Evaluation of Antibiotic Biodegradation by a Versatile and Highly Active Recombinant Laccase from the Thermoalkaliphilic Bacterium Bacillus sp. FNT.

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, ß-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20-90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL-1) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation.

Rational Design of an α-1,3-Fucosyltransferase for the Biosynthesis of 3-Fucosyllactose in Bacillus subtilis ATCC 6051a via De Novo GDP-l-Fucose Pathway.

3-Fucosyllactose (3-FL) is an important oligosaccharide and nutrient in breast milk that can be synthesized in microbial cells by α-1,3-fucosyltransferase (α-1,3-FucT) using guanosine 5'-diphosphate (GDP)-l-fucose and lactose as substrates. However, the catalytic efficiency of known α-1,3-FucTs from various sources was limited due to their low solubility. To enhance the microbial production of 3-FL, the efficiencies of α-1,3-FucTs were evaluated and in Bacillus subtilis (B. subtilis) chassis cells that had been endowed with a heterologous synthetic pathway for GDP-l-fucose, revealing that the activity of FucTa from Helicobacter pylori (H. pylori) was higher than that of any of other reported homologues. To further improve the catalytic performance of FucTa, a rational design approach was employed, involving intracellular evaluation of the mutational sites of M32 obtained through directed evolution, analysis of the ligand binding site diversity, and protein structure simulation. Among the obtained variants, the FucTa-Y218 K variant exhibited the highest 3-FL yield, reaching 7.55 g/L in the shake flask growth experiment, which was 3.48-fold higher than that achieved by the wild-type enzyme. Subsequent fermentation optimization in a 5 L bioreactor resulted in a remarkable 3-FL production of 36.98 g/L, highlighting the great prospects of the designed enzyme and the strains for industrial applications.

Phytosterol conversion into C9 non-hydroxylated derivatives through gene regulation in Mycobacterium fortuitum.

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17ß-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

Functional analysis of acyl-CoA dehydrogenases and their application to C22 steroid production.

Acyl-CoA dehydrogenase (ChsE) is involved in the steroid side-chain degradation process. However, their function in vivo remains unclear. In this study, three ChsE, ChsE1-ChsE2, ChsE3, and ChsE4-ChsE5, were identified in Mycolicibacterium neoaurum, and their functions in vivo are studied and compared with those from Mycobacterium tuberculosis in vitro. By gene knockout, complementation, and the bioconversion of phytosterols, the function of ChsE was elucidated that ChsE4-ChsE5 could utilize C27, C24, and C22 steroids in vivo. ChsE3 could utilize C27 and C24 steroids in vivo. ChsE1-ChsE2 could utilize C27, C24, and C22 steroids in vivo. What is more, the production strain of a C22 steroid, 3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (PDCE), is constructed with ChsE overexpression. This study improved the understanding of the steroid bioconversion pathway and proposed a method of the production of a new C22 steroid. KEY POINTS: • Three ChsE paralogs from M. neoaurum are identified and studied. • The function of ChsE is overlapped in vivo. • A C22 steroid (PDCE) producer was constructed with ChsE overexpression.

Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1-dehydrogenase deletions and multiple genetic modifications in Mycobacterium fortuitum.

BACKGROUND: 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1­dehydrogenation and regulating metabolic flux. RESULTS: Five 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ­dihydroxy­20­methyl­pregna­4­en­3­one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9­OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol. CONCLUSIONS: KstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

Rational design of GDP­D­mannose mannosyl hydrolase for microbial L­fucose production.

