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研究生：	郭亭君 Ting-Chun Kuo
論文名稱：	重組Candida rugosa脂肪酶在Pichia pastoris的表達量提升及其在生產生質柴油的應用 Enhanced expression of recombinant Candida rugosa lipase 2 in Pichia pastoris and its application in biodiesel production
指導教授：	李冠群 Lee, Guan-Chiun
學位類別：	博士 Doctor
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	120
中文關鍵詞：	假絲酵母脂肪酶、外泌蛋白、嗜甲醇酵母菌、生質柴油、痲瘋樹、液態脂肪酶
英文關鍵詞：	Candida rugosa lipase (CRL) isozyme, secretion, Jatropha curcas, Crude seed oil, Soluble lipase
論文種類：	學術論文
相關次數：	點閱：132 下載：3
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脂肪酶 (lipase) 在生物技術領域中，是一種極重要且具應用潛力的生物觸媒，已廣泛被應用於食品、醫藥、清潔劑、化學合成及油脂等工業。假絲酵母 (Candida rugosa) 脂肪酶具有廣效的受質特異性，是一種重要的工業用酵素，並已廣泛地被應用於生物技術領域中，該脂肪酶的組成包含了數種性質互異的同功酶 (isoenzymes)，先前的研究已將五種同功酶基因 (CRL1-5) 成功的在Pichia pastoris中表現具有活性的脂肪酶，並且證明了此五種同功酶的催化性質皆不相同。然而，在 Pichia 系統表達的脂肪酶產量尚未符合經濟效益，因此本研究選擇以CRL2 為研究目標，針對 Pichia 表達系統的轉錄、轉譯與全基因體層面設計了四個提升產量的策略，期望能提升蛋白的產量。(1) 在轉錄層面，首先我們藉由依序提高抗生素的濃度來增加LIP2 基因套數。當抗生素 Zeocin 濃度由 100 g/mL 提高至 500 g/mL，在 105 個轉型株當中，我們篩選到三個活性相較於其母株提升 2.4-5.8倍的菌株，結合低溫培養的策略，可再提升脂肪酶表達量至 32 倍，此方法可應用在提升其他四種同功酶在 Pichia 系統的產量；(2) 在轉錄層面，我們另一方面藉由隨機突變 Pichia 的 GAP 啟動子，建立一個突變庫以篩選較強的啟動子。在此策略下，我們構築一個雙同源互換的質體，利用抗生素為報導基因，成功建立啟動子篩選平台；(3) 在轉譯層面，我們希望藉由分泌蛋白胜肽 (-factor）的 mRNA 結構最適化，欲藉此提升轉譯的初始效率。透過隨機突變 -factor 的核甘酸序列但不改變氨基酸序列的情形下，我們由 1,500個突變株當中，篩選到兩個脂肪酶活性提升 1.2 倍的菌株，並且證實了突變株的5’ mRNA 結構的鍵結能量較野生株低；(4) 在全基因體層面，我們利用global transcription machinery engineering (gTME) 方法隨機突變 P. pastoris 的轉錄因子 TATA-binding protein，希望藉此改變全基因轉錄體，刺激細胞性狀改變，最後我們由 1,300個突變株當中，篩選到三個脂肪酶活性相較於野生株提升 1.5 倍的菌株。
在重組CRL2的應用方面，目前尚未有利用市售 C. rugosa 脂肪酶針對非食用性油脂進行轉酯化，成功生產生質柴油的報導。本實驗利用 P. pastoris 表達的四個重組同功酶 (CRL1-CRL4)，針對非食用油進行轉酯化生產生質柴油的研究。結果顯示 CRL2 及 CRL4 對於痲瘋樹籽油轉酯化生產脂肪酸甲酯 (FAME) 有較好的催化效果。CRL2 於最適反應條件下 (50 wt% 水，初始添加1當量甲醇，24小時再追加 0.5 當量甲醇，於 37oC 下總共反應 48 小時) 可達到最大 FAME 產量 95.3%。此結果可證實 C. rugosa 單一種同功酶可當作轉化低價的痲瘋樹籽油為生質柴油的良好生物觸媒。

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.

