Glucose-6-phosphate dehydrogenase deficiency in pleiotropic carbohydrate-negative mutant strains of Rhizobium meliloti

Several mutant strains of Rhizobium meliloti isolated after nitrosoguanidine mutagenesis were selected as unable to grow on mannose. Some of them also failed to grow on glucose, fructose, ribose, and xylose but grew on L-arabinose, galactose, and many other carbon sources. Biochemical analysis demonstrated that the mutants lacked NAD- and NADP-linked glucose-6-phosphate dehydrogenase activities that reside on a single enzyme species. One such mutant was found to accumulate glucose-6-phosphate, and this could partially explain the inhibition of growth observed on mixtures of permissive and nonpermissive carbon sources. Symbiotic properties remained unaffected in all these mutants.     

alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

A UV-induced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance

In the fermentation process of lignocellulosic biomass (such as wood and rice straw), efficient conversion of pentose (mainly xylose) into ethanol is important. Mutants of Pichia stipitis NBRC1687 were obtained after UV mutagenesis and selection of large colonies on ethanol-containing medium. One mutant, PXF58, produced 4.3% ethanol from 11.4% xylose while the parent strain only produced 3.1%. The ethanol productivities of PXF58 from glucose and fructose were about were about 1.4-fold higher than those of the parent strain. After continuous cultivation of PXF58 in YNB (yeast nitrogen base) medium containing 2% xylose and 5-7% ethanol, an ethanol-tolerant mutant, PET41, was obtained. Strain PET41 was able to produce 4.4% ethanol when first supplied with xylose then with glucose. This isolate might be thus useful for two-phase fermentation in which xylan is saccharified by xylanase to produce xylose, and glucan is saccharified later by cellulase and β-glucosidase to produce glucose.     

Effect of alanine, leucine and fructose on lysyl-transfer ribonucleic acid ligase activity in a mutant of Escherichia coli K-12

A mutant of Escherichia coli K-12 was examined which has growth medium-dependent lysyl-transfer ribonucleic acid (tRNA) ligase activity. In minimal medium or 0.5% yeast extract, the activity of the enzyme in the mutant strain was 5 to 10% of wild type. However, when the mutant was grown in a highly enriched medium, such as AC broth (Difco), the activity of the mutant ligase increased 10- to 20-fold. We found that the supplementation of 0.5% yeast extract by l-alanine plus d-fructose replaces the need for the highly enriched medium. Fructose plus l-leucine and fructose plus l-alpha-amino-n-butyric acid were also stimulatory, but not as effective as fructose and alanine. With minimal medium, a combination of carbohydrate (fructose or glucose) plus alanine and leucine was required to replace the enriched medium. The most effective combination was fructose, glucose, alanine, and leucine. Lysyl-tRNA ligase was stimulated 1.5 to 2-fold in the wild-type strain or Hfr H (Hayes) by fructose plus alanine when these strains were cultured in 0.5% yeast extract. Experiments employing the combined technique of density labeling with D(2)O and isopycnic centrifugation in cesium chloride indicated that the increased activity of lysyl-tRNA ligase observed in AC broth or in the presence of fructose, glucose, alanine, and leucine is due to the synthesis of new enzyme.     

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.     

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.     

不同碳源对大肠杆菌lpdA突变菌累积丙酮酸的影响

为了利用大肠杆菌构建模式"细胞工厂",必须了解在构建过程中各种因素的影响。本研究选用敲除了lpdA基因的大肠杆菌作为模型细胞,考察了该突变菌在合成培养基中利用葡萄糖、果糖、木糖和甘露糖累积丙酮酸的能力。结果显示,在初始糖浓度为10g/L的情况下,lpdA突变菌可以很好地利用葡萄糖、果糖、木糖和甘露糖转化丙酮酸,其得率分别达到了0.884g/g、0.802g/g、0.817g/g和0.808g/g,且在以葡萄糖、果糖和木糖发酵时,丙酮酸的积累过程与细胞生长偶联。甘露糖发酵的情况则不同:菌浓度很快达到平台期,随后丙酮酸积累和甘露糖消耗都表现为线性变化。当在考察了不同的接种量对lpdA突变菌发酵葡萄糖的影响时发现,大接种量能加快葡萄糖消耗速率、丙酮酸的积累速率和细胞生长速率,但丙酮酸得率却明显下降。这些结果对构建以大肠杆菌为母体的模式"细胞工厂"有参考价值。     

Succinate dehydrogenase mutant of Rhizobium meliloti.

