Microbial Synthesis Of Biodegradable Plastic Monomer 2-pyrone-4,6-dicarboxylic Acid

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.