Effects of CO 2 enrichment on photosynthesis,growth, and nitrogen metabolism of the seagrass Zostera noltii

Seagrass ecosystems are expected to benefit from the global increase in CO ₂ in the ocean because the photosynthetic rate of these plants may be C_i‐limited at the current CO ₂ level. As well, it is expected that lower external pH will facilitate the nitrate uptake of seagrasses if nitrate is cotransported with H⁺ across the membrane as in terrestrial plants. Here, we investigate the effects of CO ₂ enrichment on both carbon and nitrogen metabolism of the seagrass Zostera noltii in a mesocosm experiment where plants were exposed for 5 months to two experimental CO ₂ concentrations (360 and 700 ppm). Both the maximum photosynthetic rate (P_m) and photosynthetic efficiency (α) were higher (1.3‐ and 4.1‐fold, respectively) in plants exposed to CO ₂‐enriched conditions. On the other hand, no significant effects of CO ₂ enrichment on leaf growth rates were observed, probably due to nitrogen limitation as revealed by the low nitrogen content of leaves. The leaf ammonium uptake rate and glutamine synthetase activity were not significantly affected by increased CO ₂ concentrations. On the other hand, the leaf nitrate uptake rate of plants exposed to CO ₂‐enriched conditions was fourfold lower than the uptake of plants exposed to current CO ₂ level, suggesting that in the seagrass Z. noltii nitrate is not cotransported with H⁺ as in terrestrial plants. In contrast, the activity of nitrate reductase was threefold higher in plant leaves grown at high‐CO ₂ concentrations. Our results suggest that the global effects of CO ₂ on seagrass production may be spatially heterogeneous and depend on the specific nitrogen availability of each system. Under a CO ₂ increase scenario, the natural levels of nutrients will probably become limiting for Z. noltii. This potential limitation becomes more relevant because the expected positive effect of CO ₂ increase on nitrate uptake rate was not confirmed.     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.     

Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes

Antibiotic production in Streptomyces can often be increased by introducing heterologous genes into strains that contain an antibiotic biosynthesis gene cluster. A number of genes are known to be useful for this purpose. We chose three such genes and cloned them singly or in combination under the control of the strong constitutive ermE* promoter into a ?C31-derived integrating vector that can be transferred efficiently by conjugation from Escherichia coli to Streptomyces. The three genes are adpA, a global regulator from Streptomyces coelicolor, metK, encoding S-adenosylmethionine synthetase from S. coelicolor, and, VHbS, hemoglobin from Vitreoscilla. The substitutions with GC in VHbS was intended to convert codons from lower usage to higher, yet causing no change to the encoded amino acid. Plasmids containing either one of these genes or genes in various combinations were introduced into Streptomyces sp. FR-008, which produces the macrolide antibiotic FR-008-III (also known as candicidin D). The largest increase in FR-008-III production was achieved by the plasmid containing all three genes. This plasmid also increased avermectin production in Streptomyces avermitilis, and is likely to be generally useful for improving antibiotic production in Streptomyces.     

Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4)

Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3‐year‐old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild‐type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid‐insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild‐type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production.     

Glutamine‐fueled mitochondrial metabolism is decoupled from glycolysis in melanoma

In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not ‘dysfunction’ but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non‐conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. Biosynthetic networks linked with non‐essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。