Circular cell culture for sustainable food production using recombinant lactate-assimilating cyanobacteria that supplies pyruvate and amino acids.

Recently, we reported a circular cell culture (CCC) system using microalgae and animal muscle cells for sustainable culture food production. However, lactate accumulation excreted by animal cells in the system characterized by medium reuse was a huge problem. To solve the problem, as an advanced CCC, we used a lactate-assimilating cyanobacterium Synechococcus sp. PCC 7002, using gene-recombination technology that synthesises pyruvate from lactate. We found that the cyanobacteria and animal cells mutually exchanged substances via their waste media: (i) cyanobacteria used lactate and ammonia excreted by animal muscle cells, and (ii) the animal cells used pyruvate and some amino acids excreted by the cyanobacteria. Because of this, animal muscle C2C12 cells were amplified efficiently without animal serum in cyanobacterial culture waste medium in two cycles (first cycle: 3.6-fold; second cycle: 3.9-fold/three days-cultivation) using the same reuse medium. We believe that this advanced CCC system will solve the problem of lactate accumulation in cell culture and lead to efficient cultured food production.

The phototroph-specific ß-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria.

The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic ß-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the ß-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the ß-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.

The N-terminal region of the ϵ subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity.

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a ß-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.

The ß-hairpin region of the cyanobacterial F1-ATPase γ-subunit plays a regulatory role in the enzyme activity.

The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.

L-Lactate treatment by photosynthetic cyanobacteria expressing heterogeneous L-lactate dehydrogenase.

L-Lactate is a major waste compound in cultured animal cells. To develop a sustainable animal cell culture system, we aimed to study the consumption of L-lactate using a photosynthetic microorganism. As genes involved in L-lactate utilization were not found in most cyanobacteria and microalgae, we introduced the NAD-independent L-lactate dehydrogenase gene from Escherichia coli (lldD) into Synechococcus sp. PCC 7002. The lldD-expressing strain consumed L-lactate added to basal medium. This consumption was accelerated by expression of a lactate permease gene from E. coli (lldP) and an increase in culture temperature. Intracellular levels of acetyl-CoA, citrate, 2-oxoglutarate, succinate, and malate, and extracellular levels of 2-oxoglutarate, succinate, and malate, increased during L-lactate utilization, suggesting that the metabolic flux from L-lactate was distributed toward the tricarboxylic acid cycle. This study provides a perspective on L-lactate treatment by photosynthetic microorganisms, which would increase the feasibility of animal cell culture industries.

Metabolomics-based engineering for biofuel and bio-based chemical production in microalgae and cyanobacteria: A review.

Metabolomics, an essential tool in modern synthetic biology based on the design-build-test-learn platform, is useful for obtaining a detailed understanding of cellular metabolic mechanisms through comprehensive analyses of the metabolite pool size and its dynamic changes. Metabolomics is critical to the design of a rational metabolic engineering strategy by determining the rate-limiting reaction and assimilated carbon distribution in a biosynthetic pathway of interest. Microalgae and cyanobacteria are promising photosynthetic producers of biofuels and bio-based chemicals, with high potential for developing a bioeconomic society through bio-based carbon neutral manufacturing. Metabolomics technologies optimized for photosynthetic organisms have been developed and utilized in various microalgal and cyanobacterial species. This review provides a concise overview of recent achievements in photosynthetic metabolomics, emphasizing the importance of microalgal and cyanobacterial cell factories that satisfy industrial requirements.

Nitrogen Availability Affects the Metabolic Profile in Cyanobacteria.

Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.