亚油酸异构酶基因在产油真菌中的异源表达及产物的生物合成
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摘要
共轭亚油酸（Conjugated linoleic acid，CLA）是具有共轭双键的十八碳二烯酸的统称，由于其多样的生理活性而受到广泛关注。为了满足CLA在医药及营养领域的应用需求，通过生物技术生产单一、安全的CLA活性异构单体成为CLA合成技术的发展趋势。本论文将来源于痤疮丙酸杆菌（Propionibacterium acnes）的亚油酸异构酶（PAI）基因分别在大肠杆菌（Escherichia coli）、高山被孢霉（Mortierella alpina）和耶氏解脂酵母（Yarrowia lipolytica）中进行异源表达，确定耶氏解脂酵母作为合适的trans-10，cis-12-CLA生物合成系统，在此基础上，通过基因工程手段构建了一株trans-10，cis-12-CLA的高产菌株，并对发酵条件进行初步优化，进一步提高CLA产量。论文的主要研究结果如下：
(1)构建了三种重组大肠杆菌重组菌株，分别表达三种重组PAI：C端融合6×His-tag的PAI、N端融合6×His-tag的PAI和不带有6×His-tag的PAI。将经镍柱纯化后的PAI免疫兔子，得到PAI多克隆抗体。通过SDS-PAGE和western blot分析三种重组菌的蛋白质表达情况，并利用气相色谱检测胞内相对酶活。结果显示，在相同的培养及诱导表达条件下，三种重组大肠杆菌均成功表达了具有亚油酸异构酶活性的重组PAI，6×His-tag的融合表达对于重组菌株的生长及PAI的表达总量没有影响，但减少了PAI可溶性表达量，并且降低酶活，而C端添加亲和标签比N端对此影响更为显著。为了获得更多的活性PAI，在产油真菌中异源表达PAI基因时，应不添加任何亲和纯化标签。
(2)对M. alpina ATCC32222的孢子产量、孢子萌发率、孢子收集方法和孢子对抗生素敏感性进行了研究，确定最佳产孢培养条件是28℃、 PDA斜面上静置培养30天；为了去除菌丝并减少孢子的损失，采用60目滤布和20μm滤膜结合的两步法过滤孢子液；选择潮霉素作为筛选标记，潮霉素的最佳使用浓度为0.8-1.5mg/mL。构建了两种带有PAI基因的重组质粒：pD4-pai和pBIG-pai，分别用于基因枪轰击和农杆菌介导两种转化法。对转化实验条件进行摸索，最终通过农杆菌介导转化法获得稳定的转化子。以转化子的基因组DNA和cDNA为模板，通过PCR扩增证实了PAI基因成功整合在M.alpina转化子基因组上并发生转录，同时证明M. alpina表达系统构建成功。但M. alpina重组菌未能成功表达亚油酸异构酶。来源于细菌的PAI基因和真核生物M. alpina在密码子使用偏好性方面的差异可能是导致PAI异源表达失败的重要原因。
(3)鉴于PAI基因在M. alpina中异源表达失败，首先根据Y. lipolytica的密码子偏好性，优化了PAI基因，并在PAI基因的起始密码子前加上了Kozak序列。天然的PAI基因（pai）和优化后的基因（opai）分别以单拷贝整合方式整合到Y. lipolytica基因组上。通过对重组蛋白表达量分析及产物trans-10，cis-12-CLA含量测定，发现在七个整合了pai的重组Y. lipolytica菌株中，只在两个转化子中检测到了PAI蛋白，而十二个带有opai的重组菌株中全部检测到了PAI。脂肪酸分析的结果与蛋白表达结果一致，带有天然PAI基因的转化子只有两个检测到产物trans-10，cis-12-CLA。经过密码子优化后，trans-10，cis-12-CLA平均产量提高了10倍，为2.1mg/L。这些结果证明PAI基因的优化是在真核生物中成功实现异源表达的前提。通过多拷贝整合方式，进一步提高了PAI表达量和trans-10，cis-12CLA含量，其平均含量为14.3mg/L，是opai的单拷贝重组菌中CLA平均产量的6.8倍。
(4)从增加底物LA和提高PAI表达量两方面对酵母菌株进行改造。以带有单拷贝opai的Y. lipolytica重组菌株为出发菌株，将来源于M. alpina的delta-12脱氢酶基因在Y. lipolytica中进行异源表达，胞内亚油酸（LA）含量从总脂的27.8%提高至62.2%，底物的增加使得产物trans-10，cis-12CLA含量从2.3mg/mL升至3.8mg/L；在opai的原启动子hp4d上游插入12个UAS1B调控元件，得到新启动子hp16d，opai在新启动子调控下，PAI表达量提高了3倍，trans-10，cis-12CLA产量也提高至7.0mg/L；将以上两个基因改造策略结合使用后，二者的效果得到了叠加，trans-10，cis-12CLA产量提高了4.8倍，至11.0mg/L；最后通过多拷贝整合方式，进一步增强了二者的作用，trans-10，cis-12CLA产量为36.8mg/L，是出发菌株的16倍。经过分子改造后的最优菌株Polh-pINA1292-spopai-d12-16中CLA产量为44.9mg/L，占总脂肪酸的9.8%。
(5)基于对重组蛋白和脂质底物的双重需求以及Y.lipolytica高效吸收利用脂类物质的特性，采用添加了2%亚油酸的培养基YNBDL6，定向增加PAI底物。在摇瓶培养的72h，trans-10，cis-12CLA产量达到最高，而且在培养基上清中检测到产物CLA，胞内含量为0.2g/L，培养基上清中为0.1g/L，总产量是YPD培养基中CLA产量的6.7倍。通过提高培养基中氮含量，抑制了Y.lipolytica产酸，提高了菌体生长速度、脂质吸收效率及PAI表达量，在高氮培养基YN20BDL6中培养72h，trans-10，cis-12CLA的产量达到2.6g/L，为低氮培养基中CLA产量的8.7倍。在高氮培养基配方的基础上，考察不同纯度的LA对Y.lipolytica重组菌产CLA的影响。