The Experts below are selected from a list of 354 Experts worldwide ranked by ideXlab platform
Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.
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hyper secretion of thermobifida fusca β glucosidase via a tat dependent signal peptide using streptomyces lividans
Microbial Cell Factories, 2013Co-Authors: Takaya Miyazaki, Shuhei Noda, Tsutomu Tanaka, Akihiko KondoAbstract:Protein production as secretory-form is a powerful tool in Industrial Enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation Enzymes derived from Thermobifida fusca YX and S. lividans. Three candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide. In the present study, we focused on signal peptide sequences derived from biomass degradation Enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.
Igor V Grigoriev - One of the best experts on this subject based on the ideXlab platform.
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transcriptome analysis of aspergillus niger xlnr and xkia mutants grown on corn stover and soybean hulls reveals a highly complex regulatory network
BMC Genomics, 2019Co-Authors: Claire Khosravi, Joanna E Kowalczyk, Tania Chroumpi, Evy Battaglia, Mariavictoria Aguilar Pontes, Mao Peng, Ad Wiebenga, Anna Lipzen, Diane Bauer, Igor V GrigorievAbstract:Enzymatic plant biomass degradation by fungi is a highly complex process and one of the leading challenges in developing a biobased economy. Some Industrial fungi (e.g. Aspergillus niger) have a long history of use with respect to plant biomass degradation and for that reason have become ‘model’ species for this topic. A. niger is a major Industrial Enzyme producer that has a broad ability to degrade plant based polysaccharides. A. niger wild-type, the (hemi-)cellulolytic regulator (xlnR) and xylulokinase (xkiA1) mutant strains were grown on a monocot (corn stover, CS) and dicot (soybean hulls, SBH) substrate. The xkiA1 mutant is unable to utilize the pentoses D-xylose and L-arabinose and the polysaccharide xylan, and was previously shown to accumulate inducers for the (hemi-)cellulolytic transcriptional activator XlnR and the arabinanolytic transcriptional activator AraR in the presence of pentoses, resulting in overexpression of their target genes. The xlnR mutant has reduced growth on xylan and down-regulation of its target genes. The mutants therefore have a similar phenotype on xylan, but an opposite transcriptional effect. D-xylose and L-arabinose are the most abundant monosaccharides after D-glucose in nearly all plant-derived biomass materials. In this study we evaluated the effect of the xlnR and xkiA1 mutation during growth on two pentose-rich substrates by transcriptome analysis. Particular attention was given to CAZymes, metabolic pathways and transcription factors related to the plant biomass degradation. Genes coding for the main Enzymes involved in plant biomass degradation were down-regulated at the beginning of the growth on CS and SBH. However, at a later time point, significant differences were found in the expression profiles of both mutants on CS compared to SBH. This study demonstrates the high complexity of the plant biomass degradation process by fungi, by showing that mutant strains with fairly straightforward phenotypes on pure mono- and polysaccharides, have much less clear-cut phenotypes and transcriptomes on crude plant biomass.
Takaya Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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hyper secretion of thermobifida fusca β glucosidase via a tat dependent signal peptide using streptomyces lividans
Microbial Cell Factories, 2013Co-Authors: Takaya Miyazaki, Shuhei Noda, Tsutomu Tanaka, Akihiko KondoAbstract:Protein production as secretory-form is a powerful tool in Industrial Enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation Enzymes derived from Thermobifida fusca YX and S. lividans. Three candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide. In the present study, we focused on signal peptide sequences derived from biomass degradation Enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.
Yunjun Yan - One of the best experts on this subject based on the ideXlab platform.
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intracellular expression of vitreoscilla hemoglobin improves production of yarrowia lipolytica lipase lip2 in a recombinant pichia pastoris
Enzyme and Microbial Technology, 2012Co-Authors: Xiaofeng Wang, Yongchuan Sun, Xuguang Shen, Yun Liu, Heyun Zhao, Yunjun YanAbstract:The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb(+)) displayed higher biomass and YlLIP2 activity than control strains (VHb(-)). Compared with VHb(-) cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb(+) in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb(-) cells than in VHb(+) cells. The highest YlLIP2 activity of VHb(+) cells was approximately 1.84-fold higher than that of VHb(-) cells at lower DO levels. Moreover, the recombinant strain VHb(+) exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb(-) cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and Industrial Enzyme production.
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expression of vitreoscilla hemoglobin improves recombinant lipase production in pichia pastoris
Chinese Journal of Biotechnology, 2011Co-Authors: Xiaofeng Wang, Yongchuan Sun, Xuguang Shen, Yun Liu, Yunjun YanAbstract:Yarrowia lipolytica lipase Lip2 (YlLip2) is an important Industrial Enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.
Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.
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high throughput screening for Industrial Enzyme production hosts by droplet microfluidics
Lab on a Chip, 2014Co-Authors: Staffan L Sjostrom, Jens Nielsen, Yunpeng Bai, Mingtao Huang, Zihe Liu, Haakan N Joensson, Helene Andersson SvahnAbstract:A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted Industrial Enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in Enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving Enzyme protein structure. In the workflow presented, Enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted Enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved Enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.
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physiological characterization of the high malic acid producing aspergillus oryzae strain 2103a 68
Applied Microbiology and Biotechnology, 2014Co-Authors: Christoph Knuf, Intawat Nookaew, Stephen H Brown, Alan Berry, Jens Nielsen, Ilse M Remmers, Sakda KhoomrungAbstract:Malic acid is a C4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C4 dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an Industrial Enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding Enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))−1 h−1 and with a yield of 1.49 mol mol−1. Intracellular fluxes were obtained using 13C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.