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1.
Gosset G 《Microbial cell factories》2005,4(1):14
The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological
processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source.
In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations
like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport,
therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes.
Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory
constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose
of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies
of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent
uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity
for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy
to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates. 相似文献
2.
Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc 总被引:3,自引:0,他引:3
Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y
Glc values. Assuming equal Y
ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.Abbreviations PTS
phosphoenolpyruvate: carbohydrate phosphotransferase system
- HPr
histidine-containing phosphocarrier protein
- GalP
galactose permease 相似文献
3.
Adelfo Escalante Rocío Calderón Araceli Valdivia Ramón de Anda Georgina Hernández Octavio T Ramírez Guillermo Gosset Francisco Bolívar 《Microbial cell factories》2010,9(1):21
Background
Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG fbr, tktA, aroB and aroE, on SA synthesis. 相似文献4.
5.
Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTSd) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTSd phosphofructokinase defective (PFK–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTSd strain.Abbreviations BM
basal medium
- BM-G
basal medium containing glucose
- CM
complex medium
- man:PTS
phosphoenolpyruvate:mannose phosphotransferase system
- CCCP
carbonylcyanide m-chlorophenylhydrazone
- DCCD
N,N-dicyclohexyl carbodiimide
- P
proton motive force
- pH
transmembrane pH gradient
-
transmembrane electrical potential difference
- MNNG
N-methyl-N-nitro-N-nitrosoguanidine
- PIPES
piperazine-N,N-bis(-ethanesulfonic acid)
- MES
4-morpholineethanesulfonic acid
- G-6-P
glucose 6-phosphate
- F-6-P
fructose 6-phosphate
- FDP
fructose 1,6-bisphosphate
- EMP
Embden-Meyerhof-Parnas pathway
- PFK
phosphofructokinase
- GK
glucokinase
- HK
hexokinase
- IAA
iodoacetic acid
- IIman
enzyme II component of man:PTS 相似文献
6.
Background
The phosphoenolpyruvate phosphotransferase system (PTS) plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP) and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS. 相似文献7.
8.
Jongrae Kim Min Kyung Kong Sang Yup Lee Pyung Cheon Lee 《World journal of microbiology & biotechnology》2010,26(12):2231-2239
The effects of three phosphoenolpyruvate (PEP)-dependent PTS carbon sources (glucose, mannose and maltose) and three non-PTS
carbon sources (glycerol, galactose, and lactose) on the formation of four carotenoids with diverse structures and on the
cell growth of the recombinant Escherichia coli were investigated. The biosynthetic pathways of four carotenoids, C30 diapolycopene, C30 diapotorulene, C40 lycopene, and C40 beta-carotene, were engineered in E. coli. The resulting E. coli cells were grown in a mineral medium supplemented with each of the six carbon sources. Among the six carbon sources, non-PTS
glycerol showed the highest performance in production of all four carotenoid structures, whereas PTS glucose showed the lowest
performance. Based on the conversion yield, carotenoid-producing capability, and the cell density, we found that there was
no close correlation between PTS and non-PTS transport mechanism and carotenoid formations in E. coli. 相似文献
9.
A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EIIman), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (K
m=11 M) and a low-affinity proton motive force driven glucose permease (GP) (K
m=170 M). (ii) Both 9R-4 and X-160 had only the low-affinity system (K
m=181 M for 9R-4, 278 M for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EIIman) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.Abbreviations GCR
glucose-mediated catabolite repression
- FCR
fructose-mediated catabolite repression
- PEP
phosphoenolpyruvate
- man:PTS
phosphoenolpyruvate:mannose phosphotransferase system
- glc:PTS
phosphoenolpyruvate:glucose phosphotransferase system
- GP
glucose permease
- CCCP
carbonylcyanide mchlorophenylhydrazone
- DCCD
N,N-dicyclohexylcarbodiimide
- P
proton motive force
- G-6-P
glucose-6-phosphate
- 2DG
2-deoxyglucose
- IAA
iodoacetic acid
- EIIman
enzyme II component of man:PTS
- EIIIman
enzyme III component of man:PTS
- EIIglc
enzyme II component of glc:PTS
- EIIIglc
enzyme III component of glc:PTS 相似文献
10.
Characterization of Streptococcus mutans Strains Deficient in EIIABMan of the Sugar Phosphotransferase System 下载免费PDF全文
The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIABMan (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIABMan is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIABMan transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIABMan controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIABMan-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIABMan is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIABMan-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIABMan has a pleiotropic effect on gene expression. 相似文献
11.
C Aguilar A Escalante N Flores R de Anda F Riveros-McKay G Gosset E Morett F Bolívar 《BMC genomics》2012,13(1):385
ABSTRACT: BACKGROUND: Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400 % increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways. RESULTS: Whole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna) and in other putative regulatory loci (yjjU, rssA and ypdA). In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented. CONCLUSIONS: The deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased its growth rate, very likely by increasing glycolytic mRNA genes stability. Furthermore, galR inactivation allowed glucose transport by GalP into the cell. The deletion of mutH in an already stressed strain that lacks PTS is apparently responsible for the very high mutation rate observed. 相似文献
12.
13.
A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium. 总被引:5,自引:0,他引:5
M H Saier D L Wentzel B U Feucht J J Judice 《The Journal of biological chemistry》1975,250(13):5089-5096
Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome. 相似文献
14.
Muñoz AJ Hernández-Chávez G de Anda R Martínez A Bolívar F Gosset G 《Journal of industrial microbiology & biotechnology》2011,38(11):1845-1852
l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate
Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways.
Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant
version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase
from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS− gluc+ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase
in the specific rate of l-Tyr production (q
l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q
l-Tyr in the PTS+ and the PTS− gluc+ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS− gluc+
tyrR
− strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS− gluc+
tyrR
− phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h. 相似文献
15.
16.
Sigala JC Flores S Flores N Aguilar C de Anda R Gosset G Bolívar F 《Journal of molecular microbiology and biotechnology》2009,16(3-4):224-235
The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS(-)). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS(-)Glc(+)), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (mu) higher than PB12 (0.21 and 0.13 h(-1), respectively) while JM101 had a mu of 0.28 h(-1). To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS(-) strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS(-) derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12. 相似文献
17.
Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIAGlc, and enzyme IIBCGlc. In Escherichia coli, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the Vibrio cholerae PTS were coregulated with the vps genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections. 相似文献
18.
In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate. 相似文献
19.
Physiological and transcriptional characterization of Escherichia coli strains lacking interconversion of phosphoenolpyruvate and pyruvate when glucose and acetate are coutilized 下载免费PDF全文
Andrea Sabido Juan Carlos Sigala Georgina Hernández‐Chávez Noemí Flores Guillermo Gosset Francisco Bolívar 《Biotechnology and bioengineering》2014,111(6):1150-1160
20.
Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than
5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2
was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains
had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS
activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity
than 581AXY2 (1600 versus 600 nmol glucose (mg protein)−1(min)−1. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K
s
) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The
low capacity system had a Ks of 0.20 mM, but the Ks of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity
of glucose enzyme II and the velocity of glucose transport.
Received: 22 January 1996 / Accepted: 18 March 1996 相似文献