title: Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.
abstract: BACKGROUND
  We 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.
  
  
  RESULTS
  Several 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%.
  
  
  CONCLUSIONS
  Our 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.
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Jinjin
authors: J
authors: Huang
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Ji
authors: J
authors: Xia
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Zhen
authors: Z
authors: Yang
authors: Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, 5#, Panjiayuannanli Street, Beijing, 100021 China.
authors: Feifei
authors: F
authors: Guan
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Di
authors: D
authors: Cui
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Guohua
authors: G
authors: Guan
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Wei
authors: W
authors: Jiang
authors: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
authors: Ying
authors: Y
authors: Li
keywords: Biodiesel; Heterologous expression; Lipase; Microalgae oil; Pichia pastoris; Rhizomucor miehei