【作者】 周丹；

【导师】 王永忠； 王钦宏；

【作者基本信息】 重庆大学 ， 生物学， 2022， 硕士

【摘要】 2-吡喃酮-4,6-二羧酸（PDC）是木质素降解过程中多种代谢途径的中间产物,也是一种有价值的生物可降解塑料单体,可作为聚对苯二甲酸乙二醇酯（PET）中对苯二甲酸（TPA）的潜在替代物。因此,开发经济上可行的PDC生物合成方法具有重要作用。基于此,本论文的主要研究工作如下:利用已报道的原儿茶酸-4,5-双加氧酶α亚基（lig A和pmd A）酶为模板,进行氨基酸序列同源比对,构建系统发育树,筛选出16组原儿茶酸-4,5-双加氧酶（ABs）与4-羧基-2-羟基粘康酸-6-半醛脱氢酶（Cs）,构建重组质粒在大肠杆菌中表达,以原儿茶酸（PCA）为底物,在摇瓶中进行全细胞催化验证。结果显示,BL2ABC与BLlig ABC菌株得到具有良好催化性能,可在摇瓶催化6 h分别得到6.20 g/L及6.17 g/L PDC;筛选得到的BL14ABC与BL15ABC菌株具有较快的产物合成速率,摇瓶催化3h即可分别得到5.04 g/L及4.79 g/L PDC。其次,构建ABs酶与异源Cs酶组合的重组菌株,催化结果发现4-羧基-2-羟基粘康酸-6-半醛（CHMS）向PDC转化是PDC生物合成的限速步骤。由此,将BL14ABC菌株中催化限速步骤的C酶进行替换,发现BL14AB4C菌株的PDC产量可提高至5.27 g/L。构建了由两种重组大肠杆菌模块构成的多酶级联催化系统,可将生物基3-脱氢莽草酸（DHS）直接转化为PDC。其中一个模块过表达3-脱氢莽草酸脱水酶（3-dehydroshikimate dehydratase,qui C）,简称PCA模块,可转化DHS为PCA。另一个模块基于过表达上述筛选得到的ABC酶,简称PDC模块,可转化PCA为PDC。其次,对两个模块的全细胞催化条件进行了优化,对于PCA模块,BL21-qui C菌株可在最佳条件下,得到79.10 g/L PCA,摩尔转化率达100%;对于PDC模块,BLlig ABC与BL2ABC菌株可在最佳条件下,分别得到49.18 g/L及53.46 g/L PDC,摩尔转化率分别可达75%及83%。最后,将大肠杆菌WJ060通过分批补料发酵制备的DHS作为底物,利用PCA模块与PDC模块进行接力催化,在86.97 g/L生物基DHS条件下,最终得到49.19 g/L生物基PDC,摩尔转化率达63%。利用CRISPR基因编辑技术,对WJ060菌株的莽草酸合成途径进行改造,构建了PDC从头合成的细胞工厂。首先,通过将qui C基因整合到WJ060菌株基因组中,构建了PCA生产菌株PDC01。该菌株通过摇瓶发酵可得到5.40 g/L PCA,通过分批补料发酵45 h,得到38.19 g/L PCA,摩尔转化率达23.6%。由此,以PDC01作为出发菌株,通过整合2ABC基因,得到PDC09菌株,通过分批补料发酵76 h,可获得55.89 g/L PDC,摩尔转化率达29%,由此证明了该代谢路径的可行性。另外,CHMS作为PDC合成的中间产物,CHMS到PDC是整个反应的限速步骤,由此在PDC09菌株中导入了含2C基因的高拷贝质粒p BM-2C,得到的PDC092C菌株具有生长快、产量高的优点;通过分批补料发酵60 h,可得到77.57 g/L PDC,OD₆₀₀可达77,摩尔转化率达29%。为进一步获得高效稳定的PDC生产菌株,增加了2C基因在PDC09基因组的拷贝数,最终得到的PDC14可通过分批补料发酵,得到105.89 g/L PDC,摩尔转化率达39%,表明所构建的工程菌株具有广阔的研究与应用前景。更多还原

