Peihong Shen

Characterization of a novel β-glucosidase-like activity from a soil metagenome

We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed d-glucosyl-β-(l–4)-d-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-d-glucoside as the substrate. A CaCl2 concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent Km value of 0.19 mM, a Vmax value of 4.75 U/mg and a kcat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

BackgroundFumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.ResultsA plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s.ConclusionsWe isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.

Changes in microbial community structure in two anaerobic systems to treat bagasse spraying wastewater with and without addition of molasses alcohol wastewater.

This study investigates the microbial community and structure in the internal circulation (IC) reactors that treat wastewater from bagasse spraying, with (reactor B) and without (reactor A) addition of molasses alcohol wastewater (MAW). The V3 regions in the 16S rRNA of bacteria were sequenced using illumina sequencing to characterize the microbial community structures. The results showed that there were approximately 34.8% more microorganisms were reduced, while the proportions of the three most predominant bacterial populations especially some sulfate-reducing bacteria increased in reactor B. The archaeal community composition was measured by PCR-DGGE (denaturing gradient gel electrophoresis) analysis and sequencing some clones from the 16S rRNA gene library. The results showed that numerous, mostly uncharacterized, archaeal genera are present in reactors A and B; the genus Methanomethylovorans was only detected in the samples that received MAW. This study demonstrated the significant effect of MAW on microbial communities in the wastewater treatment bioreactor.

Using pig manure to promote fermentation of sugarcane molasses alcohol wastewater and its effects on microbial community structure

Molasses alcohol wastewater (MAW) is difficult to be bio-treated and converted into biogas. In this study, MAW mixed with pig manure (PM) in different ratios was co-digested. Biogas production, chemical oxygen demand (COD) removal and the structure of microbial communities were monitored in the process. Our results showed that under the optimal COD ratio of PM:MAW (1.0:1.5), CODremoval and biogas yield were the highest. And in fermentation tanks with different PM to MAW ratios, the structure and composition of bacterial communities varied in the early and late stage. Furthermore, the type of main bacterial operational taxonomic units (OTUs) have no differences, yet the relative abundance of OTUs varied. The current research showed that there was a good potential to the use of PM as a co-digested material to anaerobic treatment of MAW and provided references for further improving bio-treatment of MAW.

Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome

A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with Ki values of around 1.79 × 10−8 and 1.52 × 10−8 M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0–8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.

Overexpression of the methanol dehydrogenase gene mxaF in Methylobacterium sp. MB200 enhances L-serine production.

Increase in L‐serine production is of interest for industry. Here, we describe a metabolic engineering approach to increase the production of L‐serine in a methylotrophic bacterium. mxaF, the gene encoding the large subunit of a methanol dehydrogenase, was cloned from Methylobacterium sp. MB200 through transposon mutagenesis. Deletion of mxaF gene prevented the strain to grow on methanol, suggesting that mxaF is involved in methanol metabolism. Overexpression of mxaF gene in the strain MB200 resulted in a fivefold increase in methanol dehydrogenase activity compared to the wild‐type. Resting cell assays showed that the recombinant strain accumulated 6·6 mg ml−1 L‐serine in 72 h with 30 mg ml−1 wet cells from 50 mg ml−1 glycine and 50 mg ml−1 methanol, representing a 1·5‐fold increment for L‐serine production in contrast to the wild‐type strain. These results demonstrate that the potential for improving the production of L‐serine can be achieved by overexpressing mxaF gene in methylotrophic bacteria.

Screening of Burkholderia sp. WGB31 producing anisic acid from anethole and optimization of fermentation conditions

Anisic acid, the precursor of a variety of food flavors and industrial raw materials, can be bioconversed from anethole which extracted from star anise fruits. WGB31 strain with anisic acid molar production rate of 10.25% was isolated and identified as Burkholderia sp. Three significant influential factors, namely, glucose concentration, initial pH value, and medium volume were selected and their effects were evaluated by Box–Behnken Design (BBD). Regression analysis was performed to determine response surface methodology and the significance was tested to obtain the process model of optimal conditions for producing anisic acid. The fermentation conditions at the stable point of the model were obtained: glucose 6 g L−1, pH 6.2, culture medium volume 61 mL in a triangular flask with 250 ml volume. Verification test indicated that the production rate of anisic acid was 30.7%, which was three times of that before optimizing. The results provide a basis and reference for producing anisic acid by microbial transformation.

Deletion of gene gnd encoding 6-phosphogluconate dehydrogenase promotes l-serine biosynthesis in a genetically engineered strain of Methylobacterium sp. MB200

ObjectiveTo identify potential target genes involved in l-serine biosynthesis in Methylobacterium sp. MB200 and to evaluate the gnd genetically-engineered strains for l-serine production.ResultsFive genes that are not associated with the central metabolic pathway but with l-serine biosynthesis were identified from Methylobacterium sp. MB200 mutants. Gene gnd, encoding 6-phosphogluconate dehydrogenase (PGDH), was selected for further evaluation. The gnd deletion mutant showed a 600% increase in d-serine tolerance and an 80% decrease in PGDH activity compared to Methylobacterium sp. MB200. gnd over-expression did not affect d-serine tolerance, whereas it did increase enzyme-activity up to 136%. Additionally, analysis revealed that in Methylobacterium sp. MB200, l-serine inhibited PGDH activity. The deletion of gnd did not affect growth, whereas it did enhance the biosynthesis of l-serine, resulting in a 225% increase in production of l-serine compared to the wild-type.Conclusiongnd, one of the five genes identified here that is associated with l-serine synthesis, can be developed as a potential candidate for metabolic engineering to promote l-serine synthesis in Methylobacterium sp. MB200.

D-Amino Acid Oxidase and Metagenomics

D-amino acid oxidase (DAAO, EC 1. 4. 3. 3) converts D-amino acid to a corresponding α-keto acid via deamination. D-amino acid oxidase is one of the most important enzymes responsible for maintenance proper level of D-amino acids, which play a key role in regulation of many processes in living cells. This paper summary the applications of D-amino acid oxidase in agricultural and industry practices, as the target in human disease treatments, etc. Most important of all, we demonstrate the importance of metagenomic library in exploring the novel D-amino acid oxidase from the environmental microbiology to optimize their applications.