Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy

Abstract

Simple and cost-effective lipase expression host microorganisms are highly desirable. A combinatorial library strategy is used to improve the secretory expression of lipase from Bacillus thermocatenulatus (BTL2) in the culture supernatant of Saccharomyces cerevisiae. A plasmid library including expression cassettes composed of sequences encoding one of each 15 promoters, 15 secretion signals, and 15 terminators derived from yeast species, S. cerevisiae, Pichia pastoris, and Hansenula polymorpha, is constructed. The S. cerevisiae transformant YPH499/D4, comprising H. polymorpha GAP promoter, S. cerevisiae SAG1 secretion signal, and P. pastoris AOX1 terminator, is selected by high-throughput screening. This transformant expresses BTL2 extra-cellularly with a 130-fold higher than the control strain, comprising S. cerevisiae PGK1 promoter, S. cerevisiae α-factor secretion signal, and S. cerevisiae PGK1 terminator, after cultivation for 72 h. This combinatorial library strategy holds promising potential for application in the optimization of the secretory expression of proteins in yeast.

An improved secretion signal enhances the secretion of model proteins from Pichia pastoris

“…Second, even though BTL2 is naturally produced as an extracellular enzyme [ 30 ], it was active when expressed intracellularly in E. coli [ 32 ], indicating that the protein can fold in the cytosol. Third, the secretion of BTL2 from S. cerevisiae was recently found to be influenced by the signal sequence [ 34 ]. Fourth, although BTL2 is a monomeric protein, it tends to aggregate at high concentrations [ 29 , 51 ].…”

“…P. pastori s has been engineered to secrete BTL2 [ 33 ]. A recent study of BTL2 secretion from S. cerevisiae showed that the choice of secretion signal was particularly important [ 34 ], hinting that BTL2 might be prone to folding prior to translocation. In support of this idea, use of the improved secretion signal in P. pastoris strongly enhances BTL2 secretion.…”

Heat‐induced overexpression of the thermophilic lipase from Bacillus thermocatenulatus in Escherichia coli by fermentation and its application in preparation biodiesel using rapeseed oil

“…3,11 Lipases from different species can catalyze the synthesis of biodiesel, but their catalytic properties are quite diverse. 12 The Bacillus thermocatenulatus lipase 2 (BTL2) is a typical thermophilic enzyme with high hydrolytic activity and well tolerant to organic solvents like methanol and ethanol 13 and can overexpress under the control of the temperature promoter λP L according to the previous study. 14 If the expression level of BTL2 is increased with respect to fer-mentation and the BTL2 solution is directly used to achieve the catalytic preparation of biodiesel, the purification and immobilization of lipase can be omitted, thereby simplifying the enzymatic process, saving costs, and improving economics.…”