结果显示，LA纯度的提高对于Y.lipolytica重组菌株的生长和产脂没有影响，而且trans-10，cis-12CLA的产量随着LA纯度的增加而提高，添加了纯度为33%、65%、96%LA的培养基中Polh-pINA1292-spopai-d12-16的CLA总产量分别为0.9g/L、2.6g/L和3.7g/L。大豆油价格低廉，来源广泛，其中LA的含量占总脂肪酸的56%，用大豆油替代培养基中的亚油酸，Y.lipolytica重组菌的生长、产脂及CLA产量与添加65%LA的结果相近，trans-10，cis-12CLA的最高产量同样出现在摇瓶发酵的72h，总产量为2.5g/L，证明Y.lipolytica重组菌能够高效转化大豆油为CLA。在5-L发酵罐中，Polh-pINA1292-spopai-d12-16利用添加大豆油的培养基，在发酵38.5h，CLA产量达最高，胞内为3.1g/L，培养基中为0.9g/L。经过培养条件的初步优化，CLA的总产量达到4g/L，为YPD培养基中CLA产量的90倍，是迄今为止报道的最高水平的20倍。通过胞外酶活检测及显微镜观察细胞完整性，结果显示胞外并未检测到PAI酶活，也并未观测到细胞碎片，说明培养基上清中的产物CLA不是由PAI分泌或细胞裂解引起，推测其原因是胞内外发生脂质交换。
Conjugated linoleic acid (CLA) is a generic term used to describe a mixture of positionaland geometric isomers of linoleic acid (LA) with conjugated double bonds. In the past threedecades, CLA has attracted much attention because of its biologically beneficial functions. Tomeet the requirements for medicinal and nutritional purposes, CLA isomers need to bebiologically safe and highly specific. Therefore biosynthesis of CLA has become theinevitable technology trends. In this dissertation, the linoleic acid isomerase gene fromPropionibacterium acnes (PAI) was successively expressed in Escherichia coli, Mortierellaalpina and Yarrowia lipolytica. Yarrowia lipolytica was confirmed to be the properbiosynthesis system for trans-10，cis-12-CLA. A recombinant Yarrowia lipolytica strain withability to produce large quantities of trans-10, cis-12-CLA was constructed by geneticmodifications. The trans-10, cis-12-CLA yield was further improved by optimization ofcultivation conditions. The main results were described as follows:
(1) Three new recombinant E.coli strains were constructed and used to over-expressthree types of recombinant PAI: PAI,.PAI fused with C-terminal6×his-tag and PAI fusedwith N-terminal6×his-tag. The purified his-tag PAI was used as antigen for the generation ofPAI-specific polyclonal antibodies in rabbits. To analysis the recombinant PAI expression inE.coli, SDS-PAGE and western blot were carried out. PAI activity in recombinant E.coli strainwas confirmed by gas chromatogram. The results showed that, under the same cultivation andinducible expression conditions, three recombinant E.coli strains successfully expressedrecombinant proteins which had linoleic acid isomerase activity. The total recombinant PAIproduction levels were similar among three recombinant E.coli strains. His-tag had anoticeable negative effect on protein solubility and PAI activity. C-terminal his-tag had astronger negative effect on these aspects than N-terminal his-tag. To obtain maximum activePAI, PAI gene should be expressed in oleaginous fungus without any affinity purification tags.
