大肠杆菌中聚羟基脂肪酸酯合成基因的优化和染色体表达
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摘要
聚羟基脂肪酸酯（polyhydroxyalkanoates，简称PHA）是一类由微生物合成的高分子聚酯，具有广泛的应用范围和良好的发展前景。利用大肠杆菌合成PHA是该领域的重要研究方向，然而目前仍需依托质粒系统，且短链中长链共聚PHA（SCL-MCL PHA）产量较低。本论文从PHA合成相关基因在大肠杆菌中异源表达的优化以及染色体表达等方面展开研究，旨在通过优化过程提高PHA，特别是SCL-MCL PHA的产量，并以染色体为载体实现稳定的PHA合成，为进一步通过合成生物学手段改造生产菌、降低生产成本打下基础。论文主要研究成果包括：
通过密码子优化和引入茎环结构等手段，针对来源于施氏假单胞菌Pseudomonas stutzeri1317的广泛底物特异性聚合酶基因phaC2Ps实现异源表达的优化；优化后基因的转录产物稳定性和异源表达水平均得到显著提高，PHB和SCL-MCL PHA的含量分别由细胞干重的11.7wt%和1.79wt%升高至29.7wt%和3.93wt%。同时，所得SCL-MCL PHA的组分更加广泛，单体碳链长度覆盖4-12，细胞干重也随之上升。
证明不同染色体整合位点和拷贝数对于PHA合成基因异源表达水平的影响。选择13个染色体整合位点，发现其对于基因的异源表达有一定的影响，在yheO(ECK3333)、gltA (ECK0709)、asnB (ECK0662)等转录活跃区域表达量较高；当拷贝数增加时，PHA的含量逐渐上升，当单拷贝整合phbCAB（phbCRe，phbBRe和phbARe）或phaC2AB（优化型phbC2Ps，phbBRe和phbARe）时均无明显PHB积累，而phbCAB增加至11个拷贝时PHB含量可达细胞干重的5.18wt%，phaC2AB增加至12个拷贝时PHB含量达3.01wt%。
通过表达量较高且对生长影响小的asnB位点，利用Cre-loxP位点特异性重组构建了多拷贝整合phbCAB的重组菌，使PHB含量达34.1wt%，经qPCR测定，其染色体上整合的phbCAB操纵子的拷贝数约为50个左右。实验证明利用这种构建方法可以快速诱导靶基因在任意染色体位点上进行多拷贝整合，并且在不添加抗生素的情况下，重组菌的稳定性高于质粒系统。以大肠杆菌染色体为载体合成PHA的方式克服了质粒系统的固有缺陷，为进一步改造生产菌株的代谢通路，实现利用廉价碳源进行不同种类PHA合成提供了有利的平台。
Polyhydroxyalkanoates (PHAs) are microbial polyesters with variable mechanicalproperties and great potential for industrial applications. Current researches onmetabolic engineered Escherichia coli for PHA production mainly rely on unstableplasmid-based systems, and the yield of short-chain-length and medium-chain-lengthcopolymers SCL-MCL PHA in E. coli is relatively low. This study focused on theoptimization of PhaC2Ps, a PHA synthase with wide substrate specificities, to promoteSCL-MCL PHA production in E. coli, and on the stabilized PHA production throughchromosomal expression of PHA-related genes.
The PHA synthase PhaC2Psfrom Pseudomonas stutzeri1317along with itsmutants were used for PHA production in E. coli. Codon optimization of the doublemutant PhaC2PsQKST enhanced its protein expression level, and the introduction of ahairpin structure in the5’ untranslated region led to stabilized transcription. Both PHBand SCL-MCL PHA productions were enhanced, along with higher CDW. Compared tothe double mutant, the PHB and SCL-MCL PHA contents using optimized synthasewere increased by2.6-fold and2.2-fold, reaching29.7wt%and3.93wt%, respectively.
Investigation on13chromosomal integration sites indicated that the expressionlevels of PHA-related genes were higher when integrated into potential active sites oftranscription, such as yheO, gltA and asnB. The copy number of the genes also affectedtheir expression levels. No PHB accumulation was observed when one copy of phbCABor phaC2AB was integrated, while the presence of11copies of phbCAB or12copies ofphaC2AB led to PHB production of5.18wt%and3.01wt%, respectively.
