Effective cultivation conditions and safety evaluation of filamentous cyanobacteria producing phycocyanins with antiglycation activities.

We investigated suitable culture conditions for the production of the blue pigment phycocyanin (PC) from the unique filamentous cyanobacteria Pseudanabaena sp. ABRG5-3 and Limnothrix sp. SK1-2-1. White, green, or red LED irradiation at 30 µmol photons/m2/s was effective for phycocyanin production when compared with Arthrospira platensis (Spirulina) sp. NIES-39, which is generally grown under high light irradiation. To investigate the safety of the cyanobacteria, ABRG5-3 cells were subjected to Ames (reverse mutation) tests and single oral-dose rat studies, which revealed non-mutagenic and non-toxic properties. When three purified phycocyanins (abPC, skPC, and spPC) were subjected to agarose gel electrophoresis, they showed different mobility, indicating that each phycocyanin has unique properties. abPC exhibited strong antiglycation activities as novel function.

Expression of candidate marker genes of sugar starvation is upregulated in growth-suppressed parthenocarpic cucumber fruit. Novel gene markers for sugar starvation in growth-suppressed cucumber fruit.

To examine the physiological change in the growth suppression and abortion of parthenocarpic cucumber fruit, the expression of candidate marker genes of sugar starvation in relation to growth activity was examined. Fruits that failed to start exponential growth seemed to eventually abort. Hexose concentration of fruits was low in growth-suppressed fruit and increased in normally growing fruit consistent with the vacuolization. The correlation matrix indicated that the transcript levels of the genes, except CsaV3_6G046050 and CsaV3_5G032930, had a highly significant negative correlation with the relative growth rate in fruit length and had highly significant mutual positive correlations, suggesting that the asparagine synthetase gene, Cucumis sativus putative CCCH-type zinc finger protein CsSEF1, C. sativus BTB/POZ domain-containing protein At1g63850-like, CsaV3_3G000800, CsaV3_3G041280, and CsaV3_7G032930 are good markers of sugar starvation in cucumber fruit. The expression of candidate marker genes together with the hexose analysis strongly suggests that severe sugar starvation is occurring in growth-suppressed fruit.

Efficient pH and dissolved CO2 conditions for indoor and outdoor cultures of green alga Parachlorella.

Efficient pH and dissolved CO2 conditions for indoor (50-450 mL scale) and outdoor (100-500 L scale) culture of a green alga BX1.5 strain that can produce useful intracellular lipids and extracellular polysaccharides were investigated for the first time in Parachlorella sp. The cultures harvested under 26 different conditions were analysed for pH, dissolved CO2 concentration, and the biomass of extracellular polysaccharides. The BX1.5 strain could thrive in a wide range of initial medium pH ranging from 3 to 11 and produced valuable lipids such as C16:0, C18:2, and C18:3 under indoor and outdoor culture conditions when supplied with 2.0% dissolved CO2. Particularly, the acidic BG11 medium effectively increased the biomass of extracellular polysaccharides during short-term outdoor cultivation. The BG11 liquid medium also led to extracellular polysaccharide production, independent of acidity and alkalinity, proportional to the increase in total sugars derived from cells supplied with high CO2 concentrations. These results suggest Parachlorella as a promising strain for indoor and outdoor cultivation to produce valuable materials.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Coproduction of lipids and carotenoids by the novel green alga Coelastrella sp. depending on cultivation conditions.

A novel green alga Coelastrella sp. D3-1 was isolated, and its unique and significant lipid and carotenoid coproduction capability was characterised depending on cultivation conditions. The main component of produced lipids was triacylglycerol under nutrient depletion conditions, in which fatty-methyl-esters made up 20-44% of the dry cell weight (DCW) and consisted of abundant C16:0 and C18:1 fatty acids. The red (orange)-stage cells also produced a large portion of carotenoids (38.5% of the DCW) involving echinenone, canthaxanthin, and astaxanthin as major components accumulated over only 5-6 days under optimal conditions. Stress tests revealed resistance of the cells to pH 2-11, high temperatures (40-60 °C), ultraviolet irradiation, drought, and H2O2 treatment, thereby showing a robust nature. Both green- and red (orange)-stage cell extracts also showed antioxidant and anti-inflammatory abilities, implying that they have significant functions as useful biorefinery materials.

Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp. PCC 6803.

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram-negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli, feedback-regulate RNase E expression via the rne 5'-untranslated region (5' UTR) in cis. However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.

Development of a method for phycocyanin recovery from filamentous cyanobacteria and evaluation of its stability and antioxidant capacity.

BACKGROUND: Most commercial phycocyanins are extracted from a filamentous cyanobacterium, Arthrospira (Spirulina) platensis. Owing to the expenses of culture and complexities of the physical and chemical methods of phycocyanin purification, a more effective and simple method is required. RESULTS: We developed a new method for efficiently recovering the blue pigment protein, phycocyanin, from unique filamentous cyanobacteria, Pseudanabaena sp. ABRG5-3 and Limnothrix sp. SK1-2-1. The cells were cultivated in economy medium BG11 and lysed by adding water in a 1:16 ratio of wet cells to water. After extraction and purification, 28-30% dry cell weight of phycocyanin was obtained and its purity was confirmed. The stabilities of the phycocyanins at different pH in the presence of high temperature and light conditions and their antioxidant abilities were assessed. Results indicated that the phycocyanins were stable and possessed antioxidant properties. Interestingly, the Pseudanabaena phycocyanin was less likely to deteriorate under acidic conditions. CONCLUSIONS: Overall, we developed a promising and novel method for producing high functional phycocyanin concentrations at a low cost. The possibilities of adapting this new phycocyanin biorefinery to unique bioreactor utilization have also been discussed.

Characteristics and function of an extracellular polysaccharide from a green alga Parachlorella.

The green alga Parachlorella sp. BX1.5 is a new strain that can significantly coproduce lipids and polysaccharides, inside and outside the cells, respectively. This study aimed to investigate the characteristics and functional properties of the extracellular polysaccharide (EPS). Ethanol-extracted EPS from the cells grown in N-starved BG11 medium was found to be a novel acidic rhamnan of straight-chain type, with a high molecular mass of > 1.75 × 106 daltons. The water-soluble EPS exhibited high viscosity as sol, even at low concentrations, with 0.5 % EPS showing almost constant viscosity at 10-40 °C. Its addition to mayonnaise resulted in improved oil-off, suggesting its possible application as a thickener or food additive. Cells with added EPS were found to exhibit resistance to environmental stress conditions, such as heat, dryness, and decompression, and ultraviolet rays. Based on these rheological and biological effects, its unique properties with respect to biorefinery have been discussed here.

Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli.

OBJECTIVES: To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase (coaA) and acetyl-CoA carboxylase (acc) in Escherichia coli. RESULTS: The expression of acc, obtained from Corynebacterium glutamicum, accumulated 2.2-fold more fatty acids in E. coli. The addition of coaA from Pseudomonas putaida or fatty acid synthase (fasA) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA, or acc and fasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc, coaA, and fasA stored 691 mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. CONCLUSIONS: Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli.

Coproduction of lipids and extracellular polysaccharides from the novel green alga Parachlorella sp. BX1.5 depending on cultivation conditions.

A novel strain of microalga Parachlorella sp. BX1.5 was isolated and its unique properties of producing lipids and extracellular polysaccharides (EPS) characterized. The cells could extracellularly produce a large amount of acidic EPS, when cultured in nitrogen-deficient BG110 medium (BG11-N) with 2 % CO2-air supply. The main component of intracellularly accumulated lipids was triacylglycerol (TAG), depending on the different cultivation conditions of BG11, BG11-N, BG11-P (phosphate depleted), and BG11-N-P (nitrogen and phosphate depleted). Fatty-methyl-esters (FAMEs), methyl-esterification of total lipids, consisted of abundant saturated C16 and unsaturated C18 fatty acids under the culture conditions. Cell spot assays on BG11 plates revealed the resistance of cells to pH 2-11, high temperatures of 50-70 °C, ultraviolet irradiation, and drought, under different culture conditions, thereby suggesting the biological significance of lipid and EPS accumulation. The prospects of BX1.5 as a dual producer has also been discussed for biorefineries.

Biofuel production utilizing a dual-phase cultivation system with filamentous cyanobacteria.