(2) The production, germination rate, collection method and antibiotics sensitivity ofspores were investigated. The optimum cultivation condition for spores production is that M.alpina was cultivated on slant PDA medium at28℃for30days. To remove mycelia andminimize spores losses, the spore suspension was successively filtrated by250μm fabric and20μm membrane. Hygromycin B was used as selection marker and the optimumconcentration for transformation was0.8-1.5mg/mL. The recombinant vectors pD4-pai andpBIG-pai were constructed for microprojectile bombardment and Agrobacteriumtumefaciens-mediated transformation (ATMT) respectively. Transformation conditions wereinvestigated and the stable transformants were obtained by ATMT. PAI gene integration intochromosomal DNA and transcription were confirmed by PCR analysis, using genomic DNAand cDNA from stable transformants. The results suggested gene manipolation system for M.alpina was established. However the recombinant PAI was not detected by SDS-PAGE andwestern blot analysis. These results revealed that PAI gene was not expressed in M. alpina.Codon usage differences between PAI gene and M. alpina may be the reason for the failure ofPAI expression.
(3) Considering the failure of PAI expression in M. alpina, PAI gene was firstlyoptimized according the condon usage bias in Y. lipolytica. Additionally, the Kozak elementwas added before the first AUG codon to prevent the leaky scanning of the ribosome.Mono-copy expression cassettes of native PAI gene (pai) and optimized gene (opai) wereintegrated into genomic DNA of Y. lipolytica. The recombinant PAI is present in all yeasttransformants carrying opai. By comparison, the level of PAI expressed from the native paiwas extremely low and could not be detected by western blot except for two transformants.The average content of trans-10，cis-12-CLA in transformants carrying opai was2.1mg/L,which corresponds to a10-fold increase compared with the transformants carrying pai. Theseresults suggested that codon optimization was essential for heterologous expression ineukaryotes. The expression level of PAI and trans-10，cis-12-CLA production were furtherpromoted with a multiple-copy expression of a codon-optimized gene. The average content oftrans-10，cis-12-CLA in multiple-copy transformants carrying opai was14.3mg/L, whichcorresponds to a6.8-fold increase compared with the mono-copy transformants carrying opai.
(4) The genetic modifications of Y. lipolytica were performed to enhance the level ofsubstrate LA and PAI expression. The mono-copy integrative strain with the codon-optimizedgene opai was used as a control strain. To increase PAI substrate (LA) content, the delta-12desaturase gene was cloned from M. alpina and expressed in Y. lipolytica. As a result, PAIcontent in total fatty acids was increased from27.8%to62.2%and trans-10，cis-12-CLAproduction was increased from2.3mg/L to3.8mg/L. To enhance opai expression, thepromoter, hp4d, was replaced by a modified promoter hp16d located upstream of opai. Theexpression level of opai under hp16d was3-fold higher than that in the control strain and thetrans-10，cis-12-CLA production was increased to7.0mg/L. Furthermore, the synergisticeffect of d12expression and enhanced promoter resulted in a4.8-fold increased yield of CLA(11.0mg/L) compared to the control. The multi-copy integration also improved CLA yield:the trans-10，cis-12-CLA yield was increased to36.8mg/L, which corresponds to a total of16-fold increase. The highest CLA yield obtained by the best performing transformantPolh-pINA1292-spoPAI-D12-16was44.9mg/L, corresponding to9.8%in total fatty acids.
