Bacterial glycogen and plant starch biosynthesis

In aerobic granular sludge (AGS) system, flocs typically coexist with granules. The inherent differences between flocs and granules can result in their different behaviors in the same reactor. In this study, a flocs/granules co-existence system was investigated and differences between the flocs and granules in co-existence system were compared on proteomic level, providing an in-depth insight into co-existence of flocs and granules. Results showed that the co-existence system obtained under 4.5 kg COD m⁻³ d^-1 exhibited excellent nutrient removal (COD: 96–97 %, TN: 98–99 %) with approximately 9–11 % flocs. A comparison of flocs and granules in the co-existence system showed different functional bacteria in flocs and granules. The dominant bacteria in granules was Candidatus_Competibacter, while Azoarcus and Thauera were enriched in flocs. Furthermore, proteomic analysis suggested that the metabolic pathways associated with glycogen synthesis and denitrification were more active in granules than that in flocs, while the differentially expressed proteins (DEPs) related to aromatic compounds and xenobiotics metabolism in flocs were more abundant than that in granules. Species annotation revealed that the DEPs between granules and flocs were mainly contributed by Candidatus_Competibacter and Thauera. The in-depth insight into co-existence of flocs and granules provides important reference for the design, optimization and operation of AGS technology in further applications.

Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4–3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4–3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids β-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4–3 overexpress different proteins related to aromatic compounds catabolism and with the p-nitrophenol  degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroquinone dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4–3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.

The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 10⁵ Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.

Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting V_max values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S_0.5 values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A_0.5 ∼0.3 mM) and Pi and ADP inhibition (I_0.5 between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria.

Cyanobacteria were the first organisms to use oxygenic photosynthesis for converting carbon dioxide into useful organic chemicals. However, the chemical industry has historically relied on fossil raw materials to produce organic precursors and this has highly contributed to the current alarming levels of atmospheric CO₂. Thus, cyanobacteria have emerged as an alternative to couple solar energy with biotechnological production. This review presents the potential application of cyanobacteria as solar biofactories of valuable chemicals. Here, the photoautotrophic metabolism is described as an integrated production process, in which fixated CO₂ and N₂ are essential raw materials processed through primary and secondary metabolic reactions powered by light-derived chemical energy. Large scale application of cyanobacterial biofactories is still a technical challenge due to low yields and commoditization of biotechnological products. However, the development of feasible cyanobacterial biofactories can be enhanced through synthetic biology efforts relying on the natural ability of cyanobacteria to synthesize carbohydrates and peptides. Among these, sugars, phycobiliproteins, cyanophycin and diverse bioactive secondary metabolites are important from an economic point of view because they can be used as raw materials for specialty organic chemicals and drugs.

Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a V_max of 3350 U.mg⁻¹. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.