Using Cre-loxP recombination system, a recombinant E. coli strain with about50copies of phbCAB on its chromosome was constructed in asnB site, which benefitedgene expression with little influence on cell growth. The strain could accumulate PHBup to34.1wt%, equivalent to a moderate-copy-number plasmid system. The strain wasproven to be more stable than plasmid-based system in PHB production, indicating thatthis method can be used to engineer E. coli for industrial PHA production.
通过密码子优化和引入茎环结构等手段，针对来源于施氏假单胞菌Pseudomonas stutzeri1317的广泛底物特异性聚合酶基因phaC2Ps实现异源表达的优化；优化后基因的转录产物稳定性和异源表达水平均得到显著提高，PHB和SCL-MCL PHA的含量分别由细胞干重的11.7wt%和1.79wt%升高至29.7wt%和3.93wt%。同时，所得SCL-MCL PHA的组分更加广泛，单体碳链长度覆盖4-12，细胞干重也随之上升。
证明不同染色体整合位点和拷贝数对于PHA合成基因异源表达水平的影响。选择13个染色体整合位点，发现其对于基因的异源表达有一定的影响，在yheO(ECK3333)、gltA (ECK0709)、asnB (ECK0662)等转录活跃区域表达量较高；当拷贝数增加时，PHA的含量逐渐上升，当单拷贝整合phbCAB（phbCRe，phbBRe和phbARe）或phaC2AB（优化型phbC2Ps，phbBRe和phbARe）时均无明显PHB积累，而phbCAB增加至11个拷贝时PHB含量可达细胞干重的5.18wt%，phaC2AB增加至12个拷贝时PHB含量达3.01wt%。
通过表达量较高且对生长影响小的asnB位点，利用Cre-loxP位点特异性重组构建了多拷贝整合phbCAB的重组菌，使PHB含量达34.1wt%，经qPCR测定，其染色体上整合的phbCAB操纵子的拷贝数约为50个左右。实验证明利用这种构建方法可以快速诱导靶基因在任意染色体位点上进行多拷贝整合，并且在不添加抗生素的情况下，重组菌的稳定性高于质粒系统。以大肠杆菌染色体为载体合成PHA的方式克服了质粒系统的固有缺陷，为进一步改造生产菌株的代谢通路，实现利用廉价碳源进行不同种类PHA合成提供了有利的平台。
Polyhydroxyalkanoates (PHAs) are microbial polyesters with variable mechanicalproperties and great potential for industrial applications. Current researches onmetabolic engineered Escherichia coli for PHA production mainly rely on unstableplasmid-based systems, and the yield of short-chain-length and medium-chain-lengthcopolymers SCL-MCL PHA in E. coli is relatively low. This study focused on theoptimization of PhaC2Ps, a PHA synthase with wide substrate specificities, to promoteSCL-MCL PHA production in E. coli, and on the stabilized PHA production throughchromosomal expression of PHA-related genes.
The PHA synthase PhaC2Psfrom Pseudomonas stutzeri1317along with itsmutants were used for PHA production in E. coli. Codon optimization of the doublemutant PhaC2PsQKST enhanced its protein expression level, and the introduction of ahairpin structure in the5’ untranslated region led to stabilized transcription. Both PHBand SCL-MCL PHA productions were enhanced, along with higher CDW. Compared tothe double mutant, the PHB and SCL-MCL PHA contents using optimized synthasewere increased by2.6-fold and2.2-fold, reaching29.7wt%and3.93wt%, respectively.
Investigation on13chromosomal integration sites indicated that the expressionlevels of PHA-related genes were higher when integrated into potential active sites oftranscription, such as yheO, gltA and asnB. The copy number of the genes also affectedtheir expression levels. No PHB accumulation was observed when one copy of phbCABor phaC2AB was integrated, while the presence of11copies of phbCAB or12copies ofphaC2AB led to PHB production of5.18wt%and3.01wt%, respectively.
Using Cre-loxP recombination system, a recombinant E. coli strain with about50copies of phbCAB on its chromosome was constructed in asnB site, which benefitedgene expression with little influence on cell growth. The strain could accumulate PHBup to34.1wt%, equivalent to a moderate-copy-number plasmid system. The strain wasproven to be more stable than plasmid-based system in PHB production, indicating thatthis method can be used to engineer E. coli for industrial PHA production.
引文
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