High-level Expression Of A Heterologous Creatinase In Bacillus Subtilis

Creatinase is a widely used medical enzyme that can be used in clinical renal function testing.However,due to its shortage and high production cost,creatinase production so far can not meet the market demand that can be solved by using the traditional industrial production strains for high efficiency heterologous expression.Bacillus subtilis is generally regarded as safe strain(GRAS)for protein expressing,due to its excellent secretion ability.But,in many cases,high-level expression and secretion of heterologous proteins can't be achieved through the classical pathways,which limits the industrial application of B.subtilis.Therefore,based on the expression system of B.subtilis,comprehensive optimization was carried out at the levels of transcription,translation and transport,and we constructed the high-level expression strain 1A751-GC of creatinase that doesn't rely on the classical secretion pathway.In order to improve the expression level of creatinase in B.subtilis,the codon of creatinase was optimized,and two different types of promoter were evaluated,finally,the high expression of creatinase was successfully achieved by using maltose-induced promoter P_glv.However,creatinase can be secreted without any signal peptide.For this phenomenon,the secretory mechanism in B.subtilis was deeply explored.Firstly,we analyze the Sec pathway,the Tat pathway and the Holin pathway by knockout or overexpression related genes.Results showed that the secretion of creatinase did not depend on the pathways above,excluding the possibility of secreting creatinase as protein channels.Secondly,the Calcein-AM/PI was used to analyze the cell membrane integrity of strain 1A751-GC,results found that cell membrane was damaged to different degrees.Finally,results of scanning electron microscopy and transmission electron microscopy also confirmed that there were leakage sites on the cell surface of strain 1A751-GC,indicating that creatinase was mainly secreted by cell leakage of B.subtilis.The extracellular level of creatinase was further improved in the 5 L fermenter by high-density fermentation combined with culture medium optimization,and the total activity of creatinase reached 141.9 U/m L.In this study,high-level expression of creatinase in B.subtilis was achieved by promoter optimization,and a creatinase producing strain 1A751-GC was obtained.Meanwhile,the mechanism of creatinase secretion in B.subtilis was systematically studied.The results of this study have significance for improving the efficient production of creatinase in microbial cells,and also can provide help and guidance for the expression and secretion of heterologous proteins independent of signal peptide.