Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium

The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115. Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.     

The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for alpha4 PYC enzymes from other sources. R. etli PYC was able to restore the growth of an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the /dh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.     

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.     

Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in the flbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible beta-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of beta-galactosidase in ots-lacZ fusion mutants.     

Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield.     

Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase

In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme.     

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA),该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10．8倍,tktA活性提高了3．9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大(2．1～9．1倍),TktA的活性相对稳定(3．9～4．5倍),且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。     

Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C. glutamicum ldhA promoter were expressed at high levels in C. glutamicum, resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase (ldhA) gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase (ppc) and ldhA genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic C. glutamicum in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in C. glutamicum are correlated to oxygen-deprived metabolic flows.     

Control of gluconeogenic growth by pps and pck in Escherichia coli.

It is well-known that Escherichia coli grows more slowly on gluconeogenic carbon sources than on glucose. This phenomenon has been attributed to either energy or monomer limitation. To investigate this problem further, we varied the expression levels of pck, encoding phosphoenolpyruvate carboxykinase (Pck), and pps, encoding phosphoenolpyruvate synthase (Pps). We found that the growth rates of E. coli in minimal medium supplemented with succinate and with pyruvate are limited by the levels of Pck and Pps, respectively. Optimal overexpression of pck or pps increases the unrestricted growth rates on succinate and on pyruvate, respectively, to the same level attained by the wild-type growth rate on glycerol. Since Pps is needed to supply precursors for biosyntheses, we conclude that E. coli growing on pyruvate is limited by monomer supply. However, because pck is required both for biosyntheses and catabolism for cells growing on succinate, it is possible that growth on succinate is limited by both monomer and energy supplies. The growth yield with respect to oxygen remains approximately constant, even though the overproduction of these enzymes enhances gluconeogenic growth. It appears that the constant yield for oxygen is characteristic of efficient growth on a particular substrate and that the yield is already optimal for wild-type strains. Further increases in either Pck or Pps above the optimal levels become growth inhibitory, and the growth yield for oxygen is reduced, indicating less efficient growth.     

Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum

Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol)(-1), whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol)(-1). Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol)(-1). A further increase is possible by the application of additional C1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C1 carbon and sulfur (0.91 (C-mol) (C-mol)(-1)). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g(-1) h(-1), as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g(-1) h(-1). The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.     

Some biochemical effects of 4-deoxy-4-fluoro-D-glucose on Escherichia coli.

The uptake of 4-deoxy-4-fluoro-D-glucose (4FG), without subsequent catabolism, by resting cells of Escherichia coli (ATCC 11775) is 0.06 mg/mg dry weight. In frozen-thawed cells of this organism, 4FG is a substrate for the phosphoenolpyruvate phosphotransferase system with a rate of phosphorylation twice that found for the isomeric 3-deoxy-3-fluoro-D-glucose. 4FG is not a carbon source for growth of this organism and it inhibits the extent of growth of cells in the presence of glucose. The inhibition of growth of E. coli K12 on lactose by 4FG is also observed and this is considered to be consistent with the fact that 4FG is an uncompetitive inhibitor of beta-galactosidase (EC 3.2.1.23) activity and that 4FG or 4-deoxy-4-fluoro-D-glucose-6 phosphate repress beta-galactosidase synthesis. These results support the view that catabolite repression may be produced by compounds which are not necessarily metabolised further than hexose-6-phosphates.     

Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli

The heterologous biosynthesis of complex polyketides in Escherichia coli was recently achieved through metabolic engineering. However, it was observed that less than 10% of the propionate carbon source is transformed into the erythromycin precursor, 6-deoxyerythronolide B (6dEB), resulting in a 1.4% molar yield. Therefore, metabolic flux analysis was performed using a model of the Escherichia coli metabolism with the addition of the enzymes required for 6dEB synthesis. The analysis shows that the maximum theoretical yield for 6dEB synthesis in E. coli is 11%. The maintenance energy requirement of E. coli and limitations in the specific oxygen uptake rate can further decrease the yield, suggesting that the observed 6dEB yield of 1.4% can be the result of these two factors. In addition, the results suggest that an increase in the specific carbon and oxygen uptake rates will increase the yield of 6dEB. The use of glucose as an alternative carbon source was also evaluated using metabolic flux analysis and the results suggest that the choice of glucose as the carbon source will allow a small improvement in performance relative to a propionate-based process.