Shimpei Aikawa

Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion

Cyanobacteria represent a globally important biomass because they are responsible for a substantial proportion of primary production in the hydrosphere. Arthrospira platensis is a fast-growing halophilic cyanobacterium capable of accumulating glycogen and has the potential to serve as a feedstock in the fermentative production of third-generation biofuels. Accordingly, enhancing cyanobacterial glycogen production is a promising biofuel production strategy. However, the regulatory mechanism of glycogen metabolism in cyanobacteria is poorly understood. The aim of the present study was to determine the metabolic flux of glycogen biosynthesis using a dynamic metabolomic approach. Time-course profiling of widely targeted cyanobacterial metabolic intermediates demonstrated a global metabolic reprogramming that involves transient increases in the levels of some amino acids during the glycogen production phase induced by nitrate depletion. Also, in vivo labelling with NaH13CO3 enabled direct measurement of metabolic intermediate turnover in A. platensis, revealing that under conditions of nitrate depletion glycogen is biosynthesized with carbon derived from amino acids released from proteins via gluconeogenesis. This dynamic metabolic profiling approach provided conclusive evidence of temporal alterations in the metabolic profile in cyanobacterial cells.

Synergistic enhancement of glycogen production in Arthrospira platensis by optimization of light intensity and nitrate supply

Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium able to accumulate glycogen, was investigated for its feasibility to serve as feedstock for fermentative production of biofuels and chemicals. The culture conditions most appropriate for glycogen production were identified. Glycogen production was maximized by the depleting nitrate source under a high light intensity of 700 μmol photons m(-2) s(-1). With optimal control of both light intensity and nitrate supply, glycogen production of A. platensis reached nearly 1.03 g L(-1) (a glycogen productivity of 0.29 g L(-1) d(-1)), which is, to the best of our knowledge, the highest α-polyglucan (glycogen or starch) production performance ever reported in microalgae. The outcome of this work supports A. platensis as a promising carbohydrate source for biorefinery.

Adaptation of light-harvesting systems of Arthrospira platensis to light conditions, probed by time-resolved fluorescence spectroscopy.

Cyanobacteria change the quantity and/or quality of their pigment-protein complexes in response to light conditions. In the present study, we analyzed excitation relaxation dynamics in the cyanobacterium, Arthrospira (Spirulina) platensis, grown under lights exhibiting different spectral profiles, by means of steady-state absorption and picosecond time-resolved fluorescence spectroscopies. It was found that F760, which is the PSI red-chlorophyll characteristic of A. platensis, contributes to slower energy-transfer phase in the PSI of A. platensis. Excitation energy transfers in phycobilisome and those from PSII to PSI were modified depending on the light quality. Existence of quencher was suggested in PSI of the blue-light grown cells. Phycobilisomes in the green-light grown cells and the far-red-light grown cells transferred excitation energy from phycobilisome to chlorophyll without loss of energy. In these cells, excitation energy was shared between two photosystems. Fast energy transfer was established in phycobilisome under the yellow-light condition where only the phycobilisome can absorb the cultivation light. Differences in light-harvesting and energy-transfer processes under different cultivation-light conditions are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4.

Biodiesel production from microalgae has become a popular research topic. In this study, Chlamydomonas sp. JSC4 isolated from the southern coast of Taiwan was selected for a detailed study on cell growth and lipid accumulation under marine salinity (3.5% sea salt). Proper CO2 was supplied as the improvement of lipid productivity. Under the optimal condition, the highest lipid productivity was 169.1mg/L/d, which was significantly higher than those reported in current studies for marine green algae. To date, only very few studies have reported a marine algae strain with both high cell growth and lipid productivity. This study demonstrated that a newly isolated marine green alga Chlamydomonas sp. JSC4 would be a feasible oil producer due to its high biomass production and lipid productivity under marine salinity.

Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid.

Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.

Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2‐oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, and glyceraldehyde‐3‐phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2‐oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR.

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45–0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = –0.95 to –0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering

Photosynthetic microorganisms, such as cyanobacteria and microalgae, are currently being investigated as alternative biomass resources for bioethanol production, owing to their benefits, including high‐photosynthetic activity and whole‐year cultivation without utilization of arable land. Polyglucans comprise the major carbohydrate content of these organisms. Polyglucans can be utilized as a carbon source for microbial fermentation. Although polyglucan production has so far been promoted by nutrient limitation, it must be further enhanced to accommodate market demand. This review focuses on the recent progress in the production of α‐polyglucans such asglycogen and starch in cyanobacteria and green microalgae via cultivation design, including modifying the nutrient supply and replacing the growth medium. The control and manipulation of polyglucan metabolism necessitates the elucidation of the polyglucan production mechanism. We reviewed gene expression and metabolite accumulation profiles of cyanobacteria and green microalgae during nutrient limitation‐stimulated α‐polyglucan accumulation. We also focus on the enhancement in cyanobacterial glycogen production via the genetic engineering of glycolysis, CO2 concentration mechanism, and photosynthetic light‐harvesting protein based on the polyglucan accumulation mechanism. The combined strategies of cultivation design and genetic engineering should be considered for further enhancement of polyglucan productivity for bioethanol production.

Overexpression of flv3 improves photosynthesis in the cyanobacterium Synechocystis sp. PCC6803 by enhancement of alternative electron flow

BackgroundTo ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.ResultsIn the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O2 photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O2 evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo13C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.ConclusionsOverexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO2 incorporation is enhanced by the flv3 overexpression. Increase in O2 evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

Modification of energy-transfer processes in the cyanobacterium, Arthrospira platensis, to adapt to light conditions, probed by time-resolved fluorescence spectroscopy

In cyanobacteria, the interactions among pigment–protein complexes are modified in response to changes in light conditions. In the present study, we analyzed excitation energy transfer from the phycobilisome and photosystem II to photosystem I in the cyanobacterium Arthrospira (Spirulina) platensis. The cells were grown under lights with different spectral profiles and under different light intensities, and the energy-transfer characteristics were evaluated using steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra. The direct energy transfer from the phycobilisome to photosystem I and energy transfer from photosystem II to photosystem I were modified depending on the light quality, light quantity, and cultivation period. However, the total amount of energy transferred to photosystem I remained constant under the different growth conditions. We discuss the differences in energy-transfer processes under different cultivation and light conditions.