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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：166 下載：4
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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

                                

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