Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be...Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.展开更多
Phosphorus concentration on the surface of seawater varies greatly with different environments,especially in coastal.The molecular mechanism by which cyanobacteria adapt to fluctuating phosphorus bioavailability is st...Phosphorus concentration on the surface of seawater varies greatly with different environments,especially in coastal.The molecular mechanism by which cyanobacteria adapt to fluctuating phosphorus bioavailability is still unclear.In this study,transcriptomes and gene knockouts were used to investigate the adaptive molecular mechanism of a model coastal cyanobacterium Synechococcus sp.PCC 7002 during periods of phosphorus starvation and phosphorus recovery(adding sufficient phosphorus after phosphorus starvation).The findings indicated that phosphorus deficiency affected the photosynthesis,ribosome synthesis,and bacterial motility pathways,which recommenced after phosphorus was resupplied.Even more,most of the metabolic pathways of cyanobacteria were enhanced after phosphorus recovery compared to the control which was kept in continuous phosphorus replete conditions.Based on transcriptome,54 genes potentially related to phosphorusdeficiency adaptation were selected and knocked out individually or in combination.It was found that five mutants showed weak growth phenotype under phosphorus deficiency,indicating the importance of the genes(A0076,A0549-50,A1094,A1320,A1895)in the adaptation of phosphorus deficiency.Three mutants were found to grow better than the wild type under phosphorus deficiency,suggesting that the products of these genes(A0079,A0340,A2284–86)might influence the adaptation to phosphorus deficiency.Bioinformatics analysis revealed that cyanobacteria exposed to highly fluctuating phosphorus concentrations have more sophisticated phosphorus acquisition strategies.These results elucidated that Synechococcus sp.PCC 7002 have variable phosphorus response mechanisms to adapt to fluctuating phosphorus concentration,providing a novel perspective of how cyanobacteria may respond to the complex and dynamic environments.展开更多
Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes...Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.展开更多
The gene for the catalytic domain of thermostable endo-β-1,3-glucanase （laminarinase） LamA was cloned from Thermotoga maritima MSB8 and heterologously expressed in a bioengineered Synechococcus sp. PCC 7002. The mu...The gene for the catalytic domain of thermostable endo-β-1,3-glucanase （laminarinase） LamA was cloned from Thermotoga maritima MSB8 and heterologously expressed in a bioengineered Synechococcus sp. PCC 7002. The mutant strain was cultured in a photobioreactor to assess biomass yield, recombinant laminarinase activity, and CO2 uptake. The maximum enzyme activity was observed at a oH of 8.0 and a temoerature of 70℃. At a CO2 concentration of 5%, we obtained a maximum specific growth rate of 0.083 h^-1 a biomass productivity of 0.42 g· L^-1·d^-1 a blomass concentration of 3.697 g.L^-1 , and a specific enzyme activity of the mutant strain of 4.325 U.mg^- 1 dry mass. All parameters decreased as CO2 concentration increased from 5% to 10% and further to 15% CO2, except enzyme activity, which increased from 5% to 10% CO2. However, the mutant culture still 1 1 grew at 15% CO2 concentration, as reflected by the blomass productwlty （0.26 g.L .d ）, biomass concentration （2.416 g.L^- 1）, and specific enzyme activity （3.247 U.mg^-1 dry mass）.展开更多
基金Supported by the International S&T Cooperation Program of China(No.2012DFA30450)the National Natural Science Foundation of China(No.30871541)+1 种基金the Taishan Scholar Foundation of Shandong Province(No.tshw20091014)the Innovation Program of the University Institutes of Jinan,Shandong Province(No.201004044)
文摘Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.
基金the support from Southern Marine Science and Engineering Guangdong Laboratory(Zhuhai,No.SML2021SP204)funded by the National Natural Science Founda-tion of China(Grant Nos.32170108,and 42188102)+1 种基金the Science and Technology Innovation 2025 Major Project of Ningbo City(Grant No.2022Z189)the Ningbo Public Welfare Science and Technology Program(Grant Nos.2023S068,2023S040).
文摘Phosphorus concentration on the surface of seawater varies greatly with different environments,especially in coastal.The molecular mechanism by which cyanobacteria adapt to fluctuating phosphorus bioavailability is still unclear.In this study,transcriptomes and gene knockouts were used to investigate the adaptive molecular mechanism of a model coastal cyanobacterium Synechococcus sp.PCC 7002 during periods of phosphorus starvation and phosphorus recovery(adding sufficient phosphorus after phosphorus starvation).The findings indicated that phosphorus deficiency affected the photosynthesis,ribosome synthesis,and bacterial motility pathways,which recommenced after phosphorus was resupplied.Even more,most of the metabolic pathways of cyanobacteria were enhanced after phosphorus recovery compared to the control which was kept in continuous phosphorus replete conditions.Based on transcriptome,54 genes potentially related to phosphorusdeficiency adaptation were selected and knocked out individually or in combination.It was found that five mutants showed weak growth phenotype under phosphorus deficiency,indicating the importance of the genes(A0076,A0549-50,A1094,A1320,A1895)in the adaptation of phosphorus deficiency.Three mutants were found to grow better than the wild type under phosphorus deficiency,suggesting that the products of these genes(A0079,A0340,A2284–86)might influence the adaptation to phosphorus deficiency.Bioinformatics analysis revealed that cyanobacteria exposed to highly fluctuating phosphorus concentrations have more sophisticated phosphorus acquisition strategies.These results elucidated that Synechococcus sp.PCC 7002 have variable phosphorus response mechanisms to adapt to fluctuating phosphorus concentration,providing a novel perspective of how cyanobacteria may respond to the complex and dynamic environments.
文摘Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.
文摘The gene for the catalytic domain of thermostable endo-β-1,3-glucanase （laminarinase） LamA was cloned from Thermotoga maritima MSB8 and heterologously expressed in a bioengineered Synechococcus sp. PCC 7002. The mutant strain was cultured in a photobioreactor to assess biomass yield, recombinant laminarinase activity, and CO2 uptake. The maximum enzyme activity was observed at a oH of 8.0 and a temoerature of 70℃. At a CO2 concentration of 5%, we obtained a maximum specific growth rate of 0.083 h^-1 a biomass productivity of 0.42 g· L^-1·d^-1 a blomass concentration of 3.697 g.L^-1 , and a specific enzyme activity of the mutant strain of 4.325 U.mg^- 1 dry mass. All parameters decreased as CO2 concentration increased from 5% to 10% and further to 15% CO2, except enzyme activity, which increased from 5% to 10% CO2. However, the mutant culture still 1 1 grew at 15% CO2 concentration, as reflected by the blomass productwlty （0.26 g.L .d ）, biomass concentration （2.416 g.L^- 1）, and specific enzyme activity （3.247 U.mg^-1 dry mass）.
