Huang Jinjin, Xia Ji, Yang Zhen, Guan Feifei, Cui Di, Guan Guohua, Jiang Wei, Li Ying
State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.
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.
Biotechnol Biofuels. 2014 Aug 4;7:111. doi: 10.1186/1754-6834-7-111. eCollection 2014.
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.
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%.
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.
我们之前从米黑根毛霉中克隆了一个1,3-特异性脂肪酶基因，并在甲基营养型酵母毕赤酵母GS115菌株中进行了表达。所产生的酶（称为RML）能够催化大豆油的甲醇解反应，并表现出很强的位置特异性。然而，该酶的活性和产量对于工业应用来说并不足够。我们在本研究中的目标是改善RML的酶学性质，以便将其应用于微藻油转化为生物燃料。
通过添加目标基因的前肽、优化信号肽以及改变目标基因的拷贝数构建了几种新的表达质粒。每个质粒分别转化到毕赤酵母X-33菌株中。通过摇瓶培养筛选发现，具有两个目标基因拷贝的重组菌株的酶活性最高（提高了21.4倍）；该酶被称为脂肪酶GH2。带有前肽的表达蛋白（pRML）是一种稳定的糖基化蛋白，这是由于前肽中存在糖基化位点。实时定量逆转录聚合酶链反应分析揭示了酶活性增加的两个主要原因：（1）改良的重组表达系统使目标基因（rml）的转录水平提高，（2）该酶适合在宿主细胞中表达而不会引起内质网应激。改良后的酶具有更高的热稳定性以及对甲醇或乙醇的耐受性，并且可以直接作为游离脂肪酶（发酵上清液）应用于催化酯化和转酯反应。在30°C下反应24小时后，微藻油转化为生物燃料的转化率高于90%。
我们的实验结果表明，表达质粒中的信号肽优化、基因前肽的添加以及合适的基因剂量显著提高了RML的表达水平并增强了酶学性质。目标酶是发酵上清液的主要成分，在4°C下可稳定保存六个月以上。改良后的游离脂肪酶具有潜在的工业规模应用价值，可用于将微藻油转化为生物柴油。