Table of Contents
中文摘要    I
Abstract    III
Table of Contents    VI
List of Tables    XII
List of Figures    XIII
List of Appendixes    XVI
I.    Introduction    1
   Lipase    1
1    Lipase-catalyzed reactions    1
2    Industrial applications of lipases    2
   Candida rugosa lipase (CRL)    3
1    Multiple isoenzymes of C. rugosa lipase (CRL)    3
2    The differential expression of CRL isoenzymes    4
   Pichia pastoris Expression System    5
1    Elements in the expression vectors    6
2    Pichia strains    7
3    Recombination and integration in Pichia    8
4    How to improve the expression of recombinant protein in P. pastoris    9
4.1    Transcription level−generating multicopy strains    9
4.2    Transcription level−use of strong promoters in desired gene    10
4.3    Translation level−association between mRNA folding and translation rate    11
4.4    Whole cell level−global transcription machinery engineering    13
   Individual CRL Isoenzymes Expression in P. pastoris    14
   Biodiesel Applications    14
1    Chemical-catalyzed production of biodiesel    15
2    Lipase-catalyzed production of biodiesel    16
3    Potential feedstocks    16
4    Review: enzymatic transesterification of J. curcas oil    18
5    Liquid enzyme for biodiesel production    19
   CRL Applied in Biodiesel Production    19
   Aims of the Present Study    20
II.    Materials and Methods    22
   Strategies to Enhance Expression of CRL2    22
1    Strains, plasmid, and media    22
2    Construction of mutation library    23
2.1    GAP promoter mutant library    23
2.2    a-factor pre-sequence mutant library    24
2.3    TBP mutant library    24
3    P. pastoris transformation    25
3.1    Single crossover event    25
3.2    Double crossover event    26
4    Generation of high-expression multi-copy strains    26
5    Determination of the gene copy number    27
5.1    quantitative real-time PCR (qPCR)    27
5.2    Southern hybridization analysis.    28
6    Extraction of total RNA and reverse transcription (RT)-qPCR Analysis    30
7    High-throughput screening    31
7.1    Tributyrin qualitative screening    31
7.2    96-deepwell quantitative screening    31
8    Enzyme characterization    31
   Enzymatic Biodiesel Production    32
1    Oil extraction    32
2    Determination of the saponification value (SV)    33
3    Determination of the molecular weight of non-edible oils    34
4    Strains and lipase production    34
5    Enzymatic synthesis of fatty acid methyl ester    35
6    Sampling and analysis    35
III.    Results and Discussions    37
   Strategies to Enhance Expression of CRL2    37
1    Strategy 1−applied PTVA process for a screening-accompanied selection    37
1.1    Selection and screening of high-expression multi-copy strains    37
1.2    Analysis of the LIP2 gene copy number versus CRL2 secretion    40
1.3    The effect of temperature on lipase secretion    42
1.4    Comparison of LIP2 mRNA levels of the clones harboring different LIP2 copy numbers    43
2    Strategy 2−promoter engineering    46
2.1    Random mutagenesis of the GAP promoter    46
2.2    Screening for the high-expression strains with mutant GAP promoter    46
2.3    Southern analysis for determining the gene copy number    47
2.4    Construction of a transplacement vector to generate a single-copy integration through double crossover events in Pichia genome    48
2.5    Selection under high G418 concentrations    49
3    Strategy 3−mRNA structure-optimization of secretion signal peptide    50
3.1    Construction of a-factor pre-sequence mutant library    50
3.2    Screening for the high-expression strains with mutant a-factor pre-sequence library    51
3.3    Analysis of the mutant mRNA structure    51
4    Strategy 4−whole-cell engineering    53
4.1    Screening for the high-expression strains with mutant TBP transcription factor    54
   Biodiesel Production    56
1    Screening of CRL isoenzymes for the conversion of non-edible oils    56
2    Optimization of reaction conditions    58
2.1    Effects of water content and enzyme dosage    58
2.2    Effect of temperature    59
2.3    Effect of substrate molar ratio    60
3    Methanol feed profiles    61
4    Reusability of liquid lipase    63
IV.    Conclusions    64
V.    References    67

                                

Augustus, G.D.P.S., Jayabalan, M., Seiler, G.J. 2002. Evaluation and bioinduction of energy components of Jatropha curcas. Biomass and Bioenergy, 23, 161-164.
