Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.     

D-xylose utilization by Saccharomyces cerevisiae

Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate D-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of D-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as D-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of D-glucose, D-ribose, D-raffinose, glycerol and D-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91.3 +/- 0.8%), CO2 (2.6 +/- 2.3%) and biomass (1.7 +/- 0.6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.     

Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae.

The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated. Xylose was taken up by means of low- and high-affinity glucose transport systems. In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected. Glucose strongly inhibited the transport of xylose by both systems. Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose. A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca. 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture. No activity converting xylitol to xylulose or vice versa was found in crude extracts. Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy. Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist. However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-[1-13C]xylose was labelled only in the C-2 position. Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.     

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.     

The glucose/xylose facilitator Gxf1 from Candida intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw

Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose.     

Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.     

Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease.

We isolated 10 mannitol-positive mutants from a mannitol-negative Escherichia coli strain. These mutations mapped within ptsG, encoding the glucose permease (EIIGlc), and resulted in a G-320-to-V substitution that allows EIIGlc to transport mannitol. Gly-320 lies within a putative transmembrane helix of EIIGlc that may be involved in substrate recognition.     

皮状丝孢酵母同步利用葡萄糖/木糖的糖转运动力学简

皮状丝孢酵母（ Trichosporon cutaneum）能够同步利用葡萄糖和木糖生产油脂。以2脱氧葡萄糖（2 DOG）为底物,考察皮状丝孢酵母糖跨膜运输的转运动力学。结果表明：2 DOG转运符合米氏方程,表观米氏常数Km为0.19 mmol/L,最大转运速率Vmax为14.1 nmol/（ min＆#183;mg）。葡萄糖和木糖均竞争性抑制2 DOG转运,葡萄糖表观抑制常数Ki远低于木糖,表明存在一个共用转运体系,且该转运体系对葡萄糖亲和力更高。大量木糖与2 DOG同时转运到胞内,进一步说明木糖与葡萄糖共运输。代谢抑制剂和pH对糖转运有明显影响,说明质子/底物同向运输系统是该酵母的主要糖转运系统。     

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.     

Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium.

Transport and phosphorylation of glucose via enzymes II-A/II-B and II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system are tightly coupled in Salmonella typhimurium. Mutant strains (pts) that lack the phosphorylating proteins of this system, enzyme I and HPr, are unable to transport or to grow on glucose. From ptsHI deletion strains of S. typhimurium, mutants were isolated that regained growth on and transport of glucose. Several lines of evidence suggest that these Glc+ mutants have an altered enzyme II-BGlc as follows. (i) Insertion of a ptsG::Tn10 mutation (resulting in a defective II-BGlc) abolished growth on and transport of glucose in these Glc+ strains. Introduction of a ptsM mutation, on the other hand, which abolishes II-A/II-B activity, had no effect. (ii) Methyl alpha-glucoside transport and phosphorylation (specific for II-BGlc) was lowered or absent in ptsH+,I+ transductants of these Glc+ strains. Transport and phosphorylation of other phosphoenolpyurate:sugar phosphotransferase system sugars were normal. (iii) Membranes isolated from these Glc+ mutants were unable to catalyze transphosphorylation of methyl alpha-glucoside by glucose 6-phosphate, but transphosphorylation of mannose by glucose 6-phosphate was normal. (iv) The mutation was in the ptsG gene or closely linked to it. We conclude that the altered enzyme II-BGlc has acquired the capacity to transport glucose in the absence of phosphoenolpyruvate:sugar phosphotransferase system-mediated phosphorylation. However, the affinity for glucose decreased at least 1,000-fold as compared to the wild-type strain. At the same time the mutated enzyme II-BGlc lost the ability to catalyze the phosphorylation of its substrates via IIIGlc.     

Comparative study of xylose(glucose) isomerase synthesis in Arthrobacter strains

The substrate specificity of isomerases produced by six strains of Arthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A. ureafaciens B-6 strains showed low activity toward D-ribose, Arthrobacter sp. B-5 was slightly active toward L-arabinose, and A. ureafaciens B-6 and Arthrobacter sp. B-2239, toward L-rhamnose. In Arthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources). The synthesis of xylose/glucose isomerase induced by D-xylose in Arthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol in A. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in the Arthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Production of xylitol from D-xylose by recombinant Lactococcus lactis

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.     

Inability of Lactobacillus plantarum and other lactic acid bacteria to grow on D-ribose as sole source of fermentable carbohydrate

Lactobacillusplantarum NCIMB 8026, NCIMB 8026(s), NCIMB 8014, NCFB 1752, Lact. brevis NCIMB 4617, Leuconostoc mesenteroides NCIMB 8023, Streptococcus agalactiae NCFB 1348, Pediococcus acidilactici NCFB 1859 and Ped. pentosaceus NCFB 990 did not grow on D-ribose as the sole source of fermentable carbohydrate in a chemically defined medium but grew on D-ribose in the presence of glucose. Lactobacillus plantarum NCIMB 8026(s) also grew on D-xylose and L-arabinose in the presence but not in the absence of glucose. Enterococcus faecalis NCFB 581 grew with D-ribose as the sole fermentable carbohydrate. Leuconostoc mesenteroides NCIMB 8710 and Lactococcus lactis subsp. lactis NCFB 763 did not use ribose in the presence or absence of glucose. Lactobacillus plantarum NCIMB 8026(s) utilized ribose and glucose simultaneously in the proportion of approximately 1 ribose to 1 glucose, producing approximately 3 lactate to 1 acetate and similar yields of dry biomass from glucose and ribose. Growth of Lact. plantarum 8026(s) with glucose and excess D-ribose ceased when D-glucose was exhausted, but metabolism of D-ribose to lactic and acetic acids continued. The enzyme system for the metabolism of D-ribose in Lact. plantarum was inducible, requiring D-glucose and amino acids for adaptation.     

A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae

Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved V(max) values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.