Codon Optimization And Over-expression Of Rhizomucor Miehei Lipase (RML) In Pichia Pastoris

Lipases as biocatalysts have been widely applied in industrial fields such asfood, medicine, biodiesel, etc. Rhizomucor miehei lipase (RML) is one of the mostversatile lipases with good selectivity and high trans-esterification activity. However, thenatural yield of R. miehei lipase is relatively low and can not meet the needs of large-scaleindustrial application. Employing genetic engineering technology to integrate R.miehei lipase gene into heterologous expression system Pichia pastoris for overexpressionof R. miehei lipase to enhance production, has a vital significance to the industrialapplication of this enzyme.The full-length RML gene has been stored in our laboratory and was used for codonoptimization based on the codon usage frequency of P. pastoris. Then, the optimized genewas expressed in Pichia pastoris expression system using a FLD1as promoter. Shakingflask fermentation was used to screen the colony with the best performance and further tostudy the basic enzymatic properties of the heterologous expressed R. miehei lipase. Themain research contents and results are summarized below:1. Seven codons were optimized by inducing site mutations using the overlapextension PCR based on Pichia pastoris codon usage frequency. Both original RML andthe optimized RML were inserted into P. pastoris expression vector pFLDαA to constructpFLD-RMLoriand pFLD-RMLopt, respectively.2. The pFLD-RMLoriand pFLD-RMLoptvectors were successfully integrated intoGS115and constructed the genetic engineering strains. The positive clones were selectedon Tributyrin agar plates. The obtained positive colonies were fermented in shaking flasksand lipase activities of the supernatants were measured to screen the colony with highestactivity in respect to the two genes. The highest activities were respectively120U/mland195U/mL by the colonies of GS115/pFLD-RMLori32#and GS115/pFLD-RMLopt42#,where the optimized RML had an activity increased by60%.3. Supernatant from GS115/pFLD-RMLopt#42was used to study basic enzymaticproperties of the recombinant RML using p-nitrophenyl esters (pNP) as substrates. Itsoptimal substrate was nitrophenol butyrate, optimum temperature was45°C, and optimum pH was8.0. When incubated at temperatures from40°C to60°C, it showed significantreduction of activity to15%after2h. K+, Ca2+,Mg2+, Mn2+, Ni2+and Cu2+had a slightinhibitory effect on the recombinant RML, and Fe3+, Zn2+and Fe2+had strong inhibitoryeffect on the hydrolysis activity of RML, while Sr2+could stimulate RML to some extent.