To accurately analyze metabolites in industry-important photosynthetic microbes, LC-MS based metabolomics protocol needs to be optimized specifically for individual species. In this study, an LC-MS based metabolomics method was optimized for cyanobacterium Synechocystis sp. PCC 6803. With the optimized extraction, liquid chromatographic and mass spectral parameters, the method was capable of detecting 24 important metabolites related to central carbohydrate and energy metabolism in Synechocystis sp. PCC 6803. The study laid an important foundation for the metabolomics analysis of cyanobacteria.
Chromatography, Liquid ; Mass Spectrometry ; Metabolome ; Metabolomics ; Photosynthesis ; Synechocystis ; metabolism

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.
Bacterial Proteins/metabolism* ; Lysine/metabolism* ; Methyltransferases/metabolism* ; Photosynthesis ; Protein Processing, Post-Translational ; Synechocystis/growth & development*

To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.
Bacterial Proteins ; metabolism ; Culture Media ; Glucosides ; biosynthesis ; Glucosyltransferases ; metabolism ; Glycerol ; metabolism ; Industrial Microbiology ; Sodium Chloride ; Synechococcus ; enzymology ; Synechocystis ; enzymology ; metabolism

Ethylene is the most widely used petrochemical feedstock globally. The development of bio-ethylene is essential due to limited fossil fuels and rising oil prices. Bio-ethylene is produced primarily by the dehydration of ethanol, but can alternatively be directly produced from ethylene biosynthesis pathways in plants, algae, or microorganisms by using cheap and renewable substrates. This review addressed the biosynthesis of ethylene in plants and microorganisms, the characterization of key enzymes, genetic engineering strategies for ethylene biosynthesis in microorganisms, and evaluated its perspective and successful cases toward the industrial application. The direct production of bio-ethylene from a biological process in situ is promising to supplement and even replace the petrochemical ethylene production.
Ethylenes ; biosynthesis ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Plants ; genetics ; metabolism ; Saccharomyces cerevisiae ; metabolism ; Synechocystis ; genetics ; metabolism ; Trichoderma ; metabolism

The nucleotide sequence of pZMO1, a small cryptic plasmid of Zymomonas mobilis ATCC10988 was determined. Analysis of 1,680 bp of sequence revealed 69% identity with Shigella sonnei plasmid, pKYM and 61% identity with Nostoc sp. ss DNA replicating plasmid. Analysis of a deduced amino acid sequence of an orf of pZMO1 revealed 75% identity and 90% similarity with the repA gene of Synechocystis sp. plasmid pCA2.4. The upstream region of the repA gene of pZMO1 possesses six directed repeat sequences and two inverted repeat sequences at downstream of the IR consensus sequence of nick region of rolling circle replication (RCR) plasmid. A typical terminator hairpin structure was found at the downstream region of repA gene. Degradation of single-stranded plasmid DNA by S1 nuclease was detected by Southern hybridization. It suggests that pZMO1 replicates by a rolling circle mechanism in Z. mobilis ATCC10988 cells.
Amino Acid Sequence ; Animals ; Base Sequence ; Consensus Sequence ; DNA ; Ecthyma, Contagious ; Inverted Repeat Sequences ; Nostoc ; Plasmids* ; Shigella sonnei ; Synechocystis ; Zymomonas*

Free fatty acid profiles of wild type and fatty acyl-ACP synthase deletion mutant strain of Synechocystis sp. PCC6803 indicated that one origin of these fatty acids is the process of lipid remodeling or lipid degradation. Lipase is the key enzyme involved in this process. The gene sll1969 is the sole gene encodes a putative lipase in Synechocystis sp. PCC6803. To identify the function of this gene and its role in fatty acid metabolism, we cloned the sll1969 from genomic DNA, overexpressed it in Escherichia coli BL21 (DE3) using pET expression system and purified this recombinant enzyme with Nickel-nitrilotriacetic acid affinity chromatography. The enzyme activity was assayed by spectrophotometric with p-nitro-phenylbutyrate as substrate. The K(m) and k(cat) of the enzyme is (1.16 +/- 0.01) mmol/L and (332.8 +/- 10.0)/min, respectively toward p-nitro-phenylbutyrate at 30 degrees C. The optimal temperature of the enzyme is 55 degrees C. To investigate the biological role of Sll1969 in fatty acid metabolism in cyanobacteria, we constructed sll1969 deletion and overexpression mutant strains in the background of fatty acyl-ACP synthase deletion mutant of Synechocystis sp. PCC6803. The analyses of the content of free fatty acids in different mutant strains showed that the contents of Sll1969 and free fatty acid are positively correlated. The free fatty acid profiles of the sll1969 mutant strains suggested this enzyme is not the sole enzyme for degrading lipid in Synechocystis sp. PCC6803.
Escherichia coli ; genetics ; metabolism ; Fatty Acids, Nonesterified ; metabolism ; Lipase ; biosynthesis ; genetics ; Membrane Lipids ; genetics ; metabolism ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Synechocystis ; enzymology ; genetics ; metabolism

