Cloning,Prokaryotic Expression Of DPPS From Monascus And Study On Production Condition Of Red Yeast Rice With CoQ10 By In Solid-state Fermentation

Red yeast rice is a unique purple product from the rice fermentation with Monascus bacteria.Red yeast rice is mainly applied in wine brewing,food fermentation,pigment production and medicine industry,etc.Previous studies suggest that Monascus can be used in CoQ10 synthesis.Decaprenyl Diphosphate Synthase(DPPS)is the key limiting enzyme for the biosynthetic pathways of CoQ.However,Monascus DPPS encoding gene cloning and its heterologous expression has not been reported so far,which largely restricted the application of Monascus and its development.In our study,Monascus N4-5 was obtained and used as the bacteria strain to produce CoQ 10.Next,DPPS encoding gene cloning,Sprokaryotic recombinant expression was performed to examine the molecular mechanism of CoQ 10 synthesis with Monascus and provide a scientific basis for the development and exploitation of this new resource.The principle content of this project was illustrated as below:1.The identification parameters of reversed-phase HPLC method for coenzyme Q10 content in Monascus was determined as:Shim-pack C18 column(250×4.6mn,5μm)with mobile phase methanol-ethanol absolute(1:1).The detection was performed at 275nm with the flow rate of 1.0 mL/min.The calibration curve was linear in the range of 0.1～?300μg(R2=0.9999).The average recovery was 100.01%with the RSD being 0.33%.The method demonstrated good linearity,reproducibility,high precisionand accuracy,and efficient recovery rate.2.The full-length cDNA of DPPS gene was cloned from the Monascus by using RT-PCR technique,and the sequence analysis and function prediction were conducted.The length of cloned DPPS gene was 1188bp,containing a complete development of the reading frame(1 176bp),which encodes 392 amino acids with a protein molecular weight of about 43.57kDa.In the DNA sequence,the DPPS shows 74%,74%,and 73%homology with IPP of Aspergillus flavus,IPP of Aspergillus oryzae,and DPPS of Neosartorya fischeri,respectively.Whereas in the amino acid sequence,DPPS shows 68%,68%,68%identity and 79%,80%,80%similaritywhen compared with the DPPS of Neosartorya fischeri,IPP of Aspergillus flavus,IPP of Aspergillus oryzae,respectively.The conserved domain search of the deduced amino acid sequence was conducted.Resuts indicated that an alignment of Monascus rubber DPPS and DPPSs from other species showed a significant homology for seven highly conserved regions,including two aspartate-rich domains with oligopeptide.Based on the results,it could be inferred that DPPS resembles the ability of synthase gene in polyisoamylene phrophosphate synthesis.3.Prokaryotic Recombinant Expression,affinity chromatography purification were performed on the Monascus DPPS obtained from cloning.The expression products were identified by HPLC.Next,the DPPS prokaryotic expression vector,pET-DPPS,was constructed and transformed into Escherichia coli BL21(DE3).With IPTG induction,the target gene was successfully expressed into a fusion protein with the expected size.With further purification by affinity chromatography on DPPS,the results showed that His-DPPS fusion protein can be purified by nickel affinity chromatography to get the target protein of electrophoresis purity.The expression product was identified by HPLC and results suggested that DPPS is a key enzyme in the synthesis of CoQ10 by Monascus.4.With long-grain rice as raw material,the solid-state fermentation of CoQ10 with Monascus was optimized by using box benhnken experimental design and response surface analysis.Four key solid-state fermentation conditions which affects the Monascus color value and the content of CoQ10 were taken as independent variables,including the initial pH,inoculum,initial water ratio and linoleic acid supplementary content.Monascus color value and the content of CoQ10 in red yeast rice were used as response values to optimize the above four key fermentation parameters.On this basis,the strategies for the Monascus segmented temperature control during fermentation were explored.Results suggested that the initial pH,inoculation amount and the addition of linoleic acid had significant influences on Monascus color value and the content of CoQ10(P<0.05).The effect of initial water content was found insignificant on the color value of Monascus and the content of CoQ10(P>0.05).The optimal conditions of fermentation were determined as:the initial pH of 6.0,the inoculation quantity of 12%,the initial water content of long-grain rice as 50%,the addition of linoleic acid as 64 μ m.The optimal temperature control strategy was determined as:30℃/6d→34℃/5d→28℃/4d.Under the optimal fermentation conditions,the color value and CoQ10 content in red yeast rice reached 4521.69 U/g and 387.23 mg/kg respectively,which increased by 38.10%and 80.01%compared to the results without optimization.