Abstract
BackgroundWe previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.ResultsSeveral new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%.ConclusionsOur experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-6834-7-111) contains supplementary material, which is available to authorized users.
Highlights
We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115
Addition of the propeptide of the target gene We explored several strategies to modify R. miehei lipase (RML) and its heterologous expression system in order to improve enzyme activity and properties
The extracellular enzyme activity of zα-protein. zα-mRMLX33 (mRML) and mα-2pRML-X33 (pRML)-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL; Figure 1B). These findings indicate that in the absence of the propeptide, expression levels of the heterologous protein are consistent regardless of whether strain GS115 or X-33 is used as a host
Summary
We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. Because of its strong specificity, RML has been widely used in ester hydrolysis, ester synthesis, and transesterification reaction such as production of structured lipids [7], synthesis of butyl butyrate (pineapple flavor) [8], and synthesis of monoglyceryl esters from chiral and prochiral acid methyl esters [9]. Despite this wide applicability, cost remains a major obstacle to large-scale industrial use of RML [10]. Enhanced activity and reduced production cost of the enzyme are important goals
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