Computer‐aided rational design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli

The phosphotransferase system (PTS) is the sugar transportation machinery that is widely distributed in prokaryotes and is critical for enhanced production of useful metabolites. To increase the glucose uptake rate, we propose a rational strategy for designing the molecular architecture of the Escherichia coli glucose PTS by using a computer‐aided design (CAD) system and verified the simulated results with biological experiments. CAD supports construction of a biochemical map, mathematical modeling, simulation, and system analysis. Assuming that the PTS aims at controlling the glucose uptake rate, the PTS was decomposed into hierarchical modules, functional and flux modules, and the effect of changes in gene expression on the glucose uptake rate was simulated to make a rational strategy of how the gene regulatory network is engineered. Such design and analysis predicted that the mlc knockout mutant with ptsI gene overexpression would greatly increase the specific glucose uptake rate. By using biological experiments, we validated the prediction and the presented strategy, thereby enhancing the specific glucose uptake rate.     

Sugar transport systems in Corynebacterium glutamicum: features and applications to strain development

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. This means that worldwide amino acid production using this organism has depended exclusively on the PTS. Recently, a better understanding not only of PTS-mediated sugar uptake but also of global regulation associated with the PTS has permitted the correction of certain negative aspects of this sugar transport system for amino acid production. In addition, the recent identification of different glucose uptake systems in this organism has led to a strategy for the generation of C. glutamicum strains that express non-PTS routes instead of the original PTS. The potential practical advantages of the development of such strains are discussed.     

Carbohydrate uptake by escherichia coli

In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1- or 6- phosphate ester. The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar. Both their synthesis and their activity are controlled; in the latter, “fine” control, glucose-6-phosphate appears to play an important role. Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar-specific components of the PTS.     

Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system

Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae , regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzae ptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild-type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP-dependent sugars was restored by addition of cAMP. In wild-type cells the non-metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250-fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose-specific PTS.     

Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics

Modifying substrate uptake systems is a potentially powerful tool in metabolic engineering. This research investigates energetic and metabolic changes brought about by the genetic modification of the glucose uptake and phosphorylation system of Escherichia coli. The engineered strain PPA316, which lacks the E. coli phosphotransferase system (PTS) and uses instead the galactose-proton symport system for glucose uptake, exhibited significantly altered metabolic patterns relative to the parent strain PPA305 which retains PTS activity. Replacement of a PTS uptake system by the galactose-proton symport system is expected to lower the carbon flux to pyruvate in both aerobic and anaerobic cultivations. The extra energy cost in substrate uptake for the non-PTS strain PPA 316 had a greater effect on anaerobic specific growth rate, which was reduced by a factor of five relative to PPA 305, while PPA 316 reached a specific growth rate of 60% of that of the PTS strain under aerobic conditions. The maximal cell densities obtained with PPA 316 were approximately 8% higher than those of the PTS strain under aerobic conditions and 14% lower under anaerobic conditions. In vivo NMR results showed that the non-PTS strain possesses a dramatically different intracellular environment, as evidenced by lower levels of total sugar phosphate, NAD(H), nucleoside triphosphates and phosphoenolpyruvate, and higher levels of nucleoside diphosphates. The sugar phosphate compositions, as measured by extract NMR, were considerably different between these two strains. Data suggest that limitations in the rates of steps catalyzed by glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase may be responsible for the low overall rate of glucose metabolism in PPA316. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 583-590, 1997.     

Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum

Growth of Corynebacterium glutamicum on mixtures of glucose and fructose leads to simultaneous consumption of both sugars in which the uptake of each sugar is directly related to the expression of the corresponding sugar uptake mechanism. The overall rate of sugar uptake was higher on sugar mixtures than on either glucose or fructose alone and was similar to that observed during sucrose metabolism. The results suggest that sugar uptake limits metabolic rates though, in the case of fructose, overflow metabolism of both lactate and dihydroxyacetone was observed. Such products could reflect a higher flux through glycolysis rather than the pentose pathway during catabolism of fructose. Received: 24 October 1996 / Received revision: 10 January 1997 / Accepted: 10 January 1997     

Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr.

By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion.     

