High-Light-Induced Stress Activates Lipid Deacylation at the Sn-2 Position in the Cyanobacterium Synechocystis Sp. PCC 6803.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) produce free fatty acids (FFAs) because the FFAs generated by deacylation of membrane lipids cannot be recycled. An engineered Aas-deficient mutant of Synechocystis sp. PCC 6803 grew normally under low-light (LL) conditions (50 µmol photons m-2 s-1) but was unable to sustain growth under high-light (HL) conditions (400 µmol photons m-2 s-1), revealing a crucial role of Aas in survival under the HL conditions. Several-times larger amounts of FFAs were produced by HL-exposed cultures than LL-grown cultures. Palmitic acid accounted for ∼85% of total FFAs in HL-exposed cultures, while C18 fatty acids (FAs) constituted ∼80% of the FFAs in LL-grown cultures. Since C16 FAs are esterified to the sn-2 position of lipids in the Synechocystis species, it was deduced that HL irradiation activated deacylation of lipids at the sn-2 position. Heterologous expression of FarB, the FFA exporter protein of Neisseria lactamica, prevented intracellular FFA accumulation and rescued the growth defect of the mutant under HL, indicating that intracellular FFA was the cause of growth inhibition. FarB expression also decreased the 'per-cell' yield of FFA under HL by 90% and decreased the proportion of palmitic acid to ∼15% of total FFA. These results indicated that the HL-induced lipid deacylation is triggered not by strong light per se but by HL-induced damage to the cells. It was deduced that there is a positive feedback loop between HL-induced damage and lipid deacylation, which is lethal unless FFA accumulation is prevented by Aas.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.

Modulation of the balance of fatty acid production and secretion is crucial for enhancement of growth and productivity of the engineered mutant of the cyanobacterium Synechococcus elongatus.

BACKGROUND: Among the three model cyanobacterial species that have been used for engineering a system for photosynthetic production of free fatty acids (FFAs), Synechococcus elongatus PCC7942 has been the least successful; the FFA-excreting mutants constructed from this strain could attain lower rates of FFA excretion and lower final FFA concentrations than the mutants constructed from Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002. It has been suggested that S. elongatus PCC7942 cells suffer from toxicity of FFA, but the cause of the low productivity has remained to be determined. RESULTS: By modulating the expression level of the acyl-acyl carrier protein thioesterase and raising the light intensity during cultivation, FFA secretion rates comparable to those obtained with the other cyanobacterial species were attained with an engineered Synechococcus elongatus mutant (dAS1T). The final FFA concentration in the external medium was also higher than previously reported for other S. elongatus mutants. However, about 85 % of the total FFA in the culture was found to remain in the cells, causing severe photoinhibition. Targeted inactivation of the wzt gene in dAS1T, which gene manipulation was previously shown to result in loss of the hydrophilic O-antigen layer on the cell surface, increased FFA secretion, alleviated photoinhibition, and lead to 50 and 45 % increase in the final cell density and the total amount of FFA in the culture (i.e., the sum of the cellular and extracellular FFA), respectively. The average rate of production of total FFA by the culture of the ∆wzt strain was 2.7 mg L(-1) h(-1), being five times higher than those reported for Synechocystis sp. PCC 6803 and comparable to the rates of triacylglycerol production in green algae. CONCLUSION: Synechococcus elongatus PCC7942 has larger capacity of FFA production than Synechocystis sp. PCC6803 but accumulates most of the product in the cell because of the imbalance of the rates of FFA production and secretion. This causes severe photoinhibition and exerts adverse effects on cell growth and FFA productivity. Enhancement of FFA secretion would be required to fully exploiting the capacity of FFA production for the purpose of biofuel production.

Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.

Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.

Evaluation of the effects of P(II) deficiency and the toxicity of PipX on growth characteristics of the P(II)-Less mutant of the cyanobacterium Synechococcus elongatus.

Among the known functions of the P(II) protein (the glnB gene product) in the cyanobacterium Synechococcus elongatus, negative regulation of the activity of PipX, a transcriptional co-activator of the NtcA regulon, has been thought to be essential for cell viability, because all the P(II)-less mutants thus far constructed carry spontaneous mutations in pipX. PipX is thus deduced to be a toxic protein, but its toxicity has not been clearly defined because of the lack of P(II)-deficient mutants carrying wild-type pipX. In this study, we developed a method to construct a targeted P(II)-less mutant of S. elongatus without the pipX mutation and determined the contribution of PipX to the detrimental effects of P(II) deficiency. Growth defects of the mutant were severe under nitrogen-replete conditions, i.e. in the presence of ammonium, but were also apparent under nitrogen-limited conditions. Genetic analyses indicated that the growth impairment observed under the nitrogen-limited conditions is largely due to the toxicity of PipX. Some of the phenotypes observed under the nitrogen-replete conditions, including reduced pigmentation and death of most of the cells after transfer from nitrogen-limited conditions to nitrogen-replete conditions, were ascribed to the toxicity of PipX, but inactivation of pipX only partially rescued the growth defect observed in the presence of ammonium, indicating the presence of an as yet unknown P(II) function(s) required for normal growth. Effects of ammonium addition on the nitrite uptake activity of the glnB mutant revealed a new function for P(II) in regulation of the activity of the ABC-type cyanate/nitrite transporter.

Regulation of nitrate assimilation in cyanobacteria.

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133.

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.

Nitrite-responsive activation of the nitrate assimilation operon in Cyanobacteria plays an essential role in up-regulation of nitrate assimilation activities under nitrate-limited growth conditions.

NtcB of the cyanobacterium Synechococcus elongatus strain PCC 7942 is a LysR family protein that enhances expression of the nitrate assimilation operon (nirA operon) in response to the presence of nitrite, an intermediate of assimilatory nitrate reduction. Inactivation of ntcB in this cyanobacterium specifically abolishes the nitrite responsiveness of nirA operon expression, but under nitrate-replete conditions (wherein negative feedback by intracellularly generated ammonium prevails over the positive effect of nitrite) activity levels of the nitrate assimilation enzymes are marginally higher in the wild-type cells than in the mutant cells, raising the issue of whether the nitrite-promoted regulation has physiological importance. On the other hand, the strains carrying ntcB expressed much higher nitrate assimilation enzyme activities under nitrate-limited growth conditions than under nitrate-replete conditions whereas the ntcB-deficient strains showed levels of the enzyme activities lower than those seen under the nitrate-replete conditions. Although the ntcB mutant maintained a constant cell population in a nitrate-limited chemostat when grown as a single culture, it was diluted at a rate expected for nondividing cells when mixed with the wild-type cells and subjected to nitrate limitation in the chemostat culture system. These results demonstrated that the nitrite-promoted activation of the nitrate assimilation operon is essential for up-regulation of the nitrate assimilation activities under the conditions of nitrate limitation and for competitive utilization of nitrate.