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.
Escherichia coli ; genetics ; metabolism ; Gene Knockout Techniques ; Hydroxybutyrates ; metabolism ; Metabolic Engineering ; Mutation ; NADP ; metabolism ; Polyesters ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reducing Agents ; metabolism ; Synechocystis ; genetics ; metabolism

For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.
Genetic Vectors ; genetics ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Palmitoyl-CoA Hydrolase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Synechocystis ; genetics ; metabolism ; beta-Galactosidase ; biosynthesis ; genetics

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.
Bacterial Proteins ; chemistry ; Chromatography, Liquid ; Fatty Acid Synthases ; chemistry ; Fatty Acids, Unsaturated ; biosynthesis ; Proteome ; chemistry ; Proteomics ; Synechocystis ; enzymology ; Tandem Mass Spectrometry

Genomic DNA sequence analysis of phytochrome like photoreceptors in a number of bacteria revealed several open reading frames (ORFs) encoding proteins with amino acid sequences homologous to plant phytochromes. The phytochrome like photoreceptors, collectively called bacteriophytochromes, contain an N-terminal domain homologous to the chromophore-binding domain (CBD) of higher plants and a C-terminal domain of histidine kinase domain( HKD). Due to their simple structure, bacteriophytochromes broaden the view of phytochrome evolution and provide us with a simple model to investigate phytochrome-mediated light signal in higher plants. In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1 (C-435) were isolated from the Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) using specific primers. Then, the genes were cloned with the vector pBluescript, yielding plasmids pBlu-cphl and pBlu-cph1 ( C-435), before they are subcloned with the vector pET30, using the EcoRV and Xho I restriction sites. pBlu-cph1, pBlu-cph1 (N-435) were cleaved with Sma I and Xho I, and the released genes were ligated to the pET30a fragment. The E. coli [strain BL21 (DE3)] cells containing recombinant pET30a were grown in medium RB at 20 degrees C, and harvested 6 h later after induction with isopropyl thio-beta-D-galactoside (IPTG). Then, reconstitution systems were employed to study the characteristics of the genes. In the reconstitution system, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addition was an autocatalytic process. Reconstitution products were red/infrared (R/FR) photochromic, which was similar to that of the phytoehrome in higher plants. How ever, the spectral change ratios (deltaAmax/deltaAmin) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo-protein via Zn2+ fluoresc ence SDS-PAGE. After irradiation by light of 700 nm, the maximum absorption spectrum o f holo-Cphl was 650nm. The absorption of it after denaturatior in the dark with ur ea in the presence of hydrochloric acid (pH = 2) was 660nm, which was similar with th at of cis-PCB. In addition, after irradiation by light of 650nm, the maximum absorption spectrum of holo-Cph1 was 700nm. The absorption of it after denaturation in the dark with urea in the presence of hydrochloric acid (pH = 2) was 600nm, which was similar with that of trans-PCB. The result showed that the photochromism of phytochrome resulted from the isomerizaation of chromophore (PCB in this report). The reconstitution of Cph1 (C-435) under the same condition supported the conclusion. Fluorescence emission spectrum of the products suggested that bacteriophytochrom e structure with cis-PCB was more stable than that with trans-PCB. The new reconstitution system in this report sets a base for the application of phytochrome as photochromic biomaterials in biosensors. In addition, phytochrome shows great potential in food, cosmetic and biological engineering, etc.
Bacterial Proteins ; biosynthesis ; chemistry ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; radiation effects ; Genetic Vectors ; Photochemistry ; Phytochrome ; biosynthesis ; chemistry ; genetics ; Protein Kinases ; biosynthesis ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Synechocystis ; chemistry