| Metabolism of carotenoids and fatty acids,as the main pathways of algal production of various secondary metabolites,has been widely studied in recent years,and the functions of related genes are also being explored and discovered.In addition,the combination of genetic engineering and synthetic biology,and the production of various compounds by biological means,provides a feasible strategy to avoid environmental protection and safety problems caused by industrial production.As an ancient photosynthetic prokaryote,Synechocystis has the characteristics of clear genetic background,short culture period and strong operability,and has become a model organism in the study of cyanobacteria metabolism.This study focuses on the carotenoid and fatty acid metabolism pathways in Synechocystis PCC6803,and studies the related products in these two pathways,and on this basis,combines with synthetic biology to obtain the target product by introducing exogenous genes.The production of the aromatic compound ?-ionone in plants requires the catalysis of the carotenoid cleavage dioxygenase(CCD1).There is no report about this gene in Synechocystis.The gene was introduced into the Synechocystis genome by biological means.In addition,?-carotene serves as a substrate for CCD1 catalysis in the carotenoid metabolic pathway,and its content directly affects the conversion efficiency.In order to improve the transformation efficiency,we used methods such as knocking out the gene of apocarotenoid cleaving oxygenase(ACO),stress treatment,and adding glycerol-conducting channel protein(GlpF)to make ?-carotene accumulate continuously,and the efficiency of enzyme and substrate binding was improved.On the other hand,in the fatty acid metabolism pathway,the serine/threonine kinases(STKs)related genes spkD(sll0776)and spkG(slr0152)have been studied in depth to explore the changes in their fatty acid composition,to lay a foundation for the follow-up study on the relationship between carotenoids and fatty acid metabolism and at the same time opens up new ideas for increasing the content of related products and the function of related genes in metabolic pathways.The specific research work is divided into the following aspects:(1)In order to explore the effect of knockout of ACO(sll1541)on the carotenoid components of Synechocystis,We cloned the ACO gene from Synechocystis DNA and constructed the vector to obtain the ACO mutant strain.(2)By comparing the growth curves of ACO gene knockout mutant strains and wild-type Synechocystis,the results showed that the mutant had no significant change in growth compared with the wild type under normal light,high light,nitrogen deficiency,and combined stress of high light and nitrogen deficiency.It indicates that the knockout of the ACO gene will not have a significant impact on the growth of Synechocystis.(3)We performed stress treatment on the ACO knockout mutant strain and wild-type Synechocystis,and determined its effect on carotenoid components by comparing the three-wavelength and HPLC detection data under these conditions.The results of the three-wavelength detection showed that under the two stress treatments,the impact of high light on the ACO knockout mutants was greater than the impact caused by nitrogen deficiency.Under high light treatment conditions,the content of chlorophyll components in the ACO mutant strains changed greatly,and decreased sharply compared with normal light.At this time,the content of carotenoids in the algae relatively increased.About 2?4days after the high-light treatment,the carotenoid content of the ACO knockout mutants was slightly higher than that of the wild type.Through HPLC detection,we found that the content of ?-carotene in the ACO knockout mutant after high-light treatment was slightly higher than that of the wild type.The above results indicate that the high light treatment has a certain effect on the accumulation of carotenoids in Synechocystis,especially the?-carotene.(4)In order to successfully obtain a mutant strain of Synechocystis containing the exogenous gene CCD1,we optimized the codon of Ph CCD1 in petunia to obtain a CCD1 gene suitable for expression in Synechocystis PCC6803,and The CCD1 gene was transferred into the Synechocystis genome.Using genetic engineering methods,an expression vector was constructed,and through natural transformation and resistance screening,a mutant strain of Synechocystis containing CCD1 was finally obtained.(5)At the same time,in order to improve the binding efficiency of the enzyme and the substrate,while constructing the CCD1 mutant strain,the mutant strain added with the glycerol-conducting channel protein GlpF was simultaneously constructed,and the mutant strain was labeled CCD1+GlpF.The acquisition of the mutant strain laid the foundation for further research.(6)Through the bioinformatics analysis of CCD1,we found that the CCD1 gene encodes 546 amino acids,and the protein to be expressed has a molecular weight of about61.3 kDa and an isoelectric point of 5.87.It is a hydrophilic protein.On the other hand,we used other analysis methods to predict the three-dimensional structure of CCD1 and other related bioinformatics data.(7)In order to study the role of spkD and spkG in fatty acid synthesis,we targeted the spkD and spkG genes by inserting inactivation methods and detected the changes in fatty acid composition.The results showed that under normal light conditions,linoleic acid(C18: 2),?-linolenic acid(C18: 3n6),?-linolenic acid(C18: 3n3)and stearidonic acid(C18: 4)levels were significantly lower in spkD and spkG gene knockout mutants than in the wild type.Through qRT-PCR analysis,it was found that the expression level of fatty acid dehydrogenase gene in wild-type was significantly higher than that of knockout mutants;the expression levels of fatty acid dehydrogenase gene and other STKs genes in spkD and spkG gene knockout mutants were different.The above results indicate that spkD and spkG play an important role in the synthesis of Synechocystis polyunsaturated fatty acids. |
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