Growth engineering of Synechococcus elongatus PCC 7942 for mixotrophy under natural light conditions for improved feedstock production

Synechococcus elongatus PCC 7942 has been widely explored as cyanobacterial cell factory through genetic modifications for production of various value‐added compounds. However, successful industrial scale‐ups have not been reported for the system predominantly due to its obligate photoautotrophic metabolism and use of artificial light in photobioreactors. Hence, engineering the organism to perform mixotrophy under natural light could serve as an effective solution. Thus, we applied a genetically engineered strain of Synechococcus elongatus PCC 7942 expressing heterologous hexose transporter gene (galP) to perform mixotrophy under natural light in a temperature controlled environmental chamber (EC). We systematically studied the comparative performances of these transformants using autotrophy and mixotrophy, which showed 3.4 times increase in biomass productivity of mixotrophically grown transformants over autotrophs in EC. Chlorophyll a yield was found to have decreased in mixotrophic conditions, possibly indicating reduced dependency on light for energy metabolism. Although pigment yield decreases under mixotrophy, titer was found to have improved due to increased biomass productivity. Carotenoid analysis showed that zeaxanthin is the major carotenoid produced by the species which is essential for photoprotection. Our work thus demonstrates that mixotrophy under temperature controlled natural light can serve as the viable solution to improve biomass productivity of Synechococcus elongatus PCC 7942 and for commercial production of natural or engineered value added compounds from the system. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1182–1192, 2017     

High-Throughput Functional Analysis of the Synechococcus elongatus PCC 7942 Genome

Synechococcus elongatus PCC 7942 was the first cyanobacterialstrain to be reliably transformed by exogenously added DNA andhas become the model organism for cyanobacterial circadian rhythms.With a small genome (2.7 Mb) and well-developed genetic tools,PCC 7942 provides an exceptional opportunity to elucidate thecircadian mechanism through genetics. We describe a projectto create mutations in every locus of the genome, both to assayeach locus for its potential contribution to the circadian clockand to archive data for the cyanobacterial community. Cosmidclones that carry inserts of PCC 7942 DNA are saturated withtransposon insertions in vitro to provide sequencing templatesand substrates for mutagenesis of the PCC 7942 genome via homologousrecombination. We have mutagenized 53% of the chromosome from50 chromosome-bearing cosmids and identified the positions ofinsertions in 31 of those cosmids and the 46 kb plasmid, pANL.PCC 7942 mutants defective for 490 different genes have beenscreened for circadian phenotypes. Mutagenesis of three apparentlyessential loci, including clpPIIclpX, resulted in circadianphenotypes. We developed an effective antisense suppressionmethod to further the analysis of essential genes. When completed,the set of comprehensive mutations will provide the communitywith a unique resource whose impact will extend beyond circadianresearch.     

Phase determination of circadian gene expression in Synechococcus elongatus PCC 7942

The authors analyzed the upstream regulatory region of purF, a gene that is expressed in a minority phase that peaks at dawn (class 2 circadian phasing) in Synechococcus elongatus, to determine whether specific cis elements are responsible for this characteristic expression pattern. Fusions of various promoter-bearing fragments to luciferase reporter genes showed that normal class 2 phasing of purF expression was correlated with promoter strength. No specific cis element that is separable from the promoter was responsible for determining phase. Very weak promoter activity of unstable phasing was mapped to a 50-bp segment. Inclusion of sequences that flank this minimal promoter either upstream or downstream increased the promoter strength and stabilized the phase in class 2, but neither segment was individually necessary. Because the data suggested a role for the overall promoter context rather than a specific "phase element," the authors proposed that DNA topology is important in the phase determination of circadian gene expression in S. elongatus. To test this hypothesis, they fused the well-characterized DNA topology-dependent Escherichia coli fis promoter to luciferase and showed that it acts as a class 2 promoter in S. elongatus.     

Characterization of dnaJ multigene family in the cyanobacterium Synechococcus elongatus PCC 7942

The genome of the cyanobacterium Synechococcus elongatus PCC 7942 contains four dnaJ homologs, which are classified into three types based on domain structure. Among these, dnaJ1, dnaJ2, and dnaJ3 are essential for normal growth, and hence we analyzed them with a view to characterizing their specificity. Expression analysis indicated that dnaJ2, which encodes type II DnaJ protein, exhibited typical responses to heat and high-light stresses. Their localization and ability to prevent aggregation of luciferase were also diverse, suggesting a possible functional differentiation of these proteins. Since the expression of dnaJ1, which belongs to conserved type I DnaJ, down-regulated under heat stress, the unique structure of DnaJ2 may be involved in stress responses of S. elongatus. Based on phylogenetic analysis, the diverse dnaJ family was assumed to have evolved its own specific functions in each cyanobacterial species.     

Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942

We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.     

Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.     

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.     

Synechococcus elongatus PCC 7942 is more tolerant to chromate as compared to Synechocystis sp. PCC 6803

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC₅₀ of 12 ± 2 and 150 ± 15 μM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/10⁸ cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using ³⁵S labeled sulfate. IC₅₀ values of chromate for ³⁵sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC₅₀ values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 μM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 μM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.     

Comparative biochemical characterization of two GroEL homologs from the cyanobacterium Synechococcus elongatus PCC 7942

Unlike Escherichia coli, cyanobacteria generally contain two GroEL homologs. The chaperone function of cyanobacterial GroELs was examined in vitro for the first time with GroEL1 and GroEL2 of Synechococcus elongatus PCC 7942. Both GroELs prevented aggregation of heat-denatured proteins. The ATPase activity of GroEL1 was approximately one-sixth that of Escherichia coli GroEL, while that of GroEL2 was insignificant. The activities of both GroELs were enhanced by GroES, while that of Escherichia coli GroEL was suppressed. The ATPase activity of GroEL1 was greatly enhanced in the presence of GroEL2, but the folding activities of GroEL1 and GroEL2 were much lower than that of Escherichia coli GroEL, regardless of the co-presence of the counterpart or GroES. Both native and recombinant GroEL1 forms a tetradecamer like Escherichia coli GroEL, while GroEL2 forms a heptamer or dimer, but the GroEL1 and GroEL2 oligomers were extremely unstable. In sum, we concluded that the cyanobacterial GroELs are mutually distinct and different from Escherichia coli GroEL.     

Carbon-free production of 2-deoxy-scyllo-inosose (DOI) in cyanobacterium Synechococcus elongatus PCC 7942

Owing to their photosynthetic capabilities, there is increasing interest in utilizing cyanobacteria to convert solar energy into biomass. 2-Deoxy-scyllo-inosose (DOI) is a valuable starting material for the benzene-free synthesis of catechol and other benzenoids. DOI synthase (DOIS) is responsible for the formation of DOI from d-glucose-6-phosphate (G6P) in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as neomycin and butirosin. DOI fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source is necessary for high-yield DOI production. We constructed DOI-producing cyanobacteria toward carbon-free and sustainable DOI production. A DOIS gene derived from the butirosin producer strain Bacillus circulans (btrC) was introduced and expressed in the cyanobacterium Synechococcus elongatus PCC 7942. We ultimately succeeded in producing 400 mg/L of DOI in S. elongatus without using a carbon source. DOI production by cyanobacteria represents a novel and efficient approach for producing benzenoids from G6P synthesized by photosynthesis.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.