Baerends, R.J., Qiu, J.L., Rasmussen, S., Nielsen, H.B., Brandt, A. 2009. Impaired uptake and/or utilization of leucine by Saccharomyces cerevisiae is suppressed by the SPT15-300 allele of the TATA-binding protein gene. Appl Environ Microbiol, 75, 6055-61.
Barton, M.J., Hamman, J.P., Fichter, K.C., Calton, G.J. 1990. Enzymatic resolution of (R,S)-2-(4-hydroxyphenoxy) propionic acid. Enzyme Microb Technol, 12, 577–583.
Brierley, R.A. 1998. Secretion of recombinant human insulin-like growth factor I (IGF-I). Methods Mol Biol, 103, 149-77.
Brocca, S., Grandori, R., Breviario, D., Lotti, M. 1995. Localization of lipase genes on Candida rugosa chromosomes. Curr Genet, 28, 454-7.
Brocca, S., Schmidt-Dannert, C., Lotti, M., Alberghina, L., Schmid, R.D. 1998. Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase. Protein Sci, 7, 1415-22.
Cadwell, R.C., Joyce, G.F. 1994. Mutagenic PCR. PCR Methods Appl, 3, S136-40.
Cesarini, S., Diaz, P., Nielsen, P.M. 2013. Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock. Process Biochemistry, 48, 484-487.
Chang, R.C., Chou, S.J., Shaw, J.F. 1994. Multiple forms and functions of Candida rugosa lipase. Biotechnol.Appl.Biochem., 19, 93-97.
Chang, S.W., Huang, M., Hsieh, Y.H., Luo, Y.T., Wu, T.T., Tsai, C.W., Chen, C.S., Shaw, J.F. 2014. Simultaneous production of fatty acid methyl esters and diglycerides by four recombinant Candida rugosa lipase's isozymes. Food Chem, 155, 140-5.
Chang, S.W., Lee, G.C., Shaw, J.F. 2006a. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem, 54, 815-22.
Chang, S.W., Lee, G.C., Shaw, J.F. 2006b. Efficient production of active recombinant Candida rugosa LIP3 lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J Agric Food Chem, 54, 5831-8.
Chang, S.W., Li, C.F., Lee, G.C., Yeh, T., Shaw, J.F. 2011. Engineering the expression and biochemical characteristics of recombinant Candida rugosa LIP2 lipase by removing the additional N-terminal peptide and regional codon optimization. J Agric Food Chem, 59, 6710-9.
Chen, J.C., Tsai, S.W. 2000. Enantioselective synthesis of (S)-ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on Accurel MP1000. Biotechnol Prog, 16, 986-92.
Clare, J.J., Rayment, F.B., Ballantine, S.P., Sreekrishna, K., Romanos, M.A. 1991a. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (N Y), 9, 455-60.
Clare, J.J., Romanes, M.A., Rayment, F.B., Rowedder, J.E., Smith, M.A., Payne, M.M., Sreekrishna, K., Henwood, C.A. 1991b. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene, 105, 205-212.
Couturier, L., Taupin, D., Yvergnaux, F. 2009. Lipase-catalyzed chemoselective aminolysis of various aminoalcohols with fatty acids. Journal of Molecular Catalysis B: Enzymatic, 56, 29-33.
