A Comparative Transcriptome Analysis Unveils the Mechanisms of Response in Feather Degradation by Pseudomonas aeruginosa Gxun-7.

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT-PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming.

Comparative Transcriptome Analysis Reveals the Molecular Mechanisms of Acetic Acid Reduction by Adding NaHSO3 in Actinobacillus succinogenes GXAS137.

Acetic acid (AC) is a major by-product from fermentation processes for producing succinic acid (SA) using Actinobacillus succinogenes. Previous experiments have demonstrated that sodium bisulfate (NaHSO3) can significantly decrease AC production by A. succinogenes GXAS137 during SA fermentation. However, the mechanism of AC reduction is poorly understood. In this study, the transcriptional profiles of the strain were compared through Illumina RNA-seq to identify differentially expressed genes (DEGs). A total of 210 DEGs were identified by expression analysis: 83 and 127 genes up-regulated and down-regulated, respectively, in response to NaHSO3 treatment. The functional annotation analysis of DEGs showed that the genes were mainly involved in carbohydrates, inorganic ions, amino acid transport, metabolism, and energy production and conversion. The mechanisms of AC reduction might be related to two aspects: (i) the lipoic acid synthesis pathway (LipA, LipB) was significantly down-regulated, which blocked the pathway catalyzed by pyruvate dehydrogenase complex to synthesize acetyl-coenzyme A (acetyl-CoA) from pyruvate; (ii) the expression level of the gene encoding bifunctional acetaldehyde-alcohol dehydrogenase was significantly up-regulated, and this effect facilitated the synthesis of ethanol from acetyl-CoA. However, the reaction of NaHSO3 with the intermediate metabolite acetaldehyde blocked the production of ethanol and consumed acetyl-CoA, thereby decreasing AC production. Thus, our study provides new insights into the molecular mechanism of AC decreased underlying the treatment of NaHSO3 and will deepen the understanding of the complex regulatory mechanisms of A. succinogenes.

Streptomyces fuscus sp. nov., a brown-black pigment producing actinomycete isolated from dry mudflat sand.

A novel Gram-stain-positive, aerobic actinobacterial strain, designated GXMU-J15T, was isolated from dry mudflat sand. A polyphasic approach was employed for its taxonomic characterization. The strain developed extensively branched yellowish white to light yellow substrate mycelia and white aerial mycelia, and produced smooth cylindrical spores in a loose straight spore chain on International Streptomyces Project 2-7 agar media. Strain GXMU-J15T grew at 20-50 °C (optimum, 35 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-8 % (w/v) NaCl. Analysis of 16S rRNA gene sequences indicated that strain GXMU-J15T represents a member of the genus Streptomyces. Strain GXMU-J15T showed the highest 16S rRNA gene sequence similarity to Streptomyces lusitanus CGMCC 4.1745T (99.1 %) and Streptomyces thermocarboxydus CGMCC 4.1883T (98.8 %). Phylogenetic tree analysis based on multilocus sequence analysis (MLSA) and whole genome sequence construction revealed that strain GXMU-J15T was most closely related to Streptomyces cupreus PSKA01T, Streptomyces cinnabarinus DSM 40467T and Streptomyces davaonensis JCM 4913T. The MLSA and genome-to-genome distances between strain GXMU-J15T and its relatives were 0.0418, 0.0443 and 0.0485 and 0.1237, 0.1188 and 0.1179, respectively. The results of orthologous average nucleotide identity and digital DNA-DNA hybridization analysis corroborated the results of the MLSA and whole genome sequence evolution analysis, indicating that the novel isolate represents a distinct species of the genus Streptomyces. The whole-cell sugars of strain GXMU-J15T were xylose, glucose and galactose. The characteristic diamino acid in the cell-wall hydrolysate was ll-diaminopimelic acid. The lipids contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylglycerides, phosphatidylcholine, two phospholipids of an unknown structure containing glucosamine, one unknown phospholipid and two unknown lipids. The major cellular fatty acid components were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The main respiratory quinone types were MK-9(H6) and MK-9(H8). The whole genome size of strain GXMU-J15T was 8.68 Mbp, with 71.23 mol% G+C content. Genomic analysis indicated that strain GXMU-J15T has the potential to synthesize polyketides, terpenes and a series of important antibiotics besides the gene cluster for melanin synthesis. Based on these genotypic and phenotypic data, strain GXMU-J15T is proposed to represent a new species of the genus Streptomyces named Streptomyces fuscus sp. nov. The type strain is GXMU-J15T (=MCCC 1K08211T=JCM 35917T).