(5) The non-conventional dimorphic yeast Y. lipolytica has been efficiently cultivated onvarious hydrophobic substances such as alkanes or lipids to produce many extra-orintra-cellular metabolites of industrial significance. Considering the specific properties of Y.lipolytica and the limiting factors for CLA production, YNBDL6medium supplemented withLA was used to increase PAI substrate. At72h of cultivation in flask, the trans-10，cis-12-CLA yield in Polh-pINA1292-spoPAI-D12-16was maximum, which was6.7-foldhigher than that in YPD medium, corresponding to0.3g/L. Interestingly,0.1g/L CLA thereinwas detected in supernatant medium. Increase of nitrogen concentration in medium inhibitedacid production and promoted cell growth, lipid intake and PAI expression. In high nitrogenmedium YN20BDL6, the maximal CLA yield was2.6g/L observed at72h of cultivationwhich was8.7-fold than that in low nitrogen medium. The effect of different LAconcentration on growth characters was investigated. High LA concentration had no negativeeffect on Y. lipolytica growth and lipid accumulation. Moreover, the trans-10，cis-12-CLA yield increased according with LA concentration. The CLA production were separately0.9g/L,2.6g/L and3.7g/L in medium added with33%,65%and96%LA. The overall modifiedstrain was cultivated with YN20BDS medium supplemented with soybean oil which is wildlyavailable, low-cost and rich in LA. The growth characters, lipid accumulation and CLAproduction in YN20BDS were similar with those in the medium supplemented with65%LA.The maximum CLA production in cells was2.5g/L. Polh-pINA1292-spoPAI-D12-16wascultured in5-L fermentor with YN20BDS medium. The maximum CLA production in cellswas3.1g/L and observed at the late stage of rapid growth (38.5h of cultivation). A largeportion of CLA (0.9g/L) was detected in supernatant medium simultaneously.The total CLAproduction was4g/L, corresponding to90-fold higher than that in YPD medium and20-foldhigher than the highest production of trans-10, cis-12-CLA by biosynthesis. The extracellularactivity of PAI in medium at24h of cultivation was tested and optical microscopic analysiswas performed to evaluate the cell integrity. As a result, there was no activity of PAI to bedetected in supernatant culture and no cell fragmentation was observed. These resultssuggested that the CLA in culture supernatant was not due to PAI secretion and cell lysis. Thepossible explanation was the result of fatty acid exchange between intracellular andextracellular matrixes.
(1)构建了三种重组大肠杆菌重组菌株，分别表达三种重组PAI：C端融合6×His-tag的PAI、N端融合6×His-tag的PAI和不带有6×His-tag的PAI。将经镍柱纯化后的PAI免疫兔子，得到PAI多克隆抗体。通过SDS-PAGE和western blot分析三种重组菌的蛋白质表达情况，并利用气相色谱检测胞内相对酶活。结果显示，在相同的培养及诱导表达条件下，三种重组大肠杆菌均成功表达了具有亚油酸异构酶活性的重组PAI，6×His-tag的融合表达对于重组菌株的生长及PAI的表达总量没有影响，但减少了PAI可溶性表达量，并且降低酶活，而C端添加亲和标签比N端对此影响更为显著。为了获得更多的活性PAI，在产油真菌中异源表达PAI基因时，应不添加任何亲和纯化标签。