Cregg, J.M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., Chappell, T. 2009. Expression in the yeast Pichia pastoris. Methods Enzymol, 463, 169-89.
Dalmau, E., Montesinos, J.L., Lotti, M., Casas, C. 2000. Effect of different carbon sources on lipase production by Candida rugosa. Enzyme Microb Technol, 26, 657-663.
Damasceno, L.M., Anderson, K.A., Ritter, G., Cregg, J.M., Old, L.J., Batt, C.A. 2007. Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris. Appl Microbiol Biotechnol, 74, 381-9.
Damasceno, L.M., Huang, C.J., Batt, C.A. 2012. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, 93, 31-9.
de Oliveira, J.S., Leite, P.M., de Souza, L.B., Mello, V.M., Silva, E.C., Rubim, J.C., Meneghetti, S.M.P., Suarez, P.A.Z. 2009. Characteristics and composition of Jatropha gossypiifoliaand Jatropha curcas L. oils and application for biodiesel production. Biomass and Bioenergy, 33, 449-453.
De Schutter, K., Lin, Y.C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouze, P., Van de Peer, Y., Callewaert, N. 2009. Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol, 27, 561-6.
Diczfalusy, M.A., Hellman, U., Alexson, S.E. 1997. Isolation of carboxylester lipase (CEL) isoenzymes from Candida rugosa and identification of the corresponding genes. Arch Biochem Biophys, 348, 1-8.
Divakara, B.N., Upadhyaya, H.D., Wani, S.P., Gowda, C.L.L. 2010. Biology and genetic improvement of Jatropha curcas L.: A review. Applied Energy, 87, 732-742.
Dominguez de Maria, P., Sanchez-Montero, J.M., Sinisterra, J.V., Alcantara, A.R. 2006. Understanding Candida rugosa lipases: an overview. Biotechnol Adv, 24, 180-96.
Ferreira-Dias, S., Sandoval, G., Plou, F., Valero, F. 2013. The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries. Electron. J. Biotechnol, 16, doi:10.2225.
Ferrer, P., Alarcón, M., Ramón, R., Dolors Benaiges, M., Valero, F. 2009. Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter. Biochem Eng J, 46, 271-277.
Gasser, B., Maurer, M., Gach, J., Kunert, R., Mattanovich, D. 2006. Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng, 94, 353-61.
Gasser, B., Maurer, M., Rautio, J., Sauer, M., Bhattacharyya, A., Saloheimo, M., Penttila, M., Mattanovich, D. 2007. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics, 8, 179.
Gasser, B., Saloheimo, M., Rinas, U., Dragosits, M., Rodriguez-Carmona, E., Baumann, K., Giuliani, M., Parrilli, E., Branduardi, P., Lang, C., Porro, D., Ferrer, P., Tutino, M.L., Mattanovich, D., Villaverde, A. 2008. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact, 7, 11.
Gingold, H., Pilpel, Y. 2011. Determinants of translation efficiency and accuracy. Mol Syst Biol, 7, 481.
Gu, W., Zhou, T., Wilke, C.O. 2010. A Universal Trend of Reduced mRNA Stability near the Translation-Initiation Site in Prokaryotes and Eukaryotes. PLoS Comput Biol, 6, e1000664.
Hahn, S. 2004. Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol, 11, 394-403.
Hama, S., Kondo, A. 2013. Enzymatic biodiesel production: an overview of potential feedstocks and process development. Bioresour Technol, 135, 386-95.
Hampsey, M. 1998. Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol Mol Biol Rev, 62, 465-503.
Hartner, F.S., Ruth, C., Langenegger, D., Johnson, S.N., Hyka, P., Lin-Cereghino, G.P., Lin-Cereghino, J., Kovar, K., Cregg, J.M., Glieder, A. 2008. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Research, 36, e76-e76.
