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Ren X.,Huazhong Agricultural University | Wang J.,Huazhong Agricultural University | Yu H.,Huazhong Agricultural University | Peng C.,Huazhong Agricultural University | And 9 more authors.
Bioresource Technology | Year: 2016

In this study, a Saccharomyces cerevisiae recombinant strain 14 was constructed through genome shuffling method by transferring the whole genomic DNA of Candida intermedia strain 23 into a thermo-tolerant S. cerevisiae strain. The recombinant strain 14 combined the good natures of both parent strains that efficiently produced ethanol from glucose and single cell protein from xylose with 54.6% crude protein and all essential amino acids except cysteine at 35 °C. Importantly, the recombinant strain 14 produced 64.07 g/L ethanol from 25% (w/v) NaOH-pretreated and washed corn stover with the ethanol yield of 0.26 g/g total stover by fed-batch simultaneous saccharification and fermentation and produced 66.50 g/L dry cell mass subsequently from the residual hydrolysate and ethanol. Therefore, this study represents a feasible method to comprehensively utilize hexose and pentose in lignocellulosic materials. © 2016 Elsevier Ltd


Hu J.,Huazhong Agricultural University | Lin Y.,Huazhong Agricultural University | Zhang Z.,Huazhong Agricultural University | Xiang T.,Huazhong Agricultural University | And 8 more authors.
Bioresource Technology | Year: 2016

Because the cost of refined sugar substrate and limit of worldwide food availability, lignocellulosic materials are attractive for use in lactic acid (LA) production. In this study, we found Lactobacillus pentosus strain FL0421 produced LA with high yields (0.52-0.82 g/g stover) from five NaOH-pretreated and washed agro stovers through simultaneous saccharification and fermentation (SSF). We developed a fed-batch SSF process at 37 °C and pH 6.0 using the cellulase of 30 FPU/g stover and 10 g/L yeast extract in a 5-L bioreactor to produce LA from 14% (w/w) NaOH-pretreated and washed corn stover under non-sterile condition. The LA-titer, yield and productivity reached 92.30 g/L, 0.66 g/g stover and 1.92 g/L/h, respectively; and acetic acid titer and yield reached 34.27 g/L and 0.24 g/g stover. This study presented a feasible process for LA production from agro stovers and provided a candidate strain for genetic engineering for high-titer and -yield lignocellulosic LA production. © 2016 Elsevier Ltd.


She W.,Huazhong Agricultural University | Sun Z.,Huazhong Agricultural University | Yi L.,Huazhong Agricultural University | Zhao S.,Huazhong Agricultural University | And 3 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2016

A novel streptomycete strain, designated XY25T, was isolated from the rhizosphere soil in an alfalfa field in Jingyang, Shanxi, China. The isolate showed optimal growth at 37 8C, and was capable of growing at pH 6–10 and in the presence of 0–6% (w/v) NaCl. Mycelia of strain XY25T appeared spiral and developed into white spore chains with long-rod spores and a smooth surface. The 16S rRNA gene sequence of XY25T was determined and was found to be highly similar to those of species of the genus Streptomyces including Streptomyces silaceus DSM 41861T (99.11% 16S rRNA gene sequence similarity), Streptomyces flavofungini DSM 40366T (98.49%) and Streptomyces intermedius DSM 40372T (98.43%), all of which were used for further characterization. Each of the four streptomycetes showed distinctive patterns of carbon usage and fatty acids composition. Analysis of cellular components of strain XY25T revealed LL-diaminopimelic acid as diagnostic diamino acid and xylose as the major sugar, whereas polar lipids were determined as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid, two unknown phosphatidylinositol mannosides and several unknown lipids. Menaquinones were dominated by MK-9(H6) and MK-9(H8), and the main fatty acids were anteiso-C15: 0, iso-C16: 0 and anteiso-C17: 0. DNA–DNA hybridization studies indicated that strain XY25T showed relatedness values of 35.2–40.42% with the closest related species. Based on these results, strain XY25T represents a novel species of the genus Streptomyces, for which the name Streptomyces alfalfae sp. nov. is proposed. The type strain is XY25T (=KCTC 39571T=CCTCC AA2015019T). © 2015 IUMS.


Wu D.,Huazhong Agricultural University | Wang L.,Huazhong Agricultural University | Li Y.,Huazhong Agricultural University | Zhao S.,Huazhong Agricultural University | And 4 more authors.
Journal of Microbiology and Biotechnology | Year: 2016

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA. © 2016 by The Korean Society for Microbiology and Biotechnology.


He Y.,Huazhong Agricultural University | Shao Y.,Huazhong Agricultural University | Chen F.,Huazhong Agricultural University | Chen F.,Hubei Collaborative Innovation Center for Industrial Fermentation | Chen F.,Shihezi University
Fungal Biology | Year: 2014

Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (Mrδlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the Mrδlig4 strain increased four-fold compared with that in the wild-type strain, reached 68% and 85%, respectively. Thus, the Mrδlig4 strain is a promising host for efficient genetic manipulation. In addition, the Mrδlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate. © 2014 The British Mycological Society.


He T.,Hubei Collaborative Innovation Center for Industrial Fermentation | Hu X.,China University of Technology | Chen J.Y.,China University of Technology | Wang X.,China University of Technology
Advanced Materials Research | Year: 2014

Liquor fermentation is a complex biochemical process, to control the temperature of fermentation tank fastly and accurately in the process can improve the efficiency and quality of fermentation.Because of the nonlinear and time lag of the process, meanwhile the conventional PID control and is difficult to solve practical problems in precise control,So this paper puts forward a fuzzy predictive controlling method, by combining the advantages of fuzzy control and predictive control for big lag, nonlinear fermentation systems.After using MATLAB to make comparative data simulation, the result shows that the proposed design method can better dynamic and static characteristics of both system, with static characteristic small overshoot, fast response, high steady precision, etc, can be useful in industrial control systems. © (2014) Trans Tech Publications, Switzerland.


Liu T.,Huazhong Agricultural University | Li Y.,Huazhong Agricultural University | Wang X.,Huazhong Agricultural University | Ye Q.,Huazhong Agricultural University | And 6 more authors.
Nucleic Acids Research | Year: 2015

Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I-A of Sulfolobus islandicus REY15A. Csa3a, a MarR-like transcription factor encoded by the gene located adjacent to csa1, cas1, cas2 and cas4 cluster, but on the reverse strand, was demonstrated to specifically bind to the csa1 and cas1 promoters with the imperfect palindromic sequence. Importantly, it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence of an invading element and then activates spacer acquisition gene expression which leads to de novo spacer uptake from the invading element. © 2015 The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


Hu Y.,Huazhong Agricultural University | Hu Y.,Hubei Collaborative Innovation Center for Industrial Fermentation | Hu Y.,Hubei Normal University | Qin H.,Huazhong Agricultural University | And 10 more authors.
Biotechnology and Biotechnological Equipment | Year: 2016

Saccharomyces boulardii preparations are promising probiotics and clinical agents for animals and humans. This work focused on optimizing the nutritional conditions for the production of S. boulardii in solid-state fermentation by using classical and statistical methods. In single-factor experiments, the S. boulardii production was significantly increased by the addition of glucoamylase and the optimal carbon and nitrogen sources were found to be soluble starch and NH4Cl, respectively. The effects of the glucoamylase, soluble starch and NH4Cl on S. boulardii production were evaluated by a three-level three-factor Box-Behnken design and response surface methodology (RSM). The maximal yeast count (4.50 × 109CFU/g) was obtained under the optimized conditions (198 U/g glucoamylase, 2.37% soluble starch and 0.9% NH4Cl), which was in a good agreement with the predicted value of the model. This study has provided useful information on how to improve the accumulation of yeast cells by RSM. © 2015 The Author(s). Published by Taylor & Francis.


Yu J.,Hubei Normal University | Yu J.,Huazhong Agricultural University | Hu Y.,Hubei Normal University | Hu Y.,Huazhong Agricultural University | And 8 more authors.
Journal of Microbiology and Biotechnology | Year: 2016

The aim of this study was to isolate antioxidant peptides from an enzymatic hydrolysate of Spirulina platensis. A novel antioxidant peptide was obtained by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography, with the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay used to measure the antioxidant activity, and the sequence was determined to be Pro-Asn-Asn (343.15 Da) by electrospray ionization tandem mass spectrometry. This peptide was synthesized to confirm its antioxidant properties, and it exhibited 81.44 ± 0.43% DPPH scavenging activity at 100 μg/ml, which was similar to that of glutathione (82.63 ± 0.56%). Furthermore, the superoxide anion and hydroxyl free-radical scavenging activities and the SOD activity of the peptide were 47.84 ± 0.49%, 54.01 ± 0.82%, and 12.55 ± 0.75%, respectively, at 10 mg/ml. These results indicate that S. platensis is a good source of antioxidant peptides, and that its hydrolysate may have important applications in the pharmaceutical and food industries. © 2016 by The Korean Society for Microbiology and Biotechnology.


Hu J.,Huazhong Agricultural University | Zhang Z.,Huazhong Agricultural University | Lin Y.,Huazhong Agricultural University | Zhao S.,Huazhong Agricultural University | And 6 more authors.
Bioresource Technology | Year: 2015

Lactic acid (LA) is an important chemical with various industrial applications. Non-food feedstock is commercially attractive for use in LA production; however, efficient LA fermentation from lignocellulosic biomass resulting in both high yield and titer faces technical obstacles. In this study, the thermophilic bacterium Bacillus coagulans LA204 demonstrated considerable ability to ferment glucose, xylose, and cellobiose to LA. Importantly, LA204 produces LA from several NaOH-pretreated agro stovers, with remarkably high yields through simultaneous saccharification and fermentation (SSF). A fed-batch SSF process conducted at 50. °C and pH 6.0, using a cellulase concentration of 30. FPU (filter paper unit)/g stover and 10. g/L yeast extract in a 5-L bioreactor, was developed to produce LA from 14.4% (w/w) NaOH-pretreated non-sterile corn stover. LA titer, yield, and average productivity reached 97.59. g/L, 0.68. g/g stover, and 1.63. g/L/h, respectively. This study presents a feasible process for lignocellulosic LA production from abundant agro stovers. © 2015 Elsevier Ltd.

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