Biomass yields and biofuel production were examined in a dual (solid and liquid)-phase cultivation system (DuPHA) with the unique filamentous cyanobacteria, Pseudanabaena sp. ABRG 5-3 and Limnothrix sp. SK1-2-1. Continuous circular cultivation was driven under the indoor closed (IC) or indoor opened (IO) conditions and provided biomass yields of approximately 8-27 g dry cell weight (DCW) floor m-2 d-1. Alkanes of heptadecane (C17H36) or pentadecane (C15H32) as liquid biofuels were also recovered from the lower liquid-phase, in which cyanobacteria were dropped from the upper solid-phase and continuously cultivated with a small amount of medium. After the main cultivation in DuPHA, the upper solid-phase of a cotton cloth on which cyanobacteria grew was dried and directly subjected to a combustion test. This resulted in the thermal power (kJ s-1) of the cloth with microalgae increasing approximately 20-50% higher than that of the cloth only, suggesting a possibility of using the solid phase with microalgae as solid biofuel.

Flocculation and pentadecane production of a novel filamentous cyanobacterium Limnothrix sp. strain SK1-2-1.

OBJECTIVE: A novel filamentous cyanobacterium, a photosynthesizing microorganism, was isolated from a river, and its unique features of flocculation and pentadecane production were characterized. RESULTS: Microscopic observations and a phylogenetic analysis with 16S rDNA revealed that this strain was a Limnothrix species denoted as the SK1-2-1 strain. Auto cell-flocculation was observed when this strain was exposed to a two-step incubation involving a standing cultivation following a shaking preincubation. Flocculation was enhanced by blue light at a wavelength at 470 nm and irradiation for several hours to 1 day. Moreover, the strain exhibiting exponential cell growth may preferentially accumulate alkanes as pentadecane C15H32 alkane, which may be used as jet fuel, at a range of approximately 1% in the dry cell weight of flocculated cells. CONCLUSION: This is the first study on biofuel production using flocculated cells in which the specific manner of production may be regulated by cultivation conditions.

Complete Genome Sequence of the Nonheterocystous Cyanobacterium Pseudanabaena sp. ABRG5-3.

We report here the complete sequences of the main genome (4.8 Mb) and seven plasmids of the semifilamentous, nonheterocystous cyanobacterium Pseudanabaena sp. ABRG5-3, a strain isolated from a pond in Japan. These data are expected to enhance our understanding of the Pseudanabaena subclade near the root of cyanobacterial diversity.

The expression of a candidate cucumber fruit sugar starvation marker gene CsSEF1 is enhanced in malformed fruit induced by salinity.

The cucumber (Cucumis sativus L.) gene Cucumis sativus Somatic Embryogenesis Zinc Finger 1 (CsSEF1) was suggested to be a good marker gene for sugar starvation in fruit. The expression of this gene in fruits is dramatically upregulated in plants that have suffered either complete defoliation or prolonged darkness. CsSEF1 was initially discovered as a gene that was upregulated during somatic embryogenesis. We examined the difference in fruit parts and the effect of pollination on the upregulation of CsSEF1 induced by defoliation treatment. The results indicated that the upregulation of CsSEF1 in fruit by defoliation is not dependent on the presence of developing embryos. The expression of CsSEF1 was upregulated in malformed fruit induced by salinity in which the development of placenta was arrested. Partial cutting of the distal part of the fruit showed that if placenta tissue remained there was no upregulation of CsSEF1, whereas when placenta tissue did not remain there was a marked upregulation of CsSEF1. These results could be consistently interpreted as showing that placenta tissue induced the transport of photoassimilates to the fruit and that without developing placenta tissue, pericarp tissue suffers from severe sugar starvation. This interpretation, in turn, enforces the view that CsSEF1 is a good marker gene of fruit sugar starvation.

Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of ß-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited ß-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes.

Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis.