Hohenblum, H., Gasser, B., Maurer, M., Borth, N., Mattanovich, D. 2004. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng, 85, 367-75.
Huang, J., Xia, J., Jiang, W., Li, Y., Li, J. 2015. Biodiesel production from microalgae oil catalyzed by a recombinant lipase. Bioresour Technol, 180, 47-53.
Inan, M., Fanders, S.A., Zhang, W., Hotez, P.J., Zhan, B., Meagher, M.M. 2007. Saturation of the secretory pathway by overexpression of a hookworm (Necator americanus) Protein (Na-ASP1). Methods Mol Biol, 389, 65-76.
Jaeger, K.E., Reetz, M.T. 1998. Microbial lipases form versatile tools for biotechnology. Trends Biotechnol, 16, 396-403.
Jala, R.C., Hu, P., Yang, T., Jiang, Y., Zheng, Y., Xu, X. 2012. Lipases as biocatalysts for the synthesis of structured lipids. Methods Mol Biol, 861, 403-33.

Jegannathan, K.R., Abang, S., Poncelet, D., Chan, E.S., Ravindra, P. 2008. Production of biodiesel using immobilized lipase--a critical review. Crit Rev Biotechnol, 28, 253-64.
Joan Lin Cereghino, Cregg, J.M. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev, 24, 45-66.
Kawakami, K., Oda, Y., Takahashi, R. 2011. Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil. Biotechnol Biofuels, 4, 42.
Katiyar, M., Ali, A. 2012. Immobilization of Candida rugosa lipase on MCM-41 for the transesterification of cotton seed oil. J Oleo Sci, 61, 469-75.
Kudla, G., Murray, A.W., Tollervey, D., Plotkin, J.B. 2009. Coding-sequence determinants of gene expression in Escherichia coli. Science, 324, 255-8.
Kumari, A., Mahapatra, P., Garlapati, V.K., Banerjee, R. 2009. Enzymatic transesterification of Jatropha oil. Biotechnol Biofuels, 2, 1.
Kurtzman, C.P. 2009. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. J Ind Microbiol Biotechnol, 36, 1435-8.
Kuo, C.H., Peng, L.T., Kan, S.C., Liu, Y.C., Shieh, C.J. 2013. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour Technol, 145, 229-32.

López, C., Guerra, N.P., Rúa, M.L. 2000. Purification and characterisation of two isoforms from Candida rugosa lipase B. Biotechnol Lett, 22, 1291-1294.
Lanza, A.M., Alper, H.S. 2012. Using transcription machinery engineering to elicit complex cellular phenotypes. Methods Mol Biol, 813, 229-48.
Lara, P.V., Park, E.Y. 2004. Potential application of waste activated bleaching earth on the production of fatty acid alkyl esters using Candida cylindracea lipase in organic solvent system. Enzyme and Microbial Technology, 34, 270-277.
Lee, G.C., Lee, L.C., Sava, V., Shaw, J.F. 2002. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris. Biochem J, 366, 603-11.
Lee, L.C., Yen, C.C., Malmis, C.C., Chen, L.F., Chen, J.C., Lee, G.C., Shaw, J.F. 2011. Characterization of codon-optimized recombinant candida rugosa lipase 5 (LIP5). J Agric Food Chem, 59, 10693-8.
Li, P., Anumanthan, A., Gao, X.G., Ilangovan, K., Suzara, V.V., Duzgunes, N., Renugopalakrishnan, V. 2007. Expression of recombinant proteins in Pichia pastoris. Appl Biochem Biotechnol, 142, 105-24.
Li, X., Qian, P., Wu, S.G., Yu, H.Y. 2014. Characterization of an organic solvent-tolerant lipase from Idiomarina sp. W33 and its application for biodiesel production using Jatropha oil. Extremophiles, 18, 171-8.
