Study On The Effect Of Acyl-CoA Binding Protein On Monascus Pigments Synthesis Metabolism In Monascus Ruber

Monascus pigment is called Danqu in ancient,and has a history of more than1,000 years in China.The Monascus pigment is a natural edible pigment which was fermented by Monascus spp.with rice as substrate.The chemical structure of Monascus pigment consists of a polyketone chromophore and a fatty acid chain.The highly conserved protein acyl-coA binding proteins?ACBP?exsited in eukaryotes and prokaryotes,and involved in fatty acid metabolism.In this thesis,the relationship of the two ACBPs?MrACBP?and the Monascus pigment synthesis in Monascus CICC41233 were study.The main results were as follows:?1?Clone of Mracbp gene:The Mracbp gene was amplified by PCR using the total DNA and cDNA of Monascus ruber CICC41233 as a template.After sequencing,the Mracbp1 DNA sequence was 629 bp in length and the RNA sequence was 450 bp in length.It contains 2 introns,and encoding 149 amino acids with a predicted molecular mass of 16 kDa;the NCBI nucleic acid Blast alignment was 73.97%homologous to Aspergillus vadensis CBS 113365 ACBP-domain-containing protein?BO88DRAFT₄83611??XM₀25712234.1?and the protein Blast alignment was37.21%homologous to Aspergillus sclerotiicarbonarius CBS 121057?PYI08169.1?.The Mracbp2 DNA sequence is 1525 bp in length and the RNA sequence is 1329 bp in length.It contains 3 introns,and encoding 442 amino acids with a predicted molecular mass of 51 kDa.The NCBI nucleic acid Blast alignment has a homology of72.59%with Aspergillus nomius NRRL 13137 acyl CoA binding protein?ANOM₀09680??XM₀15554936.1?and the protein Blast alignment was 72.11%homologous to Aspergillus nomius NRRL 13137?XP₀15401904.1?.?2?Binding ability test of MrACBP to different lengths carbon chains of acyl-CoA:The intron-free Mracbp1 was constructed in the prokaryotic expression vector pMAL-c4X,transformed into E.coli Rosetta DE3 strain,and the target protein was induced by IPTG.The MBP-MrACBP1 fusion protein was obtained after purification.It has the strongest binding ability to C_14:0-CoA after tested by micro thermophoresis binding experiments.The intron-free Mracbp2 was constructed into the prokaryotic expression vector pGEX-6p-1,transformed into E.coli BL21 strain,and the target protein was induced by IPTG.The GST-MrACBP2 fusion protein was obtained after purification.It has the strongest binding ability to C_16:0-CoA after tested by micro thermophoresis binding experiments.?3?Analyze of overexpression Mracbp in Monascus ruber CICC41233 for the production of Monascus pigment:Construction of the Mracbp1 gene expression vector pNeo0380-gpdA-acbp1 and the Mracbp2 gene expression vector pNeo0380-gpdA-acbp2,and transformd into Monascus ruber CICC41233 by Agrobacterium-mediated,respectively,obtained engineering strain which named Monascus ruber ACBP5 and Monascus ruber ACBP-E.Compared with Monascus ruber CICC41233,Monascus ruber ACBP5 was fermented for 6 days,the yield of monascus pigment was increased by 12.70%.The expression of Mracbp1 and pks,mppr1,fasA and fasB which are the synthetic gene cluster of Monascus pigment was up-regulated by 77.39-fold,6.41-fold,3.21-fold,3.42-fold and 4.41-fold,respectively.Monascus ruber ACBP-E was fermented for 6 days,the yield of Monascus pigment was increased by 37.73%.The expression of Mracbp2 and pks,mppr1,fasA and fasB which are the synthetic gene cluster of Monascus pigment was up-regulated by1.74-fold,2.7-fold,2.51-fold,2.20-fold and 3.11-fold,respectively.?4?Knockout the acbp2 gene in Monascus ruber CICC41233:To improve the gene knockout efficiency of Monascus,a DNA ligase IV?ligD?knockout vector was constructed,and transformd into Monascus ruber CICC41233 by Agrobacterium-mediated and obtain a mutant that named Monascus ruber?ligD.The knockout vector of Mracbp2 gene was constructed,and was transformd into Monascus ruber?ligD by Agrobacterium-mediated.The 50 transformants were selected for screening verification,but the acbp2 gene was not successfully knocked out.?5?Express and purify MrACP protein:The subunit Acyl carrier protein?ACP?of fatty acid synthase was cloned from Monascus ruber CICC41233.The His-tag protein was introduced before the stop codon of the acp gene,and ligated into the prokaryotic expression vector pMAL-c4X to construct the MBP+His double-tag protein expression system,and then transformed into E.coli Rosetta DE3 strain and induced by IPTG.The target protein was expressed,and use the nickel column purification system to obtain the MBP-MrACP-His fusion protein.This paper explored the influence of ACBP on the synthesis of Monascus pigment,and provided new ideas for improving the yield of Monascus pigment and new directions for constructing engineering strains with high yield of Monascus pigment.