Weitao Geng

Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes.

A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillus amyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2g/L (NH(4))(2)SO(4), 0.6g/L MgSO(4), and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.

Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid

Bacillus amyloliquefaciens is one of most prevalent Gram-positive aerobic spore-forming bacteria with the ability to synthesize polysaccharides and polypeptides. Here, we report the complete genome sequence of B. amyloliquefaciens LL3, which was isolated from fermented food and presents the glutamic acid-independent production of poly-γ-glutamic acid.

Phase morphology, physical properties, and biodegradation behavior of novel PLA/PHBHHx blends.

In this study, two biodegradable polyesters [i.e., polylactic acid (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)] with complementarity in terms of mechanical performance have been combined, and a series of blends with a broad range of compositions has been prepared by thermal compounding. The evolution of phase morphologies with the variation of compositions has been characterized by using Fourier transform infrared spectroscopic imaging together with scanning electron microscope analyses. Thermal, mechanical, and biodegradation properties of the PLA/PHBHHx blends were systematically investigated. Mechanical properties were further analyzed by using theoretical models and correlated with the results of the morphology/structure and compatibility of the blends. Results indicate that PLA/PHBHHx blends are immiscible but can be compatible to some extent at certain compositions (e.g., PLA/PHBHHx (w/w) = 80/20 and 20/80). The physical properties of the blend could be fine tuned by adjusting the blend composition.

Introduction of Environmentally Degradable Parameters to Evaluate the Biodegradability of Biodegradable Polymers

Environmentally Degradable Parameter (Ed K) is of importance in the describing of biodegradability of environmentally biodegradable polymers (BDPs). In this study, a concept Ed K was introduced. A test procedure of using the ISO 14852 method and detecting the evolved carbon dioxide as an analytical parameter was developed, and the calculated Ed K was used as an indicator for the ultimate biodegradability of materials. Starch and polyethylene used as reference materials were defined as the Ed K values of 100 and 0, respectively. Natural soil samples were inoculated into bioreactors, followed by determining the rates of biodegradation of the reference materials and 15 commercial BDPs over a 2-week test period. Finally, a formula was deduced to calculate the value of Ed K for each material. The Ed K values of the tested materials have a positive correlation to their biodegradation rates in the simulated soil environment, and they indicated the relative biodegradation rate of each material among all the tested materials. Therefore, the Ed K was shown to be a reliable indicator for quantitatively evaluating the potential biodegradability of BDPs in the natural environment.

Short-cut nitrification in biological aerated filters with modified zeolite and nitrifying sludge.

Autotrophic domestication for short-cut nitrification (SCN) and microorganism immobilization was carried out in a lab-scale biological aerated filters (BAFs) system with activated sludge. Zeolite was chosen as fillings and modified to enlarge the specific surface and to remove toxic metal ions. After thirty-day domestication and immobilization, the NH4(+)-N removal capacity increased to 76.51 mg/g dry sludge (DS) and the ratio of NO2(-)-N converted from NH4(+)-N reached to 91.2% finally. The analysis of growth kinetics indicated that free ammonia should be the key factor for SCN. The abundance variation of nitrifiers, measured by qPCR, showed that AOB was enriched successfully and NOB was washed out. The results also showed that modified zeolite should be more beneficial to the specific immobilization of AOB than natural zeolite. The shift in the community structure of AOB during the domestication by DGGE profile was investigated.

Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans.

ε‐Poly‐L‐lysine (ε‐PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε‐PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε‐PL in 30 l fed‐batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε‐PL was determined by nuclear magnetic resonance (NMR) and matrix‐assisted laser desorption/ionization–time of flight mass spectrometry (MALDI‐TOF MS). Previous studies have shown that the antimicrobial activity of ε‐PL is dependent on its molecular size. In this study, the polymerization degree of the ε‐PL produced by strain NK660 ranged from 19 to 33 L‐lysine monomers, with the main component consisting of 24–30 L‐lysine monomers, which implied that the ε‐PL might have higher antimicrobial activity. Furthermore, the ε‐PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε‐PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε‐PL synthesis.

Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity

The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.

Augmented production of alginate oligosaccharides by the Pseudomonas mendocina NK-01 mutant

Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHA(MCL) synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30 L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight (M(W)). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHA(MCL) and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.

Genome Sequence of the ε-Poly-l-Lysine-Producing Strain Streptomyces albulus NK660, Isolated from Soil in Gutian, Fujian Province, China

ABSTRACT We determined the complete genome sequence of a soil bacterium, Streptomyces albulus NK660. It can produce ε-poly-l-lysine, which has antimicrobial activity against a spectrum of microorganisms. The genome of S. albulus NK660 contains a 9,360,281-bp linear chromosome and a 12,120-bp linear plasmid.