Li, Z., Xiong, F., Lin, Q., d'Anjou, M., Daugulis, A.J., Yang, D.S., Hew, C.L. 2001. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif, 21, 438-45.
Linko, Y.Y., Lamsa, M., Wu, X., Uosukainen, E., Seppala, J., Linko, P. 1998. Biodegradable products by lipase biocatalysis. J Biotechnol, 66, 41-50.
Linko, Y.Y., Wu, X.Y. 1996. Biocatalytic Production of Useful Esters by Two Forms of Lipase from Candida rugosa. J. Chem. Tech. Biotechnol., 65, 163-170.
Liu, W., Jiang, R. 2015. Combinatorial and high-throughput screening approaches for strain engineering. Appl Microbiol Biotechnol, 99, 2093-104.
Liu, Y.Y., Xu, J.H., Wu, H.Y., Shen, D. 2004. Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen. J Biotechnol, 110, 209-17.
Longhi, S., Fusetti, F., Grandori, R., Lotti, M., Vanoni, M., Alberghina, L. 1992. Cloning and nucleotide sequences of two lipase genes from Candida cylindracea. Biochim Biophys Acta, 1131, 227-32.
Lopez, N., Pernas, M.A., Pastrana, L.M., Sanchez, A., Valero, F., Rua, M.L. 2004. Reactivity of pure Candida rugosa lipase isoenzymes (Lip1, Lip2, and Lip3) in aqueous and organic media. influence of the isoenzymatic profile on the lipase performance in organic media. Biotechnol Prog, 20, 65-73.
Lotti, M., Grandori, R., Fusetti, F., Longhi, S., Brocca, S., Tramontano, A., Alberghina, L. 1993. Cloning and analysis of Candida cylindracea lipase sequences. Gene, 124, 45-55.
Lotti, M., Tramontano, A., Longhi, S., Fusetti, F., Brocca, S., Pizzi, E., Alberghina, L. 1994. Variability within the Candida rugosa lipases family. Protein Eng, 7, 531-5.
M.G. Devanesan, T. Viruthagiri, Sugumar, N. 2007. Transesterification of Jatropha oil using immobilized Pseudomonas fluorescens. Afr. J. Biotechnol, 6, 2497-2501.
Macauley-Patrick, S., Mariana L. Fazenda, McNeil, B., Harvey, L.M. 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249-270.
Makkar, H.P.S., Becker, K., Sporer, F., Wink, M. 1997. Studies on Nutritive Potential and Toxic Constituents of Different Provenances of Jatropha curcas. J Agric Food Chem, 45, 3152-3157.
Martinelle, M., Holmquist, M., Hult, K. 1995. On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta, 1258, 272-276.
Marx, H., Mecklenbrauker, A., Gasser, B., Sauer, M., Mattanovich, D. 2009. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res, 9, 1260-70.
Miranda, M., Urioste, D., Andrade Souza, L.T., Mendes, A.A., de Castro, H.F. 2011. Assessment of the Morphological, Biochemical, and Kinetic Properties for Candida rugosa Lipase Immobilized on Hydrous Niobium Oxide to Be Used in the Biodiesel Synthesis. Enzyme Res, 2011, 216435.
Modi, M.K., Reddy, J.R., Rao, B.V., Prasad, R.B. 2006. Lipase-mediated transformation of vegetable oils into biodiesel using propan-2-ol as acyl acceptor. Biotechnol Lett, 28, 637-40.
Moreno-Pirajàn, J.C., Giraldo, L. 2011. Study of immobilized candida rugosa lipase for biodiesel fuel production from palm oil by flow microcalorimetry. Arabian Journal of Chemistry, 4, 55-62.
