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Bo Wang, Qitiao Hu, Yu Zhang, Ruilin Shi, Xin Chai, Zhe Liu, Xiuling Shang, Yun Zhang, Tingyi Wen
BACKGROUND: Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation...
April 23, 2018: Microbial Cell Factories
Bin Zhu, Xiuchun Ge, Victoria Stone, Xiangzhen Kong, Fadi El-Rami, Yan Liu, Todd Kitten, Ping Xu
Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR...
December 7, 2017: Scientific Reports
Xiulin Qin, Ruijie Li, Xiang Luo, Yanmei Lin, Jia-Xun Feng
To improve the gene targeting frequency (GTF) in the lignocellulolytic filamentous fungus Penicillium oxalicum HP7-1, the non-homologous end-joining (NHEJ) gene ligD was deleted. The obtained PoligD deletion mutant ΔPoligD showed no apparent defect in cellulase production, growth rate, and sensitivity towards osmotic stress and mutagen ethyl methanesulphonate (EMS), while increased sensitivity to high concentrations of methyl methanesulfonate (MMS). Deletion of PoligD gene resulted in significantly increased GTFs at three different loci in P...
June 2017: Fungal Biology
Jing Niu, Mark Arentshorst, Felix Seelinger, Arthur F J Ram, Jean Paul Ouedraogo
To construct a set of isogenic auxotrophic strains in Aspergillus niger suited for creating multiple gene deletion mutants and executing parasexual crossings, we have combined mutations in genes involved in colour pigmentation (fwnA and olvA) with well-selectable auxotrophic markers (pyrG, nicB, argB, and adeA). All markers, except for the pyrG marker, were introduced by targeted deletion, omitting UV mutagenesis of the strains. Aspergillus oryzae orthologous genes of the argB, nicB, and adeA markers were used as heterologous selection markers, and all markers were shown to complement to respective auxotrophic A...
November 2016: Archives of Microbiology
Mattia Zampieri, Uwe Sauer
MOTIVATION: Simple forms of mutualism between microorganisms are widespread in nature. Nevertheless, the role played by the environmental nutrient composition in mediating cross-feeding in microbial ecosystems is still poorly understood. RESULTS: Here, we use mixed-integer bilevel linear programming to investigate the cost of sharing metabolic resources in microbial communities. The algorithm infers an optimal combination of nutrients that can selectively sustain synergistic growth for a pair of species and guarantees minimum cost of cross-fed metabolites...
June 1, 2016: Bioinformatics
Meijuan Xu, Jingru Qin, Zhiming Rao, Hengyi You, Xian Zhang, Taowei Yang, Xiaoyuan Wang, Zhenghong Xu
BACKGROUND: Corynebacterium crenatum SYPA 5 is the industrial strain for L-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and L-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria...
January 19, 2016: Microbial Cell Factories
Victor M Hernández, Lourdes Girard, Ismael Hernández-Lucas, Alejandra Vázquez, Catalina Ortíz-Ortíz, Rafael Díaz, Michael F Dunn
L-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG)...
August 2015: Microbiology
Dorit Eberhardt, Jaide V K Jensen, Volker F Wendisch
L-citrulline plays an important role in human health and nutrition and is an intermediate of the L-arginine biosynthetic pathway. L-citrulline is a by-product of L-arginine production by Corynebacterium glutamicum. In this study, C. glutamicum was engineered for overproduction of L-citrulline as major product without L-arginine being produced as by-product. To this end, L-arginine biosynthesis was derepressed by deletion of the arginine repressor gene argR and conversion of L-citrulline towards L-arginine was avoided by deletion of the argininosuccinate synthetase gene argG...
December 2014: AMB Express
Minliang Chen, Xuelan Chen, Fang Wan, Bin Zhang, Jincong Chen, Yonghua Xiong
BACKGROUND: L-Glutamate is an important precursor in the L-arginine (L-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and dtsR1 disruption, have been widely used to induce L-glutamate overproduction in Corynebacterium glutamicum. In this study, a novel strategy for L-Arg overproduction through Tween 40 trigger and ΔdtsR1 mutant were proposed in Corynebacterium crenatum. RESULTS: Corynebacterium crenatum mutant (CCM01) was selected as a host strain, whose argR was lethal via mutagenesis screening, the proB gene was knocked out, and argB was replaced by argB M4 (E19R, H26E, D311R, and D312R) to release L-Arg feedback resistance...
2015: Microbial Cell Factories
Yawei Chen, Shuangyan Lou, Lihai Fan, Xu Zhang, Tianwei Tan
ATP is the limiting precursor and driving force for S-adenosylmethionine (SAM) biosynthesis in Escherichia coli. In contrast to traditional optimization of fermentation processes, the synthetic sRNA-based repression strategy, which was developed as a highly efficient gene knockdown approach, has been applied for the regulation of the intracellular ATP concentration in order to enhance SAM production. In this work, proB, glnA and argB, all involved in the synthesis of ATP-dependent by-products in the S-adenosylmethionine production were selected as candidates for repression...
