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1.
Metab Eng ; 78: 148-158, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37286071

RESUMO

Focusing on the differences in the catalytic properties of two type I fatty acid synthases FasA and FasB, the fasA gene was disrupted in an oleic acid-producing Corynebacterium glutamicum strain. The resulting oleic acid-requiring strain whose fatty acid synthesis depends only on FasB exhibited almost exclusive production (217 mg/L) of palmitic acid (C16:0) from 1% glucose under the conditions supplemented with the minimum concentration of sodium oleate for growth. Plasmid-mediated amplification of fasB led to a 1.47-fold increase in palmitic acid production (320 mg/L), while fasB disruption resulted in no fatty acid production, with excretion of malonic acid (30 mg/L). Next, aiming at conversion of the palmitic acid producer to a producer of palmitoleic acid (POA, C16:1Δ9), we introduced the Pseudomonas nitroreducens Δ9-desaturase genes desBC into the palmitic acid producer. Although this resulted in failure, we noticed the emergence of suppressor mutants that exhibited the oleic acid-non-requiring phenotype. Production experiments revealed that one such mutant M-1 undoubtedly produced POA (17 mg/L) together with palmitic acid (173 mg/L). Whole genomic analysis and subsequent genetic analysis identified the suppressor mutation of strain M-1 as a loss-of-function mutation for the DtxR protein, a global regulator of iron metabolism. Considering that DesBC are both iron-containing enzymes, we investigated the conditions for increased iron availability to improve the DesBC-dependent conversion ratio of palmitic acid to POA. Eventually, supplementation of both hemin and the iron chelator protocatechuic acid in the engineered strain dramatically enhanced POA production to 161 mg/L with a conversion ratio of 80.1%. Cellular fatty acid analysis revealed that the POA-producing cells were really equipped with unnatural membrane lipids comprised predominantly of palmitic acid (85.1% of total cellular fatty acids), followed by non-native POA (12.4%).


Assuntos
Corynebacterium glutamicum , Ácido Palmítico , Ácido Palmítico/metabolismo , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica/métodos , Ácidos Graxos , Ferro/metabolismo
2.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33741612

RESUMO

l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number AAY37316) from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt, which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. Our study reports efficient methods to produce l-theanine in the absence of supplemental ethylamine.IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. In this study, we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine.


Assuntos
Escherichia coli/metabolismo , Etilaminas/metabolismo , Glutamatos/biossíntese , Vias Biossintéticas , Fermentação
3.
Appl Environ Microbiol ; 87(5)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310719

RESUMO

Fatty acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expressions of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were co-expressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruptions of them resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a fatty acid high-producer, the resulting fadDs-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose.IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has currently evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, a formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their surplusage to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a fatty acid high-producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

4.
Appl Microbiol Biotechnol ; 102(24): 10603-10612, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30276713

RESUMO

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácidos Micólicos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cerulenina/farmacologia , Corynebacterium glutamicum/efeitos dos fármacos , Inibidores da Síntese de Ácidos Graxos/farmacologia , Ácidos Graxos/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Vancomicina/farmacologia
5.
Appl Environ Microbiol ; 83(19)2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28754705

RESUMO

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.


Assuntos
Proteínas de Bactérias/metabolismo , Biotina/biossíntese , Corynebacterium glutamicum/enzimologia , Ácidos Graxos/biossíntese , Ácido Tióctico/biossíntese , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo
6.
Appl Environ Microbiol ; 79(9): 3033-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23455340

RESUMO

An L-glutamine-overproducing mutant of an Escherichia coli K-12-derived strain was selected from randomly mutagenized cells in the course of L-alanyl-L-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for L-glutamine overproduction in this mutant. Three mutations were identified that are related to the L-glutamine overproduction phenotype, namely, an intergenic mutation in the 5'-flanking region of yeiG and two nonsynonymous mutations in gyrA (Gly821Ser and Asp830Asn). Expression of yeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in gyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parC and parE), an increase in L-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in L-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an L-glutamine-hyperproducing strain by attenuating cellular L-glutamine degradation activity. Although the reconstituted mutant (with yeiG together with gyrA) produced 200 mM L-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM L-glutamine.


Assuntos
Proteínas de Bactérias/genética , DNA Girase/genética , Escherichia coli K12/enzimologia , Genoma Bacteriano/genética , Glutamina/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , DNA Girase/metabolismo , DNA Bacteriano/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo
7.
Appl Environ Microbiol ; 75(6): 1635-41, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19139237

RESUMO

Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92(up), and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (Delta argR), the production was further increased. The best mutation set, Delta argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38 degrees C.