(2)对M. alpina ATCC32222的孢子产量、孢子萌发率、孢子收集方法和孢子对抗生素敏感性进行了研究，确定最佳产孢培养条件是28℃、 PDA斜面上静置培养30天；为了去除菌丝并减少孢子的损失，采用60目滤布和20μm滤膜结合的两步法过滤孢子液；选择潮霉素作为筛选标记，潮霉素的最佳使用浓度为0.8-1.5mg/mL。构建了两种带有PAI基因的重组质粒：pD4-pai和pBIG-pai，分别用于基因枪轰击和农杆菌介导两种转化法。对转化实验条件进行摸索，最终通过农杆菌介导转化法获得稳定的转化子。以转化子的基因组DNA和cDNA为模板，通过PCR扩增证实了PAI基因成功整合在M.alpina转化子基因组上并发生转录，同时证明M. alpina表达系统构建成功。但M. alpina重组菌未能成功表达亚油酸异构酶。来源于细菌的PAI基因和真核生物M. alpina在密码子使用偏好性方面的差异可能是导致PAI异源表达失败的重要原因。
(3)鉴于PAI基因在M. alpina中异源表达失败，首先根据Y. lipolytica的密码子偏好性，优化了PAI基因，并在PAI基因的起始密码子前加上了Kozak序列。天然的PAI基因（pai）和优化后的基因（opai）分别以单拷贝整合方式整合到Y. lipolytica基因组上。通过对重组蛋白表达量分析及产物trans-10，cis-12-CLA含量测定，发现在七个整合了pai的重组Y. lipolytica菌株中，只在两个转化子中检测到了PAI蛋白，而十二个带有opai的重组菌株中全部检测到了PAI。脂肪酸分析的结果与蛋白表达结果一致，带有天然PAI基因的转化子只有两个检测到产物trans-10，cis-12-CLA。经过密码子优化后，trans-10，cis-12-CLA平均产量提高了10倍，为2.1mg/L。这些结果证明PAI基因的优化是在真核生物中成功实现异源表达的前提。通过多拷贝整合方式，进一步提高了PAI表达量和trans-10，cis-12CLA含量，其平均含量为14.3mg/L，是opai的单拷贝重组菌中CLA平均产量的6.8倍。
(4)从增加底物LA和提高PAI表达量两方面对酵母菌株进行改造。以带有单拷贝opai的Y. lipolytica重组菌株为出发菌株，将来源于M. alpina的delta-12脱氢酶基因在Y. lipolytica中进行异源表达，胞内亚油酸（LA）含量从总脂的27.8%提高至62.2%，底物的增加使得产物trans-10，cis-12CLA含量从2.3mg/mL升至3.8mg/L；在opai的原启动子hp4d上游插入12个UAS1B调控元件，得到新启动子hp16d，opai在新启动子调控下，PAI表达量提高了3倍，trans-10，cis-12CLA产量也提高至7.0mg/L；将以上两个基因改造策略结合使用后，二者的效果得到了叠加，trans-10，cis-12CLA产量提高了4.8倍，至11.0mg/L；最后通过多拷贝整合方式，进一步增强了二者的作用，trans-10，cis-12CLA产量为36.8mg/L，是出发菌株的16倍。经过分子改造后的最优菌株Polh-pINA1292-spopai-d12-16中CLA产量为44.9mg/L，占总脂肪酸的9.8%。
(5)基于对重组蛋白和脂质底物的双重需求以及Y.lipolytica高效吸收利用脂类物质的特性，采用添加了2%亚油酸的培养基YNBDL6，定向增加PAI底物。在摇瓶培养的72h，trans-10，cis-12CLA产量达到最高，而且在培养基上清中检测到产物CLA，胞内含量为0.2g/L，培养基上清中为0.1g/L，总产量是YPD培养基中CLA产量的6.7倍。通过提高培养基中氮含量，抑制了Y.lipolytica产酸，提高了菌体生长速度、脂质吸收效率及PAI表达量，在高氮培养基YN20BDL6中培养72h，trans-10，cis-12CLA的产量达到2.6g/L，为低氮培养基中CLA产量的8.7倍。在高氮培养基配方的基础上，考察不同纯度的LA对Y.lipolytica重组菌产CLA的影响。结果显示，LA纯度的提高对于Y.lipolytica重组菌株的生长和产脂没有影响，而且trans-10，cis-12CLA的产量随着LA纯度的增加而提高，添加了纯度为33%、65%、96%LA的培养基中Polh-pINA1292-spopai-d12-16的CLA总产量分别为0.9g/L、2.6g/L和3.7g/L。大豆油价格低廉，来源广泛，其中LA的含量占总脂肪酸的56%，用大豆油替代培养基中的亚油酸，Y.lipolytica重组菌的生长、产脂及CLA产量与添加65%LA的结果相近，trans-10，cis-12CLA的最高产量同样出现在摇瓶发酵的72h，总产量为2.5g/L，证明Y.lipolytica重组菌能够高效转化大豆油为CLA。在5-L发酵罐中，Polh-pINA1292-spopai-d12-16利用添加大豆油的培养基，在发酵38.5h，CLA产量达最高，胞内为3.1g/L，培养基中为0.9g/L。经过培养条件的初步优化，CLA的总产量达到4g/L，为YPD培养基中CLA产量的90倍，是迄今为止报道的最高水平的20倍。通过胞外酶活检测及显微镜观察细胞完整性，结果显示胞外并未检测到PAI酶活，也并未观测到细胞碎片，说明培养基上清中的产物CLA不是由PAI分泌或细胞裂解引起，推测其原因是胞内外发生脂质交换。
Conjugated linoleic acid (CLA) is a generic term used to describe a mixture of positionaland geometric isomers of linoleic acid (LA) with conjugated double bonds. In the past threedecades, CLA has attracted much attention because of its biologically beneficial functions. Tomeet the requirements for medicinal and nutritional purposes, CLA isomers need to bebiologically safe and highly specific. Therefore biosynthesis of CLA has become theinevitable technology trends. In this