Shuyi Zhang

The Tricarboxylic Acid Cycle in Cyanobacteria

Contrary to expectations, many photosynthetic cyanobacteria maintain a metabolic flexibility that works in the dark. It is generally accepted that cyanobacteria have an incomplete tricarboxylic acid (TCA) cycle because they lack 2-oxoglutarate dehydrogenase and thus cannot convert 2-oxoglutarate to succinyl–coenzyme A (CoA). Genes encoding a novel 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase were identified in the cyanobacterium Synechococcus sp. PCC 7002. Together, these two enzymes convert 2-oxoglutarate to succinate and thus functionally replace 2-oxoglutarate dehydrogenase and succinyl-CoA synthetase. These genes are present in all cyanobacterial genomes except those of Prochlorococcus and marine Synechococcus species. Closely related genes occur in the genomes of some methanogens and other anaerobic bacteria, which are also thought to have incomplete TCA cycles.

Biochemical validation of the glyoxylate cycle in the cyanobacterium Chlorogloeopsis fritschii strain PCC 9212

Background: Conflicting claims exist concerning the occurrence of the glyoxylate cycle in cyanobacteria. Results: The genes for isocitrate lyase and malate synthase were identified in Chlorogleopsis fritschii PCC 9212 and the purified enzymes were characterized. Conclusion: C. fritschii has a functional glyoxylate cycle and can grow in the dark on acetate. Significance: These results clarify the occurrence of the glyoxylate cycle in cyanobacteria. Cyanobacteria are important photoautotrophic bacteria with extensive but variable metabolic capacities. The existence of the glyoxylate cycle, a variant of the TCA cycle, is still poorly documented in cyanobacteria. Previous studies reported the activities of isocitrate lyase and malate synthase, the key enzymes of the glyoxylate cycle in some cyanobacteria, but other studies concluded that these enzymes are missing. In this study the genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were identified, and the recombinant enzymes were biochemically characterized. Consistent with the presence of the enzymes of the glyoxylate cycle, C. fritschii could assimilate acetate under both light and dark growth conditions. Transcript abundances for isocitrate lyase and malate synthase increased, and C. fritschii grew faster, when the growth medium was supplemented with acetate. Adding acetate to the growth medium also increased the yield of poly-3-hydroxybutyrate. When the genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the acetate assimilation capacity of the resulting strain was greater than that of wild type. Database searches showed that the genes for the glyoxylate cycle exist in only a few other cyanobacteria, all of which are able to fix nitrogen. This study demonstrates that the glyoxylate cycle exists in a few cyanobacteria, and that this pathway plays an important role in the assimilation of acetate for growth in one of those organisms. The glyoxylate cycle might play a role in coordinating carbon and nitrogen metabolism under conditions of nitrogen fixation.

Vipp1 Is Essential for the Biogenesis of Photosystem I but Not Thylakoid Membranes in Synechococcus sp. PCC 7002

Background: Vipp1 was previously thought to be essential for viability and biogenesis of thylakoid membranes. Results: A vipp1 null mutant of cyanobacterium Synechococcus sp. PCC 7002 is viable and assembles thylakoid membranes but lacks Photosystem I. Conclusion: Vipp1 is not essential but is required for biogenesis of Photosystem I. Significance: Normal thylakoid biogenesis and structure requires Photosystem I but not Vipp1. The biogenesis of thylakoid membranes in cyanobacteria is presently not well understood, but the vipp1 gene product has been suggested to play an important role in this process. Previous studies in Synechocystis sp. PCC 6803 reported that vipp1 (sll0617) was essential. By constructing a fully segregated null mutant in vipp1 (SynPCC7002_A0294) in Synechococcus sp. PCC 7002, we show that Vipp1 is not essential. Spectroscopic studies revealed that Photosystem I (PS I) was below detection limits in the vipp1 mutant, but Photosystem II (PS II) was still assembled and was active. Thylakoid membranes were still observed in vipp1 mutant cells and resembled those in a psaAB mutant that completely lacks PS I. When the vipp1 mutation was complemented with the orthologous vipp1 gene from Synechocystis sp. PCC 6803 that was expressed from the strong PcpcBA promoter, PS I content and activities were restored to normal levels, and cells again produced thylakoids that were indistinguishable from those of wild type. Transcription profiling showed that psaAB transcripts were lower in abundance in the vipp1 mutant. However, when the yfp gene was expressed from the PpsaAB promoter in the presence and the absence of Vipp1, no difference in YFP expression was observed, which shows that Vipp1 is not a transcription factor for the psaAB genes. This study shows that thylakoids are still produced in the absence of Vipp1 and that normal thylakoid biogenesis in Synechococcus sp. PCC 7002 requires expression and biogenesis of PS I, which in turn requires Vipp1.