A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis. It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates. The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective. A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.     

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.     

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.     

Catabolism of carbohydrates and organic acids and expression of nitrogenase by azospirilla

Fructose, galactose, L-arabinose, gluconate, and several organic acids support rapid growth and N2 fixation of Azospirillum brasiliense ATCC 29145 (strain Sp7) as a sole source of carbon and energy. Growth of Azospirillum lipoferum ATCC 29707 (strain Sp59b) is also supported by glucose, mannose, mannitol, and alpha-ketoglutarate. Oxidation of fructose and gluconate by A. brasiliense Sp7 and of glucose, gluconate, and fructose by A. lipoferum Sp59b was achieved through inducible enzymatic mechanisms. Both strains exhibited all of the enzymes of the Embden-Meyerhof-Parnas pathway, and strain Sp59b also possesses all the enzymes of the Entner-Doudoroff pathway. Fluoride inhibited growth on fructose (strains Sp7 and Sp59b) or on glucose (strain Sp59b) but not on malate. There was no activity via the oxidative hexose monophosphate pathway in either strain. There was greater activity with 1-phosphofructokinase than with 6-phosphofructokinase in both strains. Strain Sp59b formed fructose-6-phosphate via hexokinase, an enzyme that is lacking in strain Sp7. A. brasiliense and A. lipoferum exhibited the enzymes both of the tricarboxylic acid cycle and of the glyoxylate shunt; iodoacetate, fluoropyruvate, and malonate were inhibitory. A. brasiliense Sp7 could not transport [14C]glucose and alpha-[14C]ketoglutarate into its cells.     

NADPH-dependent pgi-gene knockout Escherichia coli metabolism producing shikimate on different carbon sources

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+) strain and its mixed glucose/fructose fermentation grew well. Interestingly, when fructose was used as a carbon source, the pgi(-) mutant showed the enhanced SA production compared with the pgi(+) strain. In silico analysis of a genome-scale E. coli model was then conducted to characterize the cellular metabolism and quantify NAPDH regeneration, which allowed us to understand such experimentally observed attenuated cell growth and enhanced SA production in glucose- and fructose-consuming pgi(-) mutant, respectively with respect to cofactor regeneration.     

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.     

Single point mutations in either gene encoding the subunits of the heterooctameric yeast phosphofructokinase abolish allosteric inhibition by ATP

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger -subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wild-type strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37 degrees C. The latter mutant was shown to be respiration-dependent for growth on glucose.     

Reduction of xylose to xylitol catalyzed by glucose–fructose oxidoreductase from Zymomonas mobilis

Genetic improvements of Zymomonas mobilis for pentose utilization have a huge potential in fuel ethanol production. The production of xylitol and the resulting growth inhibition by xylitol phosphate have been considered to be one of the important factors affecting the rates and yields from xylose metabolism by the recombinant Z. mobilis , but the mechanism of xylitol formation is largely unknown. Here, we reported that glucose–fructose oxidoreductase (GFOR), a periplasmic enzyme responsible for sorbitol production, catalyzed the reduction of xylose to xylitol in vitro , operating via a ping-pong mechanism similar to that in the formation of sorbitol. However, the specific activity of GFOR for sorbitol was higher than that for xylitol (68.39 vs. 1.102 μmol min⁻¹ mg⁻¹), and an apparent substrate-induced positive cooperativity occurred during the catalyzed formation of xylitol, with the Hill coefficient being about 2. While a change of the potential acid–base catalyst Tyr269 to Phe almost completely abolished the activity toward xylose as well as fructose, mutant S116D, which has been shown to lose tight cofactor binding, displayed an even slower catalytic process against xylose.