Heterologous Expression of Leghemoglobin in Saccharomyces cerevisiae

Abstract

Leghemoglobin is a kind of plant-derived hemoglobin, which can be used as an important flavor catalyst and colorant in the processing of plant protein-based meat because of the property of increasing the fidelity of the product greatly. In order to realize the heterologous expression of leghemoglobin in food-grade microorganisms, the plasmid PESC-TRP carrying leghemoglobin LBC2 gene was transformed into S. cerevisiae CEN.PK2-1C. Kozak sequence was added to promote protein translation, and different promoter sequences were selected to improve the expression of leghemoglobin. The recombinant strain was fermented, and the expression level of LegH protein was analyzed by western blot. The results showed that the inducible promoter GAL1,10 had a significant advantage over the three constitutive promoters TEF1, ADH1 and GAP in LegH expression ability, which was 3.93 times higher than the lowest yield ADH1 promoter. Among the constitutive promoters, TEF1 promoter had the best ability, which was 2.91times that of the ADH1 promoter and 1.2 times that of the GAP promoter. Finally, the protein was concentrated and purified by Ni-column affinity chromatography, and the fermentation concentration of LegH was 2.79 mg/L. This study successfully achieved the heterologous expression of leghemoglobin in Saccharomyces cerevisiae, and after subsequent optimization, it would become another way of leghemoglobin heterologous expression.

Keywords

• saccharomyces cerevisiae
• leghemoglobin
• promoter optimization
• heterologous expression
• western blot