Streptomyces beihaiensis sp. nov., a chitin-degrading actinobacterium, isolated from shrimp pond soil.

A Gram-stain-positive actinobacterium, designated strain GXMU-J5T, was isolated from a sample of shrimp pond soil collected in Tieshangang Saltern, Beihai, PR China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. The organism formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. Phylogenetic analysis of 16S rRNA gene sequences showed that strain GXMU-J5T was most related to Streptomyces kunmingensis DSM 41681T (similarity 97.74 %) and Streptomyces endophyticus YIM 65594T (similarity 96.80 %). However, the values of digital DNA-DNA hybridization, average nucleotide identity and evolutionary distance of multilocus sequence analysis between strain GXMU-J5T and its closest relatives indicated that it represented a distinct species. Strain GXMU-J5T contained ll-diaminopimelic acid and the major whole-cell hydrolysates were xylose and galactose. The predominant menaquinones of strain GXMU-J5T were revealed as MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides and phospholipids of unknown structure containing glucosamine. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. The whole genome size of strain GXMU-J5T was 6.79 Mbp with a 71.39 mol% G+C content. Genomic analysis indicated that strain GXMU-J5T had the potential to degrade chitin. On the basis of these genotypic and phenotypic data, it is supported that strain GXMU-J5T represents a novel species of the genus Streptomyces, for which the name Streptomyces beihaiensis sp. nov. is proposed. The type strain is strain GXMU-J5T (=MCCC 1K08064T=JCM 35629T).

Identification and Surveys of Promoting Plant Growth VOCs from Biocontrol Bacteria Paenibacillus peoriae GXUN15128.

The role of microbial volatile organic compounds (MVOCs) in promoting plant growth has received much attention. We isolated Paenibacillus peoriae from mangrove rhizosphere soil, which can produce VOCs to promote the growth of Arabidopsis thaliana seedlings, increase the aboveground biomass of A. thaliana, and increase the number of lateral roots of A. thaliana. The effects of different inoculation amounts and different media on the composition of MVOCs were studied by solid-phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) and headspace sampler/GC-MS. We found that the growth medium influences the function and composition of MVOCs. To survey the growth-promoting functions, the transcriptome of the receptor A. thaliana was then determined. We also verified the inhibitory effect of the soluble compounds produced by P. peoriae on the growth of 10 pathogenic fungi. The ability of P. peoriae to produce volatile and soluble compounds to promote plant growth and disease resistance has shown great potential for application in the sustainability of agricultural production. IMPORTANCE Microbial volatile organic compounds (MVOCs) have great potential as "gas fertilizers" for agricultural applications, and it is a promising research direction for the utilization of microbial resources. This study is part of the field of interactions between microorganisms and plants. To study the function and application of microorganisms from the perspective of VOCs is helpful to break the bottleneck of traditional microbial application. At present, the study of MVOCs is lacking; there is a lack of functional strains, especially with plant-protective functions and nonpathogenic application value. The significance of this study is that it provides Paenibacillus peoriae, which produces VOCs with plant growth-promoting effects and broad-spectrum antifungal activity against plant-pathogenic fungi. Our study provides a more comprehensive, new VOC component analysis method and explains how MVOCs promote plant growth through transcriptome analysis. This will greatly increase our understanding of MVOC applications as a model for other MVOC research.

Reduced acetic acid formation using NaHSO3 as a steering agent by Actinobacillus succinogenes GXAS137.