Many deaths of wild birds that have drunk water contaminated with hepatotoxic microcystin-producing cyanobacteria have been reported. A mass death of egrets and herons with steatitis were found at the agricultural reservoir occurring cyanobacterial waterblooms. This study aimed to verify a hypothesis that the egrets and herons which died in the reservoir drink microcystin-producing cyanobacteria and microcystin involves in the cause of death as well as the symptoms of steatitis. The cyanobacterial community in gastric contents of egrets and herons that died from steatitis was assessed using cyanobacterial 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) profiling and a cyanobacterial 16S rRNA-based clone library analysis. In addition, PCR amplification of the mcyB-C region and the mcyG gene, involved in microcystin biosynthesis, was examined. The cyanobacterial community in the gastric contents of two birds showed a simplistic composition. A comparison of cyanobacterial T-RFLP profiling and cloned sequences suggested that the genus Microcystis predominated in both samples of egrets died. Although we confirmed that two egrets which died in the reservoir have taken in cyanobacterial waterblooms containing the genus Microcystis, no mcy gene was detected in both samples according to the mcy gene-based PCR analysis. This study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds by molecular analysis. Additionally, we consider causing symptoms of steatitis in the dead egrets.

Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria.

Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

Overproduction and easy recovery of biofuels from engineered cyanobacteria, autolyzing multicellular cells.

The semi-filamentous multicellular cyanobacterium Limnothrix/Pseudanabaena sp. strain ABRG5-3 undergoes autolysis, which involves the accumulation of polyphosphate compounds and disintegration of thylakoid membranes in cells, as a unique feature that occurs due to growth conditions. In this study, the overexpression and easy recovery of alkane (a saturated hydrocarbon, C17H36) as a biofuel were examined in recombinants of the cyanobacteria ABRG5-3 and Synechocystis sp. strain PCC6803. The results obtained indicated that the accumulated mass of alkane accounted for ∼50 or 60% of the dry weight of ABRG5-3 or PCC6803 recombinant cells, respectively. Furthermore, cultivating cells in liquid medium BG11 in which the nitrogen resource had been depleted promoted the production of alkane and cell lysis, resulting in the easy recovery of target products from the supernatant.

Metabolomic analysis reveals rewiring of Synechocystis†sp. PCC 6803 primary metabolism by ntcA overexpression.

NtcA is a cAMP receptor protein-type transcription factor conserved among cyanobacteria and is essential for gene expression in response to nitrogen status. NtcA has been widely studied; however, no metabolomic analysis has been conducted using the ntcA mutant. Here, we generated a strain that overexpresses ntcA in Synechocystis sp. PCC 6803, named NOX10, and performed physiological, transcriptomic and metabolomic analyses. NOX10 grew faster than the wild-type strain under photoautotrophic conditions, but slower under light-activated heterotrophic conditions. Transcriptome analysis revealed that the expression of genes related to primary metabolism was altered by ntcA overexpression particularly under nitrogen-depleted conditions. Metabolomic analysis revealed that metabolite levels in sugar, purine/pyrimidine nucleotide, organic acid and amino acid metabolism were widely altered by ntcA overexpression. The protein levels of nitrogen-regulated transcriptional regulators were altered by ntcA overexpression during nitrogen starvation. These results demonstrate the alteration of primary metabolism by genetic engineering of NtcA, and they contribute to the current understanding of metabolic regulation of unicellular cyanobacteria.

Role of prokaryotic type I and III pantothenate kinases in the coenzyme A biosynthetic pathway of Bacillus subtilis.

Pantothenate kinases (CoaAs) catalyze the phosphorylation of pantothenate in the first step of the coenzyme A (CoA) biosynthetic pathway. These bacterial enzymes have been categorized into 3 types, the prokaryotic type I, II, and III CoaAs. Bacteria typically carry a single CoaA gene on their genome, but Bacillus subtilis possesses 2 proteins homologous to type I and III CoaAs, known as BsCoaA and BsCoaX, respectively. Both recombinant proteins exhibited the expected kinase activity and the characteristic properties of type I and III CoaAs, i.e., regulation by CoASH and acyl-CoAs in BsCoaA and the requirement of a monovalent cation in BsCoaX. Both gene disruptants appeared to grow in a manner similar to the wild-type strain. With the BsCoaX disruptant, the BsCoaA had the ability to completely fill the intracellular CoA pool, whereas the BsCoaA disruptant did not. These findings clearly indicate that these 2 CoaAs are employed together in the CoA biosynthetic pathway in B. subtilis and that the contribution of the type I CoaA (BsCoaA) to the formation of the intracellular CoA pool is larger than that of the type III CoaA (BsCoaX).