Moser, B.R. 2008. Influence of Blending Canola, Palm, Soybean, and Sunflower Oil Methyl Esters on Fuel Properties of Biodiesel. Energy Fuels, 22, 4301-4306
Nackley, A.G., Shabalina, S.A., Tchivileva, I.E., Satterfield, K., Korchynskyi, O., Makarov, S.S., Maixner, W., Diatchenko, L. 2006. Human catechol-O-methyltransferase haplotypes modulate protein expression by altering mRNA secondary structure. Science, 314, 1930-3.
Nevoigt, E., Fischer, C., Mucha, O., Matthaus, F., Stahl, U., Stephanopoulos, G. 2007. Engineering promoter regulation. Biotechnol Bioeng, 96, 550-8.
Nogueira, L.A.H. 2011. Does biodiesel make sense? Energy, 36, 3659-3666.
Nordblad, M., Silva, V.T., Nielsen, P.M., Woodley, J.M. 2014. Identification of critical parameters in liquid enzyme-catalyzed biodiesel production. Biotechnol Bioeng, 111, 2446-53.
Noureddini, H., Gao, X., Philkana, R.S. 2005. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol, 96, 769-77.
Park, E.Y., Sato, M., Kojima, S. 2008. Lipase-catalyzed biodiesel production from waste activated bleaching earth as raw material in a pilot plant. Bioresour Technol, 99, 3130-5.
Pinto, A.C., Guarieiro, L.L.N., Rezende, M.J.C., Ribeiro, N.M., Torres, E.A., Lopes, W.A., Pereira, P.A.d.P., Andrade, J.B.d. 2005. Biodiesel: an overview. J. Braz. Chem. Soc., 16, 1313-1330.
Qin, X., Qian, J., Yao, G., Zhuang, Y., Zhang, S., Chu, J. 2011. GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol, 77, 3600-8.
Rúa, M.L., Diaz-Maurino, T., Fernandez, V.M., Otero, C., Ballesteros, A. 1993. Purification and characterizations of two distinct lipases from Candida cylindracea. Biochim Biophys Acta 1156, 181- 189.
Ranganathan, S.V., Narasimhan, S.L., Muthukumar, K. 2008. An overview of enzymatic production of biodiesel. Bioresour Technol, 99, 3975-81.
Rathore, V., Madras, G. 2007. Synthesis of biodiesel from edible and non-edible oils in supercritical alcohols and enzymatic synthesis in supercritical carbon dioxide. Fuel, 86, 2650-2659.
Reis, M.d., Savva, R., Wernisch, L. 2004. Solving the riddle of codon usage preferences: a test for translational selection. Nucleic Acids Res, 32, 5036-5044.
Resina, D., Maurer, M., Cos, O., Arnau, C., Carnicer, M., Marx, H., Gasser, B., Valero, F., Mattanovich, D., Ferrer, P. 2009. Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. N Biotechnol, 25, 396-403.
Sagiroglu, A. 2008. Conversion of sunflower oil to biodiesel by alcoholysis using immobilized lipase. Artif Cells Blood Substit Immobil Biotechnol, 36, 138-49.
Sayyar, S., Abidin, Z.Z., Yunus, R., Muhammad, A. 2009. Extraction of Oil from Jatropha Seeds-Optimization and Kinetics. Am. J. Applied Sci, 6, 1390-1395.
Shah, S., Gupta, M.N. 2007. Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system. Process Biochem, 42, 409-414.
Shah, S., Sharma, S., Gupta, M.N. 2004. Biodiesel Preparation by Lipase-Catalyzed Tranesterification of Jatropha oil. Energy & Fuels, 18, 154-159.
Sharma, R., Chisti, Y., Banerjee, U.C. 2001. Production, purification, characterization, and applications of lipases. Biotechnol Adv, 19, 627-62.
Sharp, P.M., Li, W.H. 1987. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res, 15, 1281-95.
Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y. 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B-Enzym, 17, 133-142.
Smith, S.W. 2009. Chiral toxicology: it's the same thing...only different. Toxicol Sci, 110, 4-30.
Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol, 98, 503-517.