The high production of acetic acid (AC) as a by-product leads to difficult separation and purification of succinic acid (SA) and increases production costs in SA fermentation by Actinobacillus succinogenes. NaHSO3 as a steering agent was used to reduce AC production. Herein, the optimum fermentation conditions were achieved by single-factor and orthogonal tests as follows: glucose 60 g/L; MgCO3 60 g/L; NaHSO3 0.15% (w/v); and NaHSO3 addition time, 8 h after inoculation. After optimization, the SA and AC contents were 44.42 and 5.73 g/L. The SA improved by 100.72%, the AC decreased by 21.18% compared with the unfermented. The acetate kinase activity decreased by 14.36% and acetyl-CoA content improved by 97.55% in the group of NaHSO3 addition compared with control check (CK). The mechanism of NaHSO3 is formation acetaldehyde-sodium bisulfite compound and reduction the activity of acetate kinase. These findings indicated a new way of using NaHSO3 as a steering agent to reduce AC generation and may help promote the development of SA industrial production.

Comparative Transcriptome Analysis Unravels the Response Mechanisms of Fusarium oxysporum f.sp. cubense to a Biocontrol Agent, Pseudomonas aeruginosa Gxun-2.

Banana Fusarium wilt, which is caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FOC TR4), is one of the most serious fungal diseases in the banana-producing regions in east Asia. Pseudomonas aeruginosa Gxun-2 could significantly inhibit the growth of FOC TR4. Strain Gxun-2 strongly inhibited the mycelial growth of FOC TR4 on dual culture plates and caused hyphal wrinkles, ruptures, and deformities on in vitro cultures. Banana seedlings under pot experiment treatment with Gxun-2 in a greenhouse resulted in an 84.21% reduction in the disease. Comparative transcriptome analysis was applied to reveal the response and resistance of FOC TR4 to Gxun-2 stress. The RNA-seq analysis of FOC TR4 during dual-culture with P. aeruginosa Gxun-2 revealed 3075 differentially expressed genes (DEGs) compared with the control. Among the genes, 1158 genes were up-regulated, and 1917 genes were down-regulated. Further analysis of gene function and the pathway of DEGs revealed that genes related to the cell membrane, cell wall formation, peroxidase, ABC transporter, and autophagy were up-regulated, while down-regulated DEGs were enriched in the sphingolipid metabolism and chitinase. These results indicated that FOC TR4 upregulates a large number of genes in order to maintain cell functions. The results of qRT-PCR conducted on a subset of 13 genes were consistent with the results of RNA-seq data. Thus, this study serves as a valuable resource regarding the mechanisms of fungal pathogen resistance to biocontrol agents.

Enzymatic properties and biological activity of resuscitation-promoting factor B of Rhodococcus sp. (GX12401).

Resuscitation-promoting factor B (RpfB) is one of the five members of Rpf-like family in Mycobacteriales, which have the resuscitation-promoting activity. Most strains of Rhodococcus also have RpfB gene, but the study of rpfB gene in Rhodococcus is not thorough. Here, we amplified the rpfB gene of intact Rhodococcus sp. (GX12401) and cloned it into pET30a (+) expression vector. Then a recombinant form of soluble RpfB was expressed in Escherichia coli BL21. The soluble recombinant RpfB was purified by Ni-Sepharose affinity chromatography and molecular weight of the protein was 55 kDa, determined by 12% SDS-PAGE stained with Coomassie brilliant blue R-250. When 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitoside was used as enzyme substrate to test lysozyme activity, the recombinant protein RpfB had good stability and enzyme activity, and the lysozyme activity was low (4.74 U), among which Mg2+, Na+, Al3+ and DMSO could significantly increase the activity of RpfB. The purified recombinant protein was added to Rhodococcus VBNC cells, and the VBNC cells were resuscitated at the concentration of 1 picomolar concentrations, which increased by 18% compared with the control, while the cell resuscitation was inhibited at the concentration of 1,000 picomolar concentrations. Therefore, RpfB can improve the survival ability of Rhodococcus in extreme or harsh environment and enhance the corresponding biological activity.

Isolation and identification of a feather degrading Bacillus tropicus strain Gxun-17 from marine environment and its enzyme characteristics.

