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.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

果糖和麦芽糖作为有氧碳源对大肠杆菌NZN111两阶段发酵中厌氧发酵的影响

为了了解磷酸转移酶转运系统 (PTS) 依赖和非PTS依赖代谢的糖类对大肠杆菌生产琥珀酸的影响,进行了两阶段培养,有氧阶段采用PTS依赖型的果糖或非PTS依赖型的麦芽糖作为丙酮酸甲酸裂解酶 (PFL) 和乳酸脱氢酶 (LDH) 双突变株NZN111的碳源,研究其对NZN111厌氧阶段代谢葡萄糖的影响。5 L罐发酵结果表明,以果糖和麦芽糖为碳源有氧培养的细胞恢复了在厌氧条件下快速代谢葡萄糖的能力,琥珀酸和丙酮酸成为主要代谢产物,最终琥珀酸得率分别为0.84和0.75 mol/mol,丙酮酸得率分别达到了0.65和0.83 mol/mol,琥珀酸和丙酮酸终浓度比分别为1.73∶1和1.21∶1。果糖和麦芽糖培养的NZN111与葡萄糖培养的菌体代谢的明显差异推测是cyclic AMP (cAMP) 依赖型和非cAMP依赖型的分解代谢物阻遏调控这两种机制共同作用的结果。     

Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.     

Aerobic and anaerobic metabolism in a facultative anaerobe Ep01 lacking cytochrome, quinone and catalase

Abstract A facultative anaerobe, strain Ep01 produced a mixture of pyruvate, formate, acetate and ethanol from glucose anaerobically, and acetate and pyruvate aerobically. Cell extract of anaerobic-grown cells contained active pyruvate formatelyase, aldehyde dehydrogenase and alcohol dehydrogenase, while cell extract of aerobic grown cells contained an active pyruvate dehydrogenase system, NaDH oxidase and NADH peroxidase. Levels of acetate kinase and phosphate acetyltransferase activities were not significantly different in cells grown under either condition. Based on the metabolic products and the emzyme activities, we propose the presence of two metabolic pathways in strain Ep01, namely, a pathway to form formate, acetate and ethanol under anaerobic conditions, and a pathway to form under aerobic conditions. This explains why strain Ep01 can grow well both under strictly anaerobic conditions and well-aerated conditions.     

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.     

Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.     

Evidence for the Presence of an Alternative Glucose Transport System in Clostridium beijerinckii NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.     

Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.     

Occurrence and Characterization of a Phosphoenolpyruvate: Glucose Phosphotransferase System in a Marine Bacterium, Serratia marinorubra

The mechanism of d-glucose transport in the marine bacterium Serratia marinorubra was investigated. Uptake is mediated by a single, constitutive phosphoenolpyruvate:sugar phosphotransferase system (PTS), resulting in phosphorylation of d-glucose to d-glucose phosphate during transport. The system is saturable (K(m) = 6.4 x 10 M) and highly temperature dependent, with a Q(10) of 3.5 between 5 and 15 degrees C. The system is highly specific for d-glucose; structurally related sugars and sugar alcohols did not significantly compete with d-glucose for transport. The PTS requires Mg (K(m) = 2.5 x 10 M), but its activity is otherwise unaffected by salinity changes over the range tested (0 to 35 per thousand). S. marinorubra differs from other gram-negative organisms (Escherichia coli and Salmonella typhimurium) in that its glycerol (non-PTS substrate) permease is not regulated by the presence of glucose (PTS substrate).     

Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system.

Streptococcus mutans transports glucose via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Earlier studies indicated that an alternate glucose transport system functions in this organism under conditions of high growth rates, low pH, or excess glucose. To identify this system, S. mutans BM71 was transformed with integration vector pDC-5 to generate a mutant, DC10, defective in the general PTS protein enzyme I (EI). This mutant expressed a defective EI that had been truncated by approximately 150 amino acids at the carboxyl terminus as revealed by Western blot (immunoblot) analysis with anti-EI antibody and Southern hybridizations with a fragment of the wild-type EI gene as a probe. Phosphotransfer assays utilizing 32P-PEP indicated that DC10 was incapable of phosphorylating HPr and EIIAMan, indicating a nonfunctional PTS. This was confirmed by the fact that DC10 was able to ferment glucose but not a variety of other PTS substrates and phosphorylated glucose with ATP and not PEP. Kinetic assays indicated that the non-PTS system exhibited an apparent Ks of 125 microM for glucose and a Vmax of 0.87 nmol mg (dry weight) of cells-1 min-1. Sugar competition experiments with DC10 indicated that the non-PTS transport system had high specificity for glucose since glucose transport was not significantly by a 100-fold molar excess of several competing sugar substrates, including 2-deoxyglucose and alpha-methylglucoside. These results demonstrate that S. mutans possesses a glucose transport system that can function independently of the PEP PTS.     

Osmotic stress,glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum

Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km=0.35 mM). Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks=11 mM) non-PTS uptake system. During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished. Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions. However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity. This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity. The secondary glucose transporter was, however, not influenced by medium osmolarity. During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed. This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields.     

Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI

We have determined the role played by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in carbon catabolite repression (CCR) of xylose utilization in Bacillus megaterium. For that purpose we have cloned, sequenced and inactivated the genes ptsH and ptsl of B. megaterium, encoding HPr and EI of the PTS, respectively. While glucose uptake of a ptsHI mutant is not affected at 12.5 mM of glucose, CCR of the xyl operon is reduced in this mutant from 16-fold to 3-fold. This may be attributed to the loss of the corepressor of CcpA, HPr(Ser-P), or could result from the slower growth rate of the mutant. In contrast, CCR exerted by fructose or mannitol is completely abolished. We conclude that glucose triggers additional mechanisms of CCR than fructose or mannitol. The remaining 3-fold glucose repression is relieved in a strain in which ptsHI and glk, encoding glucokinase, are inactivated. This result indicates that glucose metabolism is necessary for CCR. The ability of the ptsHI mutant to take up glucose suggests the existence of a second, non-PTS glucose uptake system. The Km and vmax values of this transporter ranged between 2 and 5 mM and 154 to 219 nmol/[(mg protein)*min], respectively.     

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.