BACKGROUND: L­Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5'­diphosphate (GDP)­L­fucose. Therefore, a more direct approach for biomanufacturing L­fucose could be the enzymatic degradation of GDP­L­fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP­L­fucose. RESULTS: Herein, we constructed a de novo L­fucose synthetic route in Bacillus subtilis by introducing heterologous GDP­L­fucose synthesis pathway and engineering GDP­mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP­L­fucose, therefore, a substrate simulation­based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP­L­fucose­splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield L­fucose. WcaHR36Y/N38R was found to produce 1.6 g/L L­fucose during shake­flask growth, which was 67.3% higher than that achieved by wild­type WcaH. The accumulated L­fucose concentration in a 5 L bioreactor reached 6.4 g/L. CONCLUSIONS: In this study, we established a novel microbial engineering platform for the fermentation production of L­fucose. Additionally, we found an efficient GDP­mannose mannosyl hydrolase mutant for L­fucose biosynthesis that directly hydrolyzes GDP­L­fucose. The engineered strain system established in this study is expected to provide new solutions for L­fucose or its high value­added derivatives production.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17ß-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

Dynamic Microstructure Assembly Driven by Lysinibacillus sp. LF-N1 and Penicillium oxalicum DH-1 Inoculants Corresponds to Composting Performance.

The effects of Lysinibacillus sp. LF-N1 and Penicillium oxalicum DH-1 inoculants (LFPO group) on compost succession and the microbial dynamic structure of co-composting wheat straw and cow manure composting were investigated. The inoculants contributed to longer thermophilic stages, higher temperatures (62.8 °C) and lower microbial diversity in the LFPO treatment compared to the control group (CK). Moreover, LFPO inoculation increased the germination index and accelerated organic matter and lignocellulose degradation in the compost. Microbial analysis confirmed that the inoculants effectively altered the microbial communities. The predominant biomarkers for bacteria and fungi in inoculated compost were members of Lysinibacillus and Penicillium, respectively. Functional prediction showed greater lignocellulose degradation and less pathogen accumulation in the LFPO group. The cooccurrence network analysis showed that the network structure in LFPO compost was greatly simplified compared to that in CK. Bacterial cluster A was dominated by Lysinibacillus, and fungal cluster B was represented by Penicillium, which were significantly correlated with temperature and lignocellulose degradation, respectively (p < 0.05). These results demonstrated that the LF-N1 and DH-1 inoculants drove the bacterial and fungal assemblies to induce physicochemical property changes during cocomposting.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Quantum Monte Carlo study of topological phases on a spin analogue of Benalcazar-Bernevig-Hughes model.

We study the higher-order topological spin phases based on a spin analogue of Benalcazar-Bernevig-Hughes model in two dimensions using large-scale quantum Monte Carlo simulations. A continuous Néel-valence bond solid quantum phase transition is revealed by tuning the ratio between dimerized spin couplings, namely, the weak and strong exchange couplings. Through the finite-size scaling analysis, we identify the phase critical points, and consequently, map out the full phase diagrams in related parameter spaces. Particularly, we find that the valence bond solid phase can be a higher-order topological spin phase, which has a gap for spin excitations in the bulk while demonstrates characteristic gapless spin modes at corners of open lattices. We further discuss the connection between the higher-order topological spin phases and the electronic correlated higher-order phases, and find both of them possess gapless spin corner modes that are protected by higher-order topology. Our result exemplifies higher-order physics in the correlated spin systems and will contribute to further understandings of the many-body higher-order topological phenomena.

Engineering Bacillus subtilis ATCC 6051a for the production of recombinant catalases.

Catalases are a large group of enzymes that decompose hydrogen peroxide to oxygen and hydrogen, and have been applied widely in numerous areas. Bacillus subtilis ATCC 6051a is a well-known host strain for high level secretion of heterologous peptides. However, the application of 6051a was seriously hampered by insufficient transformation efficiency. In this study, D-xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, generating 164S, a mutant owns a transformation efficiency of 1 000-fold higher than its parent strain, thus allowing gene replacement by double crossover recombination using linear dsDNAs. The efficiency of the flanking arms for homologous recombination was then analyzed. We found that 400 bp was the minimal length of homologous fragments required to initiate efficient recombination in the 164S strain. In addition, DNA cassettes encoding two mesophilic catalases (Orf 2-62 and Orf 2-63) from B. licheniformis were integrated onto 164S. The catalytic properties of recombinant Orf 2-62 and Orf 2-63 were analyzed, and were found to be predominantly secreted into the fermentation broth, although they obviously lack any known secretory signal peptide. This work demonstrated that B. subtilis 164S is an excellent cell tool, not only for its superior secretion capacity, but also for its convenience in genetic modification.

