Production of γ-linolenic acid and stearidonic acid by Synechococcus sp.PCC7002 containing cyanobacterial fatty acid desaturase genes

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.

Genetic transformation of marine cyanobacterium Synechococcus sp.CC9311(Cyanophyceae) by electroporation

Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation(CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.

Insertal Orientation Has No Influence on the Expression of gfp Gene and the Growth of the Host Synechococcus sp.PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, the gfp reporter gene was inserted in the isiAB locus of Synechococcus sp. PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that the gfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.

Biosorption mechanism of Cr (Ⅵ) onto cells of Synechococcus sp.

The biosorption mechanism of Cr （Ⅳ） ions on Synechococcus sp. biosorbent was studied by analyzing the biosorption kinetics as well as speciation change and bond formation during the biosorption process. The kinetics study shows that the adsorption process of Cr （Ⅳ） consists of a very fast stage in the first several minutes, in which more than half of the saturation adsorption is attained, and a slower stage that approximately follows the first order kinetic model, basically Freundlich isotherm models were observed. Comparative studies of FT-LR spectra of K2Cr2O7, free cells of Synechococcus sp., and Cr-bound cells of Synechococcus sp show that the speciation of chromium that binds to the cells ofSynechococcus sp. is Cr （Ⅲ）, instead of Cr （Ⅳ）, and the carboxylic, alcoholic, amido and amino groups may be involved in the binding of Cr （Ⅲ）. Integrative analyses of the surface electric potential, the effect of pH value on adsorption behavior of Cr （Ⅵ）, and the results of FT-IR show that the biosorption of Cr （Ⅵ） follows two subsequent steps, biosorption of Cr2O7 ^2- by electrostatical force at the protonated active sites and reduction of Cr2O7^2- to Cr^3＋ by the reductive groups on the surface of the biosorbents.

Analysis of a Synechococcus sp. Bloom in the East China Sea and Its Causes

In the spring of 2007,a Synechococcus sp. bloom was monitored in station A1( 30° N,123° E) in the East China Sea. The abundance of Synechococcus sp. was nearly 2×10~6 cells/ml,and the contribution of Synechococcus sp. to chlorophyll a was nearly 90%. According to the abundance of Synechococcus sp. in the East China Sea and adjacent Changjiang River estuary in the past tow decades,the main reasons why Synechococcus sp. could form a bloom are listed below: the rising level of nutrients and the further eutrophication of the water body provided sufficient nutrients for Synechococcus sp.;with the global warming,the sea water temperature in the East China Sea rose continuously;the number of major predator heterotrophic flagellates was at a low level,reducing predation pressure.

Molecular mechanism of a coastal cyanobacterium Synechococcus sp.PCC 7002 adapting to changing phosphate concentrations

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.

The First Isolation of a Cyanophage-Synechococcus System from the East China Sea

A cyanophage strain and its host Synechococcus were isolated from the East China Sea. The host Synechococcus sp. S J01 was characterized by its 16S rRNA, ITS, andpsbA gene sequences as well as by its morphological appearance and pigmentation. The cyanophage, strain S-SJ2, was able to cause a lytic infection of the coastal Synechococcus. TEM of negative-stained specimens showed that the phage isolate has an isometric head with a diameter of 68 nm and a long tail with a length of 280 nm. The cyanophage-Synechococcus system from the East China Sea shares many properties with other marine cyanophage-Synechocoecus systems worldwide.

铁限制诱导对聚球藻转录组的影响

为探究Fe元素限制对聚球藻转录组的影响,该文以聚球藻Synechococcus sp.PCC 7002为研究对象,采用高通量测序技术对经过铁限制处理的聚球藻Synechococcus sp.PCC 7002进行转录组分析.以铁的3种摩尔浓度进行处理,其中每组数据重复3次实验,一共获得9组实验数据;对照组摩尔浓度为10.900 nmol·L^(-1),铁限制组摩尔浓度分别为0.410、0.003 nmol·L^(-1).实验发现:在铁限制环境下,聚球藻通过增加对胞外铁摄取、上调光合作用相关蛋白的表达和调整代谢通路来应对缺铁环境,且铁限制程度与差异基因数量成正相关关系.此外,在强缺铁时通过下调CRISPR系统中某些核酸酶的表达来影响聚球藻抵御外源病毒感染的能力,其转运磷酸基团ABC转运蛋白表达水平下降,从而极大地影响了聚球藻的生存.

Heterologous expression of LamA gene encoded endo-β-1,3- glucanase and CO2 fixation by bioengineered Synechococcus sp. PCC 7002

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）.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

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.