【Abstract】 2-pyranone-4,6-dicarboxylic acid(PDC)is an intermediate product of various metabolic pathways in the process of lignin degradation.It is also a valuable biodegradable plastic monomer.And it can be used as a potential substitute for terephthalic acid(TPA)in polyethylene terephthalate(PET).Therefore,the development of economically feasible PDC production process plays an important role in the preparation of bio-based plastics.Based on this,the main research results of this paper are as follows:Using the reported protocatechuate-4,5-dioxygenase α subunit(lig A and pmd A)enzymes as templates,the homology alignment of amino acid sequences was carried out to construct a phylogenetic tree,and 16 groups of protocatechuic acid-4,5-dioxygenase(ABs)and 4-carboxyl-2-hydroxymuconic acid-6-semialdehyde dehydrogenase(Cs)were screened.The recombinant plasmids were constructed and expressed in Escherichia coli(E.coli),using protocatechuic acid(PCA)as the substrate,and the whole-cell catalytic verification was carried out in shake flasks.The results showed that the BL2 ABC and BLlig ABC strains had good catalytic performance,and 6.20 g/L and6.17 g/L PDC could be obtained after 6 h of shaking flask catalysis,respectively;The screened BL14 ABC and BL15 ABC strains had faster product synthesis rate,and 5.04g/L and 4.79 g/L PDC could be obtained by shaking flask catalysis for 3h,respectively.Secondly,a recombinant strain with a combination of ABs enzyme and heterologous Cs enzyme was constructed,and it was found that the conversion of4-carboxy-2-hydroxymuconate-6-semialdehyde(CHMS)to PDC was the rate-limiting step in PDC biosynthesis.Therefore,the C enzyme that catalyzes the rate-limiting step in the BL14 ABC strain was replaced,and it was found that the PDC yield of the BL14AB4 C strain could be increased to 5.27 g/L.A multi-enzyme cascade catalytic system consisting of two recombinant E.coli modules was constructed to directly convert biobased 3-dehydroshikimic acid(DHS)to PDC.One of the modules overexpresses 3-dehydroshikimate dehydratase(qui C),referred to as the PCA module,which converts DHS to PCA.Another module is based on the ABC enzyme constructed above,referred to as the PDC module,which can convert PCA to PDC.Secondly,the whole-cell catalytic conditions of the two modules were optimized.For the PCA module,the BL21-qui C strain could obtain 79.10 g/L PCA under the optimal conditions,with a molar conversion rate of 100%;for the PDC module,the BLlig ABC and BL2 ABC strains could obtain 49.18 g/L and 53.46 g/L PDC under optimal conditions,respectively,and the molar conversion rates could reach 75%and 83%,respectively.Finally,the DHS prepared by batch fed fermentation of strain WJ060 was used as the substrate,and the PCA module and the PDC module were used for relay catalysis.Under the condition of 86.97 g/L bio-based DHS,49.19 g/L bio-based PDC was finally obtained,and the molar conversion was 63%.The shikimic acid synthesis pathway of WJ060 strain was modified by CRISPR gene editing technology,and a cell factory for de novo synthesis of PDC was constructed.First,the PCA-producing strain PDC01 was constructed by integrating the qui C gene into the WJ060 strain genome.The strain could obtain 5.40 g/L PCA through shake flask fermentation,and 38.19 g/L PCA through fed-batch fermentation for 45 h,with a molar conversion rate of 23.6%.Thus,taking PDC01 as the starting strain,PDC09 strain was obtained by integrating 2ABC gene.The strain could obtain 55.89 g/L PDC by batch fed fermentation for 76 h,and the molar conversion was 29%,which proved the feasibility of this metabolic pathway.In addition,CHMS is an intermediate product of PDC synthesis,and CHMS to PDC is the rate-limiting step of the entire reaction.Thus,a high-copy plasmid p BM-2C containing the 2C gene was introduced into the PDC09 strain,and the obtained PDC092 C strain can obtain 77.57 g/L PDC through fed-batch fermentation for 60 hours,the OD600 can reach 77,and the molar conversion rate can reach 29%,which has the advantages of fast growth and high yield.In order to get an efficient and stable PDC producing strain,the copy number of 2C gene in PDC09 genome was increased.The strain PDC14 could produce 105.89 g/L PDC by batch fed fermentation to obtain,with a molar conversion rate of 39%,indicating that the constructed engineering strain has broad research and application prospects.更多还原