Assuntos
Arginina/biossíntese , Citrulina/biossíntese , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Genes Bacterianos , Redes e Vias Metabólicas/genética , Mutação , Recombinação Genética
8.
ACS Synth Biol ; 4(4): 383-92, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25171735

RESUMO

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.


Assuntos
Proteínas de Bactérias , Mesorhizobium/enzimologia , Ácidos Pipecólicos , Prolil Hidroxilases , Sinorhizobium meliloti/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Hidroxilação , Ácidos Pipecólicos/química , Ácidos Pipecólicos/metabolismo , Prolil Hidroxilases/química , Prolil Hidroxilases/metabolismo , Estrutura Terciária de Proteína
9.
FEMS Microbiol Lett ; 304(1): 12-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20067529

RESUMO

L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.


Assuntos
Dipeptídeos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Meios de Cultura , Dipeptídeos/biossíntese , Dipeptídeos/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligases/genética , Ligases/metabolismo , Proteínas de Membrana Transportadoras/genética
10.
J Ind Microbiol Biotechnol ; 33(7): 610-5, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16506038

RESUMO

Based on the progress in genomics, we have developed a novel approach that employs genomic information to generate an efficient amino acid producer. A comparative genomic analysis of an industrial L-lysine producer with its natural ancestor identified a variety of mutations in genes associated with L-lysine biosynthesis. Among these mutations, we identified two mutations in the relevant terminal pathways as key mutations for L-lysine production, and three mutations in central metabolism that resulted in increased titers. These five mutations when assembled in the wild-type genome led to a significant increase in both the rate of production and final L-lysine titer. Further investigations incorporated with transcriptome analysis suggested that other as yet unidentified mutations are necessary to support the L-lysine titers observed by the original production strain. Here we describe the essence of our approach for strain reconstruction, and also discuss mechanisms of L-lysine hyperproduction unraveled by combining genomics with classical strain improvement.


Assuntos
Corynebacterium glutamicum/genética , Engenharia Genética , Genoma Bacteriano/genética , Microbiologia Industrial/métodos , Lisina/biossíntese , Corynebacterium glutamicum/metabolismo , Análise Mutacional de DNA , Lisina/genética , Mutagênese , Mutação
11.
Biosci Biotechnol Biochem ; 70(11): 2803-6, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17090916

RESUMO

Genomic analysis of a classically derived L-lysine-producing mutant, Corynebacterium glutamicum B-6, identified a nonsense mutation in the mqo gene, which encodes malate:quinone oxidoreductase (MQO). The effect of mqo disruption on L-lysine production was investigated in a defined L-lysine producer, C. glutamicum AHP-3, showing approximately 18% increased production. To explore the underlying mechanisms of the increase, the mqo-disrupted strain was analyzed from the viewpoints of redox balance, activities of membrane-bound dehydrogenases, and transcriptome. The intracellular [NADH]/[NAD] ratio in the strain remained unchanged. Also, there were no significant differences in the activities of the membrane-bound dehydrogenases examined. However, transcriptome analysis showed that some TCA cycle genes, such as acn, sucC, and sucD, were down-regulated in the strain. These results suggest that the loss of MQO activity down-regulates the flux of the TCA cycle to maintain the redox balance and results in redirection of oxaloacetate into L-lysine biosynthesis.


Assuntos
Corynebacterium glutamicum/metabolismo , Lisina/biossíntese , Oxirredutases/deficiência , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Oxirredutases/genética , Oxirredutases/metabolismo
12.
Biosci Biotechnol Biochem ; 70(2): 546-50, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16495679

RESUMO

Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Lisina/biossíntese , Transcrição Gênica/genética , Carbono/metabolismo , Perfilação da Expressão Gênica , Indústrias , RNA Mensageiro/genética
13.
Appl Microbiol Biotechnol ; 72(4): 783-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16944136

RESUMO

We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner.


Assuntos
Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Engenharia Genética , Lisina/biossíntese , Transcrição Gênica/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Fermentação , Genes rel/fisiologia , Genoma Bacteriano , Microbiologia Industrial/métodos , Mutação
14.
Biosci Biotechnol Biochem ; 66(6): 1337-44, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12162556

RESUMO

Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a "metabolic array", was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.


Assuntos
Acetatos/metabolismo , Corynebacterium/genética , Corynebacterium/metabolismo , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica/genética , Clonagem Molecular , DNA Complementar/genética , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Dados de Sequência Molecular , RNA Bacteriano/análise , RNA Bacteriano/genética
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