dissertation, the linoleic acid isomerase gene fromPropionibacterium acnes (PAI) was successively expressed in Escherichia coli, Mortierellaalpina and Yarrowia lipolytica. Yarrowia lipolytica was confirmed to be the properbiosynthesis system for trans-10，cis-12-CLA. A recombinant Yarrowia lipolytica strain withability to produce large quantities of trans-10, cis-12-CLA was constructed by geneticmodifications. The trans-10, cis-12-CLA yield was further improved by optimization ofcultivation conditions. The main results were described as follows:
(1) Three new recombinant E.coli strains were constructed and used to over-expressthree types of recombinant PAI: PAI,.PAI fused with C-terminal6×his-tag and PAI fusedwith N-terminal6×his-tag. The purified his-tag PAI was used as antigen for the generation ofPAI-specific polyclonal antibodies in rabbits. To analysis the recombinant PAI expression inE.coli, SDS-PAGE and western blot were carried out. PAI activity in recombinant E.coli strainwas confirmed by gas chromatogram. The results showed that, under the same cultivation andinducible expression conditions, three recombinant E.coli strains successfully expressedrecombinant proteins which had linoleic acid isomerase activity. The total recombinant PAIproduction levels were similar among three recombinant E.coli strains. His-tag had anoticeable negative effect on protein solubility and PAI activity. C-terminal his-tag had astronger negative effect on these aspects than N-terminal his-tag. To obtain maximum activePAI, PAI gene should be expressed in oleaginous fungus without any affinity purification tags.
(2) The production, germination rate, collection method and antibiotics sensitivity ofspores were investigated. The optimum cultivation condition for spores production is that M.alpina was cultivated on slant PDA medium at28℃for30days. To remove mycelia andminimize spores losses, the spore suspension was successively filtrated by250μm fabric and20μm membrane. Hygromycin B was used as selection marker and the optimumconcentration for transformation was0.8-1.5mg/mL. The recombinant vectors pD4-pai andpBIG-pai were constructed for microprojectile bombardment and Agrobacteriumtumefaciens-mediated transformation (ATMT) respectively. Transformation conditions wereinvestigated and the stable transformants were obtained by ATMT. PAI gene integration intochromosomal DNA and transcription were confirmed by PCR analysis, using genomic DNAand cDNA from stable transformants. The results suggested gene manipolation system for M.alpina was established. However the recombinant PAI was not detected by SDS-PAGE andwestern blot analysis. These results revealed that PAI gene was not expressed in M. alpina.Codon usage differences between PAI gene and M. alpina may be the reason for the failure ofPAI expression.
