appropriate enzyme sources, constructing three modules of the key enzymes, and codon-optimizing for Osa4CL、OsaCHS and MsCHI.Once this metabolic balance was achieved, the yield of optimum strain was 1.66 folder than the first strain after taking combinatorially strategies.4. Cloning and characterization of farnesyl pyrophosphate synthaseAn 1327 bp long unigene with an open reading frame of 1044 bp was retrieved from the transcriptome sequencing of O.caudatum.The full-length FPPS cDNA,designated OsaFPPS, was isolated from O.caudatum with gene-specific primers. The resultant OsaFPPS encodes a 347-amino acids protein with a calculated molecular mass of 4.0 kDa, and a theoretical isoelectric point of 5.01.Phylogenetic tree analysis indicated that OsaFPPS belongs to the plant FPPS super-family. Expression of soluble OsaFPPS in E.coli was verified by SDS-PAGE and western-blot analysis. Functional analysis of the purified OsaFPPS protein was carried out using IPP and DMAPP as substrates, and the product was determined by GC-MS analyses.