Metabolic engineering of Synechococcus sp. PCC 7002 to produce poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-4-hydroxybutyrate

Cyanobacteria are an important group of photoautotrophic bacteria that have been engineered and used to produce a wide range of biomaterials and biofuels, which are usually derived from important intermediates of the central metabolic pathways. In this study, the production of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-4-hydroxybutyrate in cyanobacteria was studied, and metabolic engineering strategies to improve the yields were also investigated. The genes involved in the biosynthetic pathway for poly-3-hydroxybutyrate from Chlorogloeopsis fritschii PCC 9212 were introduced into Synechococcus sp. PCC 7002, and the resulting strain was able to accumulate 2.77% of total cell dry weight as poly-3-hydroxybutyrate. When the ccmR gene was deleted in this strain, the yield of poly-3-hydroxybutyrate increased to 3.97% of total cell dry weight. A biosynthetic pathway for the production of 4-hydroxybutyryl-CoA was also constructed and introduced into the poly-3-hydroxybutyrate-producing strain. The resulting strain was able to produce ~4.5% of total cell dry weight as poly-3-hydroxybutyrate-co-4-hydroxybutyrate, in which 4-hydroxybutyrate accounted for ~12% of the co-polymer. These results demonstrate that poly-3-hydroxybutyrate-co-4-hydroxybutyrate can be produced in cyanobacteria and confirm that succinic semialdehyde is an important TCA cycle metabolite in cyanobacteria. This study also demonstrates the potential for future metabolic engineering in cyanobacteria that is based on recently discovered metabolites.

Effect of mono- and dichromatic light quality on growth rates and photosynthetic performance of Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 was grown to steady state in optically thin turbidostat cultures under conditions for which light quantity and quality was systematically varied by modulating the output of narrow-band LEDs. Cells were provided photons absorbed primarily by chlorophyll (680 nm) or phycocyanin (630 nm) as the organism was subjected to four distinct mono- and dichromatic regimes. During cultivation with dichromatic light, growth rates were generally proportional to the total incident irradiance at values <275 μmol photons m−2 · s−1 and were not affected by the ratio of 630:680 nm wavelengths. Notably, under monochromatic light conditions, cultures exhibited similar growth rates only when they were irradiated with 630 nm light; cultures irradiated with only 680 nm light grew at rates that were 60–70% of those under other light quality regimes at equivalent irradiances. The functionality of photosystem II and associated processes such as maximum rate of photosynthetic electron transport, rate of cyclic electron flow, and rate of dark respiration generally increased as a function of growth rate. Nonetheless, some of the photophysiological parameters measured here displayed distinct patterns with respect to growth rate of cultures adapted to a single wavelength including phycobiliprotein content, which increased under severely light-limited growth conditions. Additionally, the ratio of photosystem II to photosystem I increased ~40% over the range of growth rates, although cells grown with 680 nm light only had the highest ratios. These results suggest the presence of effective mechanisms which allow acclimation of Synechococcus sp. PCC 7002 acclimation to different irradiance conditions.

Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that N-acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gadA gene from Synechocystis sp. PCC 6803 was heterologously expressed in Synechococcus sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven Synechococcus sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in Synechococcus sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.

To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations—both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))—were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d‐lactate, by 6‐fold (2.8% yield) and 2‐fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7‐fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49‐fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways. Biotechnol. Bioeng. 2016;113: 979–988.

Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002.

CcmR, a LysR‐type transcriptional regulator, represses the genes encoding components of the high‐affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30–50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild‐type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium (“milking”) to suppress H2 uptake. Consistent with this greater reductant flux to H2, the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6–8‐fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells−1 h−1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase‐dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation. Biotechnol. Bioeng. 2016;113: 1448–1459.