(3) Considering the failure of PAI expression in M. alpina, PAI gene was firstlyoptimized according the condon usage bias in Y. lipolytica. Additionally, the Kozak elementwas added before the first AUG codon to prevent the leaky scanning of the ribosome.Mono-copy expression cassettes of native PAI gene (pai) and optimized gene (opai) wereintegrated into genomic DNA of Y. lipolytica. The recombinant PAI is present in all yeasttransformants carrying opai. By comparison, the level of PAI expressed from the native paiwas extremely low and could not be detected by western blot except for two transformants.The average content of trans-10，cis-12-CLA in transformants carrying opai was2.1mg/L,which corresponds to a10-fold increase compared with the transformants carrying pai. Theseresults suggested that codon optimization was essential for heterologous expression ineukaryotes. The expression level of PAI and trans-10，cis-12-CLA production were furtherpromoted with a multiple-copy expression of a codon-optimized gene. The average content oftrans-10，cis-12-CLA in multiple-copy transformants carrying opai was14.3mg/L, whichcorresponds to a6.8-fold increase compared with the mono-copy transformants carrying opai.
(4) The genetic modifications of Y. lipolytica were performed to enhance the level ofsubstrate LA and PAI expression. The mono-copy integrative strain with the codon-optimizedgene opai was used as a control strain. To increase PAI substrate (LA) content, the delta-12desaturase gene was cloned from M. alpina and expressed in Y. lipolytica. As a result, PAIcontent in total fatty acids was increased from27.8%to62.2%and trans-10，cis-12-CLAproduction was increased from2.3mg/L to3.8mg/L. To enhance opai expression, thepromoter, hp4d, was replaced by a modified promoter hp16d located upstream of opai. Theexpression level of opai under hp16d was3-fold higher than that in the control strain and thetrans-10，cis-12-CLA production was increased to7.0mg/L. Furthermore, the synergisticeffect of d12expression and enhanced promoter resulted in a4.8-fold increased yield of CLA(11.0mg/L) compared to the control. The multi-copy integration also improved CLA yield:the trans-10，cis-12-CLA yield was increased to36.8mg/L, which corresponds to a total of16-fold increase. The highest CLA yield obtained by the best performing transformantPolh-pINA1292-spoPAI-D12-16was44.9mg/L, corresponding to9.8%in total fatty acids.
(5) The non-conventional dimorphic yeast Y. lipolytica has been efficiently cultivated onvarious hydrophobic substances such as alkanes or lipids to produce many extra-orintra-cellular metabolites of industrial significance. Considering the specific properties of Y.lipolytica and the limiting factors for CLA production, YNBDL6medium supplemented withLA was used to increase PAI substrate. At72h of cultivation in flask, the trans-10，cis-12-CLA yield in Polh-pINA1292-spoPAI-D12-16was maximum, which was6.7-foldhigher than that in YPD medium, corresponding to0.3g/L. Interestingly,0.1g/L CLA thereinwas detected in supernatant medium. Increase of nitrogen concentration in medium inhibitedacid production and promoted cell growth, lipid intake and PAI expression. In high nitrogenmedium YN20BDL6, the maximal CLA yield was2.6g/L observed at72h of cultivationwhich was8.7-fold than that in low nitrogen medium. The effect of different LAconcentration on growth characters was investigated. High LA concentration had no negativeeffect on Y. lipolytica growth and lipid accumulation. Moreover, the trans-10，cis-12-CLA yield increased according with LA concentration. The CLA production were separately0.9g/L,2.6g/L and3.7g/L in medium added with33%,65%and96%LA. The overall modifiedstrain was cultivated with YN20BDS medium supplemented with soybean oil which is wildlyavailable, low-cost and rich in LA. The growth characters, lipid accumulation and CLAproduction in YN20BDS were similar with those in the medium supplemented with65%LA.The maximum CLA production in cells was2.5g/L. Polh-pINA1292-spoPAI-D12-16wascultured in5-L fermentor with YN20BDS medium. The maximum CLA production in cellswas3.1g/L and observed at the late stage of rapid growth (38.5h of cultivation). A largeportion of CLA (0.9g/L) was detected in supernatant medium simultaneously.The total CLAproduction was4g/L, corresponding to90-fold higher than that in YPD medium and20-foldhigher than the highest production of trans-10, cis-12-CLA by biosynthesis. The extracellularactivity of PAI in medium at24h of cultivation was tested and optical microscopic analysiswas performed to evaluate the cell integrity. As a result, there was no activity of PAI to bedetected in supernatant culture and no cell fragmentation was observed. These resultssuggested that the CLA in culture supernatant was not due to PAI secretion and cell lysis. Thepossible explanation was the result of fatty acid exchange between intracellular andextracellular matrixes.
引文
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