BACKGROUND: Feathers are the most abundant agricultural waste produced by poultry farms. The accumulation of a large number of feathers not only seriously pollutes the environment but also causes the waste of protein resources. The degradation of feather waste by keratinase-producing strains is currently a promising method. Therefore, screening high-producing keratinase strains from marine environment and studying the fermentation conditions, enzymatic properties and feather degradation mechanism are crucial for efficient degradation of feathers. RESULTS: A novel efficient feather-degrading bacteria, Gxun-17, isolated from the soil sample of a marine duck farm of Beibu Gulf in Guangxi, China, was identified as Bacillus tropicus. The optimum fermentation conditions were obtained by single factor and orthogonal tests as follows: feather concentration of 15 g/L, maltose concentration of 10.0 g/L, MgSO4 concentration of 0.1 g/L, initial pH of 7.0 and temperature of 32.5 °C. The strain completely degraded the feathers within 48 h, and the highest keratinase activity was 112.57 U/mL, which was 3.18-fold that obtained with the basic medium (35.37 U/mL). Detecting the keratinase activity and the content of sulphur-containing compounds in the fermentation products showed that the degradation of feathers by the strain might be a synergistic effect of the enzyme and sulphite. The keratinase showed optimal enzyme activity at pH 7.0 and temperature of 60 °C. The keratinase had the best performance on the casein substrate. When casein was used as the substrate, the Km and Vmax values were 15.24 mg/mL and 0.01 mg/(mL·min), respectively. Mg2+, Ca2+, K+, Co2+, Al3+, phenylmethylsulphonyl fluoride and isopropanol inhibited keratinase activity, which indicated that it was a serine keratinase. Conversely, the keratinase activity strongly increased with the addition of Mn2+ and ß-mercaptoethanol. CONCLUSIONS: A novel feather-degrading B. tropicus Gxun-17 was obtained from marine environment. The strain adapted the extreme conditions such as low temperature, high salt and high pressure. Thus, the keratinase had high activity, wide range of temperature and pH, salt tolerance and other characteristics, which had potential application value.

Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002.

BACKGROUND: Chitinases are enzymes which degrade ß-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. METHODS: Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. RESULTS: The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg-1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0-10.0 and 25-45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and ß-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. CONCLUSIONS: This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.

Ottowia flava sp. nov., isolated from fish intestines.

A novel Gram-negative bacterium, non-motile and short rod-shaped, designated strain GY511T, was isolated from the intestines of fish collected from Maowei Sea, China. Growth occurred at pH 6.0-9.0 (optimum 7.0), 4-37 °C (optimum 28 °C) and at 0-2.5% (w/v) NaCl (optimum 1.0%). The result of 16S rRNA gene sequence analysis showed that strain GY511T is closely related to O. oryzae NBRC 113109T (97.6%), O. konkukae DSM 105395T (97.4%), Ottowia beijingensis CGMCC 1.12324T (95.9%), Ottowia pentelensis DSM 21699T (95.2%) and Ottowia thiooxydans DSM 14619T (95.0%). The DNA-DNA hybridization values of strain GY511T with O. oryzae NBRC 113109T and O. konkukae DSM 105395T were 35.4 ± 3.1% and 26.3 ± 1.8%, respectively. The major fatty acids (> 10%) were identified as summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0 and summed feature 8 (C18:1ω7c and/or C18:1ω6c) and the major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, two unidentified aminolipids and an unidentified phospholipid. The G+C content of the genomic DNA was 62.9 mol%. Thiosulfate could be utilized as co-substrate for aerobic growth and was oxidised to sulfate. On the basis of phenotypic, chemotaxonomic and molecular data, strain GY511T is considered to represent a novel species of the genus Ottowia, for which the name Ottowia flava sp. nov. is proposed. The type strain is GY511T (= NBRC 113500T = DSM 107425T = CGMCC 1.13650T).

Falsibacillus albus sp. nov., isolated from mangrove soil.

A novel Gram-stain-positive, catalase- and oxidase-positive, endospore-forming bacterium, designated GY 10110T, was isolated from mangrove soil collected from Qinzhou, Guangxi province, China. Cells were aerobic, motile with peritrichous flagella and rod-shaped. The strain grew at 15-37 °C (optimum, 28 °C), at 0-3 %(w/v) NaCl (1 %) and at pH 6.0-9.0 (pH 7.0). The major fatty acids of strain GY 10110T were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0. The predominant menaquinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphoglycolipid, glycolipid, two unidentified aminophospholipids and three unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain GY 10110T was closely related to Falsibacillus pallidus CCTCC AB 207188T (98.0 % sequence similarity) and Bacillus oceanisediminis CGMCC 1.10115T (96.9 %), respectively. The G+C content of strain GY 10110T based on the whole genome sequence was 42.3 mol%. The novel strain showed an average nucleotide identity (ANI) value of 77.8 % and a digital DNA-DNA hybridization (dDDH) value of 15.6 % with Falsibacillus pallidus CCTCC AB 207188T based on draft genome sequences, followed by Bacillus oceanisediminis CGMCC 1.10115T with ANI and dDDH values of 75.2 and 12.8 %, respectively. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analysis, showed that strain GY 10110T represents a novel species of the genus Falsibacillus, for which the name Falsibacillus albus sp. nov. is proposed. The type strain is GY 10110T (=CGMCC 1.13648T=NBRC 113502T).