Bacterial sugar utilization gives rise to distinct single‐cell behaviours

Inducible utilization pathways reflect widespread microbial strategies to uptake and consume sugars from the environment. Despite their broad importance and extensive characterization, little is known how these pathways naturally respond to their inducing sugar in individual cells. Here, we performed single‐cell analyses to probe the behaviour of representative pathways in the model bacterium Escherichia coli. We observed diverse single‐cell behaviours, including uniform responses (d ‐lactose, d ‐galactose, N‐acetylglucosamine, N‐acetylneuraminic acid), ‘all‐or‐none’ responses (d ‐xylose, l ‐rhamnose) and complex combinations thereof (l ‐arabinose, d ‐gluconate). Mathematical modelling and probing of genetically modified pathways revealed that the simple framework underlying these pathways – inducible transport and inducible catabolism – could give rise to most of these behaviours. Sugar catabolism was also an important feature, as disruption of catabolism eliminated tunable induction as well as enhanced memory of previous conditions. For instance, disruption of catabolism in pathways that respond to endogenously synthesized sugars led to full pathway induction even in the absence of exogenous sugar. Our findings demonstrate the remarkable flexibility of this simple biological framework, with direct implications for environmental adaptation and the engineering of synthetic utilization pathways as titratable expression systems and for metabolic engineering.     

Fructose transport by Escherichia coli

The utilization of fructose by Escherichia coli involves, as first step, the uptake of the sugar, normally via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This fructose-specific PTS differs in several ways from that effecting the uptake of other sugars that also possess the 3,4,5-D-arabino-hexose configuration: these differences are discussed. Mutants that lack the genes ptsI and ptsH, which specify components of the PTS common to most PT-sugars, can mutate further to regain the ability to utilize fructose when this is present in relatively high concentration (i.e. greater than 2 mM) in the medium. Some of the properties of this unusual uptake system is discussed.     

The transport and mediation mechanisms of the common sugars in Escherichia coli

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.     

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.     

Histidine phosphocarrier protein regulates pyruvate kinase A activity in response to glucose in Vibrio vulnificus

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) consists of two general energy‐coupling proteins [enzyme I and histidine phosphocarrier protein (HPr)] and several sugar‐specific enzyme IIs. Although, in addition to the phosphorylation‐coupled transport of sugars, various regulatory roles of PTS components have been identified in Escherichia coli, much less is known about the PTS in the opportunistic human pathogen Vibrio vulnificus. In this study, we have identified pyruvate kinase A (PykA) as a binding partner of HPr in V. vulnificus. The interaction between HPr and PykA was strictly dependent on the presence of inorganic phosphate, and only dephosphorylated HPr interacted with PykA. Experiments involving domain swapping between the PykAs of V. vulnificus and E. coli revealed the requirement for the C‐terminal domain of V. vulnificus PykA for a specific interaction with V. vulnificus HPr. Dephosphorylated HPr decreased the K_m of PykA for phosphoenolpyruvate by approximately fourfold without affecting V_max. Taken together, these findings indicate that the V. vulnificus PTS catalyzing the first step of glycolysis stimulates the final step of glycolysis in the presence of glucose through the direct interaction of dephospho‐HPr with the C‐terminal domain of PykA.     

Improvement of <Emphasis Type="Italic">Escherichia coli</Emphasis> production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

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.     

Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum

The phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) catalyzes transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. Enzyme I and HPr, encoded in ptsI and ptsH, respectively, are cytoplasmic proteins commonly used for transport of variety of PTS sugars. In this study, we investigated the role of SugR on the expression of the ptsI and ptsH which increases in the presence of PTS sugars in Corynebacterium glutamicum. Disruption of sugR resulted in the increased expression of ptsI and ptsH in the absence of PTS sugar. Introduction of a plasmid containing sugR gene complemented the effect of sugR disruption. SugR was purified and binding to the promoter regions of ptsI and ptsH was indicated by EMSA. DNase I footprinting analysis indicated the binding sites of SugR on the promoter region of divergently transcribed ptsI gene and fructose-pts operon. The binding sites contain a possible SugR binding motif which is conserved in the promoter regions of general and sugar-specific pts genes. Mutations in this motif resulted in the decrease of SugR binding to the ptsI promoter. These results suggest that SugR represses ptsI and ptsH in the absence of PTS sugar and derepression is the mechanism for the induction of the general components of PTS.