Steensels, J., Snoek, T., Meersman, E., Picca Nicolino, M., Voordeckers, K., Verstrepen, K.J. 2014. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiol Rev, 38, 947-95.
Su, E.-Z., Xu, W.-Q., Gao, K.-L., Zheng, Y., Wei, D.-Z. 2007. Lipase-catalyzed in situ reactive extraction of oilseeds with short-chained alkyl acetates for fatty acid esters production. J Mol Catal B-Enzym, 48, 28-32.
Su, E., You, P., Wei, D. 2009. In situ lipase-catalyzed reactive extraction of oilseeds with short-chained dialkyl carbonates for biodiesel production. Bioresour Technol, 100, 5813-7.
Sunga, A.J., Tolstorukov, I., Cregg, J.M. 2008. Posttransformational vector amplification in the yeast Pichia pastoris. FEMS Yeast Res, 8, 870-6.
Tamalampudi, S., Talukder, M.R., Hama, S., Numata, T., Kondo, A., Fukuda, H. 2008. Enzymatic production of biodiesel from Jatropha oil: A comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. Biochem Eng J, 39, 185-189.
Tang, S.J., Shaw, J.F., Sun, K.H., Sun, G.H., Chang, T.Y., Lin, C.K., Lo, Y.C., Lee, G.C. 2001. Recombinant expression and characterization of the Candida rugosa lip4 lipase in Pichia pastoris: comparison of glycosylation, activity, and stability. Arch Biochem Biophys, 387, 93-8.
Tenkanen, M., Kontkanen, H., Isoniemi, R., Spetz, P., Holmbom, B. 2002. Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa. Appl Microbiol Biotechnol, 60:, 120-127.
Toftgaard Pedersen, A., Nordblad, M., Nielsen, P.M., Woodley, J.M. 2014. Batch production of FAEE-biodiesel using a liquid lipase formulation. J Mol Catal B-Enzym, 105, 89-94.
Tuller, T., Waldman, Y.Y., Kupiec, M., Ruppin, E. 2010. Translation efficiency is determined by both codon bias and folding energy. Proc Natl Acad Sci U S A, 107, 3645-50.
Vassileva, A., Chugh, D.A., Swaminathan, S., Khanna, N. 2001. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. J Biotechnol, 88, 21-35.
Vogl, T., Glieder, A. 2013. Regulation of Pichia pastoris promoters and its consequences for protein production. N Biotechnol, 30, 385-404.
Wang, L., Wessler, S.R. 2001. Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc(1,2). Plant Physiol, 125, 1380-7.
Wang, Y., Liu, J., Gerken, H., Zhang, C., Hu, Q., Li, Y. 2014. Highly-efficient enzymatic conversion of crude algal oils into biodiesel. Bioresour Technol, 172, 143-9.
Waterham, H.R., Digan, M.E., Koutz, P.J., Lair, S.V., Cregg, J.M. 1997. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 186, 37-44.
Xie, W., Wang, J. 2014. Enzymatic Production of Biodiesel from Soybean Oil by Using Immobilized Lipase on Fe3O4/Poly(styrene-methacrylic acid) Magnetic Microsphere as a Biocatalyst. Energy & Fuels, 28, 2624-2631.
Xu, L., Jiang, X., Yang, J., Liu, Y., Yan, Y. 2010. Cloning of a novel lipase gene, lipJ08, from Candida rugosa and expression in Pichia pastoris by codon optimization. Biotechnol Lett, 32, 269-76.
You, Q., Yin, X., Zhao, Y., Zhang, Y. 2013. Biodiesel production from Jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite. Bioresour Technol, 148, 202-7.
Yu, M., Wen, S., Tan, T. 2010. Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris. Eng. Life Sci., 10, 458-464.
Zur, H., Tuller, T. 2012. Strong association between mRNA folding strength and protein abundance in S. cerevisiae. EMBO Rep, 13, 272-7.

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