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations.

BACKGROUND: Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. OBJECTIVE: In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). METHODS: Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. RESULTS: According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and ß-sheet, ß-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. CONCLUSION: This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

[In vivo synthesis of csypyrone derivatives by exploring the substrate diversity of start units of type â ¢ polyketide synthase CsyB].

As a novel fungal type Ⅲ polyketide synthase, CsyB from Aspergillus oryzae can sequentially accept one molecular short chain fatty acyl CoA as start unit, one molecular malonyl-CoA and one molecular acetoacetyl-CoA as extend unit to produce the short chain csypyrone B1-3. On the basis of crystal structure of CsyB, a fatty acyl CoA binding tunnel of a length of about 16 Šis located in its active center that is proposed to accept diversified start units. In order to examine the substrate diversity of CsyB, CsyB gene was introduced and expressed in Escherichia coli that contained a number of precursors of long chain fatty acyl CoA in vivo. The results of HPLC revealed that a series of long chain csypyrone derivatives were detected in the recombinant strain in comparison with the control strain. These new csypyrone compounds were preliminarily analyzed by UV-visible spectroscopy and LC-HRMS. Three hydroxylated csypyrones were intensively determined by 1D and 2D NMR experiments, especially the position of the hydroxyl group in these compounds. These results demonstrate that CsyB exhibits a broad substrate specificity, which not only can accept the long chain saturated or unsaturated fatty acyl CoA as substrate, but also accept hydroxylated long chain fatty acyl CoA.

Complete Genome Sequence of Actinobacillus succinogenes GXAS137, a Highly Efficient Producer of Succinic Acid.

The bacterium Actinobacillus succinogenes GXAS137, an efficient producer of succinic acid, was isolated from bovine rumen in Nanning, Guangxi Province, China. Here, we present the 2.3-Mb genome assembly of this strain, which consists of 2,314,479 bp (G+C content of 44.89%) with a circular chromosome, 2,235 DNA coding sequences, 57 tRNAs, and 15 rRNAs.

Efficient production of succinic acid from duckweed (Landoltia punctata) hydrolysate by Actinobacillus succinogenes GXAS137.

A novel process of enzyme pretreatment and semi-simultaneous saccharification and fermentation (SSSF) was developed in this work to improve succinic acid (SA) productivity from duckweed (Landoltia punctata) and achieve low viscosity. Viscosity (83.86%) was reduced by the pretreatment with combined enzymes at 50 °C for 2 h to a greater extent than that by single enzyme (26.19-71.75%). SSSF was an optimal combination with 65.31 g/L of SA content, which was remarkably higher than those obtained through conventional separate hydrolysis and fermentation (62.12 g/L) and simultaneous saccharification and fermentation (52.41 g/L). The combined approach was effective for SA production. Approximately 75.46 g/L of SA content with a yield of 82.87% and a productivity of 1.35 g/L/h was obtained after 56 h in a 2 L bioreactor. Further studies will focus on increasing the working scale of the proposed method.

Succinic acid production from duckweed (Landoltia punctata) hydrolysate by batch fermentation of Actinobacillus succinogenes GXAS137.

Duckweed is potentially an ideal succinic acid (SA) feedstock due to its high proportion of starch and low lignin content. Pretreatment methods, substrate content and nitrogen source were investigated to enhance the bioconversion of duckweed to SA and to reduce the costs of production. Results showed that acid hydrolysis was an effective pretreatment method because of its high SA yield. The optimum substrate concentration was 140g/L. The optimum substrate concentration was 140g/L. Corn steep liquor powder could be considered a feasible and inexpensive alternative to yeast extract as a nitrogen source. Approximately 57.85g/L of SA was produced when batch fermentation was conducted in a 1.3L stirred bioreactor. Therefore, inexpensive duckweed can be a promising feedstock for the economical and efficient production of SA through fermentation by Actinobacillus succinogenes GXAS137.