A wheat bran inducible expression system for the efficient production of α-L-arabinofuranosidase in Bacillus subtilis.

α-l-arabinofuranosidases (EC 3.2.1.55; AFs) cause the release of arabinosyl residues from hemicellulose polymers such as xylans, and are receiving increased levels of research attention as they could be applied in a range of processes that involve the enzymatic degradation of xylans. The secretory production of bacterial AFs has not been attempted previously. In this study, we designed a unique induction system for the production of a recombinant AF in Bacillus subtilis in order to exploit its enzymic degradation of wheat bran. We found that non-starch phytochemicals were more efficient than d-xylose when inducing the expression of T7 RNA polymerase and driving the transcription of AF by the T7 promoter. The host cell, B. subtilis (ATCC 6051a-derived strain 164T7P) was engineered to incorporate a DNA cassette that expressed T7 RNA polymerase under the control of a d-xylose inducible promoter (PxylA). The T7 promoter engineered into 164T7P was initially tested and compared with P43 in terms of GFP expression; we found that the expression level of GFP by the T7 promoter was ten-fold higher than that achieved by P43. When cultured in a flask with gentle shaking, and with d-xylose as an inducer, the recombinant strain successfully expressed arbf, a family 51 (GH 51) glycoside hydrolase from Bacillus licheniformis, and secreted 141.4 ±â€¯4.8 U/mL of enzyme, with a Km of 1.4 ±â€¯0.1 mM and a kcat of 139.4 s-1. However, the protein was devoid of a secretary signal peptide. When cultures were supplemented with wheat bran, the maximal yield of the secreted AF reached 194.8 ±â€¯4.1 U/mL. The results provide a foundation for the high level production of heterologous proteins using wheat bran as the inducer in B. subtilis.

A novel and highly active recombinant spore-coat bacterial laccase, able to rapidly biodecolorize azo, triarylmethane and anthraquinonic dyestuffs.

Laccases are enzymes able to catalyze the oxidation of a wide array of phenolic and non-phenolic compounds using oxygen as co-substrate and releasing water as by-product. They are well known to have wide substrate specificity and in recent years, have gained great biotechnological importance. To date, most well studied laccases are from fungal and mesophilic origin, however, enzymes from extremophiles possess an even greater potential to withstand the extreme conditions present in many industrial processes. This research work presents the heterologous production and characterization of a novel laccase from a thermoalkaliphilic bacterium isolated from a hot spring in a geothermal site. This recombinant enzyme exhibits remarkably high specific activity (>450,000 U/mg) at 70 °C, pH 6.0, using syringaldazine substrate, it is active in a wide range of temperature (20-90 °C) and maintains over 60% of its activity after 2 h at 60 °C. Furthermore, this novel spore-coat laccase is able to biodecolorize eight structurally different recalcitrant synthetic dyes (Congo red, methyl orange, methyl red, Coomassie brilliant blue R250, bromophenol blue, malachite green, crystal violet and Remazol brilliant blue R), in just 30 min at 40 °C in the presence of the natural redox mediator acetosyringone.

[Microbial Community Succession in Industrial Composting with Livestock Manure and Peach Branches and Relations with Environmental Factors].