August 2015: FEMS Microbiology Letters
Lifeng Xiong, Jade L L Teng, Rory M Watt, Cuihua Liu, Susanna K P Lau, Patrick C Y Woo
The betaproteobacterium Laribacter hongkongensis is associated with invasive bacteremic infections and gastroenteritis. Its genome contains two adjacent arc gene cassettes (arc1 and arc2) under independent transcriptional control, which are essential for acid resistance. Laribacter hongkongensis also encodes duplicate copies of the argA and argB genes from the arginine biosynthesis pathway. We show that arginine enhances the transcription of arcA2 but suppresses arcA1 expression. We demonstrate that ArgR acts as a transcriptional regulator of the two arc operons through binding to ARG operator sites (ARG boxes)...
November 2015: Environmental Microbiology
Xue Lan Chen, Bin Zhang, Li Tang, Hai Tao Jiao, Heng Yi Xu, Feng Xu, Hong Xu, Hua Wei, Yong Hua Xiong
OBJECTIVE: Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain. METHODS: Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer...
June 2014: Biomedical and Environmental Sciences: BES
Ling-Lin Fu, Rui Wang, Yanbo Wang, Junda Lin
BACKGROUND: High hydrostatic pressure (HHP) processing is currently being used as a treatment for certain foods to inhibit spoilage organisms and control the presence of foodborne pathogens. In this study proteome profiles were performed by two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification to determine the effects of HHP (50, 100, 150 and 200 MPa, each for 10 min) on Vibrio parahaemolyticus ATCC 17802 (∼8 log CFU mL⁻¹) in order to understand how it responds to mechanical stress injury...
October 2014: Journal of the Science of Food and Agriculture
Hongmei Sun, Zhiming Rao, Xiupeng Li, Meijuan Xu, Xian Zhang, Zhenghong Xu
OBJECTIVE: We constructed an integrative recombinant Corynebacterium crenatum that could directly convert glucose in gamma-aminobutyric acid (GABA). METHODS: Using overlap PCR, we obtained delta argB fragment that lacked 491 bp of N-acetylglutamate kinase (NAGK) gene. The glutamate decarboxylase (GAD) gene attached tac promoter was amplified and inserted into delta argB fragment. Using the upstream and downstream of delta argB as homologous arms, we constructed pK18-delta argB::tacgad which was used for the integration of tacgad gene onto the C...
August 4, 2013: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
Wenqing Chen, Daofeng Dai, Changchun Wang, Tingting Huang, Lipeng Zhai, Zixin Deng
BACKGROUND: Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the "pathway redundancy" of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. RESULTS: Here we report the genetic characterization of CPOAA biosynthesis with revealing a "pathway redundancy" in metabolic networks...
2013: Microbial Cell Factories
Georg Schendzielorz, Martin Dippong, Alexander Grünberger, Dietrich Kohlheyer, Ayako Yoshida, Stephan Binder, Chiharu Nishiyama, Makoto Nishiyama, Michael Bott, Lothar Eggeling
Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine...
January 17, 2014: ACS Synthetic Biology
Nicola Beckmann, Lukas Schafferer, Markus Schrettl, Ulrike Binder, Heribert Talasz, Herbert Lindner, Hubertus Haas
The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively...
2013: PloS One
Shuji Tani, Atsushi Tsuji, Emi Kunitake, Jun-Ichi Sumitani, Takashi Kawaguchi
Auxotrophic mutants of Aspergillus can be isolated in the presence of counter-selective compounds, but the process is laborious. We developed a method to enable reversible impairment of the ku80 gene (Aaku80) in the imperfect fungus Aspergillus aculeatus. Aaku80 was replaced with a selection marker, orotidine 5'-phosphate decarboxylase (pyrG), followed by excision of pyrG between direct repeats (DR) to yield the Aaku80 deletion mutant (MR12). The gene-targeting efficiency at the ornithine carbamoyltransferase (argB) locus was drastically elevated from 3% to 96% in MR12...
2013: AMB Express
Austin S Nuxoll, Steven M Halouska, Marat R Sadykov, Mark L Hanke, Kenneth W Bayles, Tammy Kielian, Robert Powers, Paul D Fey
Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate...
2012: PLoS Pathogens
Takayuki Itoh, Tetsuo Kushiro, Isao Fujii
Expression of multiple genes involved in a particular metabolic pathway in a heterologous host facilitates the study of fungal secondary metabolite biosynthesis and production of useful compounds. Two plasmids with different selection markers, argB and the pyrithiamine resistance marker, are used to transform Aspergillus oryzae allowing for expression of up to three genes simultaneously.
2012: Methods in Molecular Biology
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