Applied Microbiology and Biotechnology

Synthesis of silver nanoparticles and its contribution to the capability of Bacillus subtilis to deal with polluted waters Abstract Bacillus subtilis widely exists in environment and shows a capability to deal with heavy metals and dyes in polluted waters by adsorption or biological oxidation and reduction. Little is known about the roles of lipopeptides in this capability of B. subtilis. In this study, we found that the lipopeptides produced by B. subtilis could reduce silver ions to silver nanoparticles (AgNPs) and iturin was identified as the major effective fraction. Furthermore, the synthesized AgNPs was successfully used to catalyze the reduction of organic dyes and reduce Pb2+ contamination in water. The formation of AgNPs was confirmed by the features analyzed by UV-vis spectroscopy, dynamic light scattering, high-resolution transmission electron microscopy (HR-TEM), and selected area electron diffraction (SAED). The formed AgNPs showed crystalline, with small size (~ 20 nm) and spherical shape. The biosynthesis of AgNPs was significantly accelerated by UV irradiation. A pH of 10 resulted in the highest formation rate, while pH 9.2 provided the most stability of AgNPs. In mechanisms, tyrosine and the polypeptide were identified as the major groups in iturin-A to form AgNPs via Ar–OH groups. The study revealed that iturin played important roles for the capability of B. subtilis to treat polluted water via a possible way by synthesizing AgNPs and then catalyzing the reduction of organic dyes and reducing the contamination of Pb2+.

Secreted protein MoHrip2 is required for full virulence of Magnaporthe oryzae and modulation of rice immunity Abstract MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What’s more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.

Effect of SDS on release of intracellular pneumocandin B 0 in extractive batch fermentation of Glarea lozoyensis Abstract Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelia of Glarea lozoyensis and inhibits fungal 1,3-β-glucan synthase. Extractive batch fermentation can promote the release of intracellular secondary metabolites into the fermentation broth and is often used in industry. The addition of extractants has been proven as an effective method to attain higher accumulation of hydrophobic secondary metabolites and circumvent troublesome solvent extraction. Various extractants exerted significant but different influences on the biomass and pneumocandin B0 yields. The maximum pneumocandin B0 yield (2528.67 mg/L) and highest extracellular pneumocandin B0 yield (580.33 mg/L) were achieved when 1.0 g/L SDS was added on the 13th day of extractive batch fermentation, corresponding to significant increases of 37.63 and 154% compared with the conventional batch fermentation, respectively. The mechanism behind this phenomenon is partly attributed to the release of intracellular pneumocandin B0 into the fermentation broth and the enhanced biosynthesis of pneumocandin B0 in the mycelia.

Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner Abstract Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.

Prophylactic effects of probiotic Bifidobacterium spp. in the resolution of inflammation in arthritic rats Abstract In the present study, the modulatory effects of bifidobacterial spp. (Bifidobacterium breve NCIM 5671, Bifidobacterium longum NCIM 5672 and Bifidobacterium bifidum NCIM 5697) on adjuvant induced arthritis in rats were evaluated. Arthritis was induced in male Wistar rats by injecting 250 μg of Freund’s adjuvant directly into the paw. Fifteen days before and 15 days after the induction of arthritis, suspended cultures of bifidobacteria (109 cfu/ml) were administered by oral gavage. Paw volume, bone mineral content, oxidative stress markers, antioxidant enzymes, cytokines, eicosanoids and expression of COX2, as well as bone hydrolytic enzymes, were assessed by RT PCR. Although piroxicam-treated groups (drug control) had better effects than bifidobacteria-treated groups, bifidobacteria probiotics administration exhibited significant (P < 0.05) prophylactic effects in terms of downregulating arthritis markers. Parameters including paw volume, bone mineral content, cytokines, and eicosanoids level were significantly (p < 0.05) modulated in bifidobacteria administered groups compared to arthritic control group. Among the three strains tested, B. breve NCIM 5671 exhibited superior prophylactic effects as assessed in the experimental rat model of arthritis. In conclusion, bifidobacteria probiotics administration can downregulate the markers of arthritis and hence can be a potential therapeutic regimen in the treatment of arthritis.