This study sets out to understand the evolution of the microbial community structure in industrial composting with livestock manure and peach branches. Pig manure, peach branches, and decomposed organic fertilizer were used as materials for composting. Changes in physical and chemical indicators and the evolution in the structure of the compost microbial community, determined by high-throughput sequencing, were analyzed. The results of physical and chemical parameters show that the pile reached the high-temperature stage on day 2, and the thermophilic period lasted for 30 days. The changes in total carbon were volatile, and there was an overall decline in the amount of TOC in the whole process of composting; The final content of TN was 20.58 g·kg-1, which was 5.90% lower compared to the initial compost. Alpha analysis indicated that a different microbial community diversity existed at different times during aerobic composting periods. At the bacterial phyla level, Firmicutes and Actinobacteria were the dominant phyla, and the proportion of relative abundance were 79.31%-95.09% and 2.98%-19.70%, respectively, in the entire compost. The relative abundance of Firmicutes and Actinobacteria were 87.36% and 9.66%, respectively, and their respective relative abundances were 79.38% and 19.70% at the end of composting. At the bacterial genus level, the dominant group changed from Clostridium_sensu_stricto_1, Terrisporobacter, and Bacillus to norank_f_Bacillaceae, Bacillus, Oceanbacillus, and Pseudogracilibacillus; Regarding the fungus phyla, the Ascomycota was the dominant phylum. For the fungus genus, the relative abundance of norank_c_Sordariomycetes gradually increased during composting, and finally was predominant group. The redundancy analysis (RDA) showed that the correlation rank between environmental factors and microbial community structure was:pH > NH4+-N > T > TOC > TN, where pH had the greatest impact on the microbial community composition. norank_c_Sordariomycetes, norank_o_Sordariales, and norank_c_Agaricomycetes may be related to the volatilization of ammonium nitrogen.

Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.

Isolation and characterization of violacein from an Antarctic Iodobacter: a non-pathogenic psychrotolerant microorganism.

Violacein is an intensely purple pigment synthesized by various genera of bacteria that has been discovered to have a wide range of interesting biological activities which range from anticarcinogenic to antibacterial. One of the hindrances for its real-life application is that the first microorganisms found to produce the compound may act as opportunistic pathogens. Here, we report the isolation and characterization of violacein from a non-pathogenic Antarctic Iodobacter strain. Its anti-microbial properties were also tested. The method proposed here for the purification of violacein shows high yields, indicating that this Antarctic microorganism could be a valuable source for this important pigment. This is the first characterization of violacein from an Antarctic Iodobacter strain and here we also present a viable method to obtain this pigment for potential biotechnological applications.

Activation of colanic acid biosynthesis linked to heterologous expression of the polyhydroxybutyrate pathway in Escherichia coli.

The biosynthesis of colanic acid (CA) in Escherichia coli was known to be activated during growth at low temperature using sub-optimal medium. However, in this study, an E. coli transformant S173-H (S17-3 with plasmid pBhya-CAB) was found to be able to excrete high amount of CA (10.39 g/L) in glucose supplemented Luria-Bertani medium (LBG) when growing at 37 °C. Inoculation of cells in low pH medium was required for the derepression of the CA regulon, another indispensable requirement was the use of high copy number plasmid for over-expression of the heterologous polyhydroxybutyrate (PHB) biosynthesis pathway in S17-3. In addition, S173-H exhibited superior growth performance in LBG, the maximal cell density (OD600) of cultures reached 40.0, far exceeding that of any known E. coli strains cultivated under similar conditions. Genomic data mining and transcriptional analysis hinted that the persistent growth or CA production might be modulated by interplaying regulation networks that signal the level of messenger substrate, acetyl-CoA or acetylphosphate. Depletion of these messenger substrates may be triggered by efficient PHB biosynthesis that links to enhanced capability in NADPH regeneration in S17-3, due to mutations on loci at pgi, csrA, or other sites.

A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2 ). Here, the authors expressed the four αßγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons.

BACKGROUND: 3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation. RESULTS: The recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg-1, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L-1 AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L-1 phytosterols to 14.18 g L-1 4HP. CONCLUSIONS: In vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate.