Enhanced expression of recombinant Candida rugosa lipase 2 in Pichia pastoris and its application in biodiesel production

• Main Authors: Ting-Chun Kuo, 郭亭君
• Other Authors: Guan-Chiun Lee
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/43334489394737520781

博士 === 國立臺灣師範大學 === 生命科學系 === 103 === Lipase is an important group of versatile enzymes in the field of biotechnology. It has been widely used in detergents, foods, pharmaceuticals, cosmetics, leathers, and paper industries. The secretory lipase of non-sporogenic Candida rugosa has been widely applied in industry. It contains a mixture of lipase isoenzymes (CRLs), which have different catalytic properties. Five CRL genes (lip1 to lip5) have been identified and are expressed in Pichia pastoris. These recombinant isoenzymes exhibit distinct substrate preferences and catalytic activities. However, the expression levels did not qualify for economical industrial applications. In the present study, the expression of recombinant CRL2 in P. pastoris was improved by employing four approaches through modulating the transcriptional, translational, and even the whole-cell levels. At the transcriptional level, two strategies including the introduction of multiple-copies of the LIP2 gene and the promoter engineering were used. (1) The LIP2 gene copy number of the Pichia transformants was increased by sequential selections at gradually increasing Zeocin concentrations. After the first selection at 500 μg/mL of Zeocin, three clones (500-clones) with 2.4-fold to 5.8-fold improvement in CRL2 secretion were identified from 105 survival clones through lipase activity screening. Combining the low culture temperature effect, a maximal 32-fold increase in CRL2 secretion was obtained. (2) For promoter engineering, we will select stronger mutant promoters from a random-mutagenesis promoter library. We have constructed a double-homologous recombination plasmid and used antibiotics resistant gene as a reporter. Through this system, we have successfully established a functional Pichia GAP promoter assay platform. (3) At the translational level, we firstly attempted to optimize the structure of the mRNA encoding secretion signal peptide (-factor) to enhance the initiation of the translation. We randomly mutate the nucleotides sequence of -factor but do not alter the amino acids sequence. Two slightly-improved strains were obtained with 1.2 folds lipase production higher than the wild-type strain screening from 1,500 mutants. We considered that the hydrogen bonding energy of 5’ mRNA structure near start codon of mutants was lower than that of wild-type strain. (4) For the whole-cell engineering, the global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes. We have improved the lipase production through random mutagenesis of the P. pastoris transcription factor-TATA binding protein gene. Three engineered strains were obtained with 1.5 folds lipase production higher than the wild-type strain screening from 1,300 mutants. There have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isoenzymes. In the present study, four liquid recombinant CRL isoenzymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24 h for a total reaction time of 48 h at 37°C). We concluded that specific recombinant CRL isoenzymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.