Tumebacillus flagellatus sp. nov., an α-amylase/pullulanase-producing bacterium isolated from cassava wastewater.

A novel α-amylase/pullulanase-producing bacterium, designated strain GST4(T), was isolated from samples collected from the wastewater of a cassava starch factory in Nanning, Guangxi Autonomous Region, southern China. Cells of strain GST4(T) were rod-shaped bacilli containing ellipsoidal terminal spores and found to be Gram-reaction-positive, aerobic, motile, oxidase-positive, catalase-negative and formed light yellow colonies on agar plates. Strain GST4(T) was able to grow at pH 4.5-8.5 (optimum at pH 5.5), temperatures ranging from 20 to 42 °C (optimum at 37 °C) and salt concentrations of 0-1% (w/v) NaCl (optimum at 0.5%, w/v) on R2A medium. Strain GST4(T) grew heterotrophically on complex carbon substrates and chemolithoautotrophically on inorganic sulfur compounds, as demonstrated by growth on sodium thiosulfate and sulfite as sole electron donors. It can reduce nitrate and nitrite. Strain GST4(T) contained iso-C(15:0) and anteiso-C(15:0) as the major cellular fatty acids and menaquinone 7 (MK-7) as the major respiratory quinone. The cell-wall peptidoglycan was of type A1γ. The genomic DNA G+C content of strain GST4(T) was 53.7 mol%. Physiological and chemotaxonomic characteristics combined with phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GST4(T) was a member of the genus Tumebacillus and most closely related to Tumebacillus permanentifrigoris DSM 18773(T) and Tumebacillus ginsengisoli DSM 18389(T) with 97.3 and 94.5% sequence similarity, respectively. The DNA-DNA relatedness values between strain GST4(T) and T. permanentifrigoris DSM 18773(T), and strain GST4(T) and T. ginsengisoli DSM 18389(T) were 44.0 and 60.4%, respectively. The new isolate differed from those species of the genus Tumebacillus in that it has peritrichous flagella for motility. Based on the evidence obtained from this study, strain GST4(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus flagellatus sp. nov. is proposed. The type strain is GST4(T) ( =CGMCC 1.12170(T) =DSM 25748(T)).

[Optimization of succinic acid fermentation with Actinobacillus succinogenes by response surface methodology].

Succinic acid is an important C4 platform chemical in the synthesis of many commodity and special chemicals. In the present work, different compounds were evaluated for succinic acid production by Actinobacillus succinogenes GXAS 137. Important parameters were screened by the single factor experiment and Plackeet-Burman design. Subsequently, the highest production of succinic acid was approached by the path of steepest ascent. Then, the optimum values of the parameters were obtained by Box-Behnken design. The results show that the important parameters were glucose, yeast extract and MgCO3 concentrations. The optimum condition was as follows (g/L): glucose 70.00, yeast extract 9.20 and MgCO3 58.10. Succinic acid yield reached 47.64 g/L at the optimal condition. Succinic acid increased by 29.14% than that before the optimization (36.89 g/L). Response surface methodology was proven to be a powerful tool to optimize succinic acid production.

[Very high gravity ethanol fermentation with cassava flour and sugarcane juice].

We optimized the conditions of mixed fermentation of very high gravity ethanol with cassava flour and sugarcane juice. Based on the single factor experiment, we screened the important parameters for very high gravity ethanol fermentation with cassava flour and sugarcane juice by the Plackeet-burman design. Then, we obtained the optimum values of the important parameters by the orthogonal experiments: the mixing ratio of cassava flour to sugarcane juice, 1:5; initial pH of fermentation, 4.0-4.5; the concentrations of urea and MgSO4, 0.25% and 0.04% (W/W), respectively. Finally, we used a gradient temperature control strategy with the optimized conditions, and ethanol concentration of 17.84% (V/V) and fermentation efficiency of 91.82% were achieved, correspondingly.