Key sites insight on the stereoselectivity of four mined aldo-keto reductases toward α-keto esters and halogen-substituted acetophenones Abstract Biocatalytic reduction catalyzed by aldo-keto reductases (AKRs) is a valuable approach for asymmetric synthesis of chiral alcohols. In this study, four novel aldo-keto reductases with significant activity and stereoselectivity toward a variety of α-keto esters and halogen-substituted acetophenones were identified by genome mining. Through analysis of the crystal structure and multiple-sequence alignment of the starting AKR YvgN from Bacillus subtilis, residues F25 and W113 were proposed as the key positions that might control the stereoselectivity of YvgN. F25S and F25S/W113F variants of YvgN were able to improve its activity and stereoselectivity toward some α-keto ester compounds and halogen-substituted acetophenone derivatives. In addition, similar enhancement of catalytic activity and stereoselectivity was also found in the other three AKRs with corresponding mutations of starting YvgN.

The substrate specificity of aniline dioxygenase is mainly determined by two of its components: glutamine synthetase-like enzyme and oxygenase Abstract The residues of aniline and its derivatives are serious environment pollutants. Aniline dioxygenase (AD) derived from aerobic bacteria catalyzes the conversion of aniline to catechol, which has potential use in the bioremediation of aromatic amines and biorefining process. AD contains four components: a glutamine synthetase (GS)-like enzyme, a glutamine amidotransferase (GAT)-like enzyme, oxygenase, and reductase. ADs from diverse hosts exhibit different substrate specificities against aniline derivatives. However, what component of AD determines AD’s substrate specificity is still unknown which limits the effects of extending AD’s substrate spectrum through mutagenesis. Here, each component of two ADs (AtdA1A2A3A4A5 and AdoQTA1A2B) which have different substrate ranges was heterologously expressed and purified. The activity of both ADs was successfully constructed in vitro using the purified components. To identify the component that affects the substrate specificity of the ADs, the substrate specificity of each component was studied. The inability of AtdA1A2A3A4A5 to catalyze 4-methylaniline was determined with GS-like enzyme AtdA1; its inability to convert 2-isopropylaniline was caused by the oxygenase component, and its inability to convert 4-isopropylaniline was caused by both GS-like enzyme AtdA1 and oxygenase components. The inability of AdoQTA1A2B to catalyze 2-methylaniline was determined by GS-like enzyme AdoQ; its inability to convert 2-isopropylaniline was caused by both GS-like enzyme AdoQ and oxygenase components. Together, these results show that GS-like enzyme and oxygenase but not GAT-like enzyme or reductase play dominant roles in the substrate specificity of AD, and this finding will facilitate the engineering of AD to expand its substrate range.

Metabolism of sucrose in a non-fermentative Escherichia coli under oxygen limitation Abstract Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.

Biosynthesis of D-danshensu from L-DOPA using engineered Escherichia coli whole cells Abstract D-Danshensu (D-DSS), a traditional Chinese medicine, is used to treat cardiovascular and cerebrovascular diseases. However, current isolation protocols for D-DSS both natural and synthetic are not ideal; therefore, in this study, we have developed a whole-cell biotransformation method to produce D-DSS from L-DOPA. This was done by co-expressing L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD), and glucose dehydrogenase (gdh). To begin to optimize the production of D-DSS, varying copy number plasmids were used to express each of the required genes. The resulting strain, Escherichia coli ALG7, which strongly overexpressed aadL, ldhD, and weakly overexpressed gdh, yielded a 378% increase in D-DSS production compared to E. coli ALG1. Furthermore, the optimal reaction conditions for the production of D-DSS were found to be a pH of 7.5, temperature at 35 °C, and 50 g/L wet cells for 12 h. Under these optimized conditions, the D-DSS amount achieved 119.1 mM with an excellent ee (> 99.9%) and a productivity of 9.9 mM/h.

Biological nitrogen removal from wastewater using sulphur-driven autotrophic denitrification Abstract Biological denitrification process in mainstream wastewater treatment often needs dosing supplemental electrons, consequently adding a remarkable operating cost. Organic carbon compounds are nowadays the most intensively used electron sources in full-scale wastewater treatment, corresponding with the well-understood carbon-nitrogen biogeochemistry for heterotrophic denitrification process. In the twenty-first century, the low-carbon technology is on calling to reduce the carbon footprint and relieve climate changing threatens. Autotrophic denitrification is highly recommended for mainstream wastewater treatment. The reduced-sulphur compounds (such as sulphide, elemental sulphur, and thiosulphate) could be utilised as electron donors, to drive sulphur cycle reactions to reduce nitrate and nitrite to dinitrogen gas. Based on the literature review and our own research experiences, this paper presents our perspectives on sulphur-driven autotrophic denitrification. It particularly focuses on the functional enzymes, sulphur bioreactors, and influential operating factors. Overall, this paper provides new insights on sulphur-nitrogen biogeochemistry and application as a low-carbon technology for nitrogen removal during municipal wastewater treatment.