Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

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.     

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 7.5 g l⁻¹ and 0.36 g (g xylose consumed)⁻¹. These parameters improved to 13.4 g l⁻¹ and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l⁻¹ glucose and 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 29.8 g l⁻¹ and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l⁻¹ and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield. Received: 14 May 1999 / Received revision: 27 September 1999 / Accepted: 2 October 1999     

Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae

Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation

Xylose isomerase (XI) is a key enzyme in the conversion of d ‐xylose, which is a major component of lignocellulosic biomass, to d ‐xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI‐related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell‐surface display system, and the xylose assimilation and fermentation properties of this XI‐displaying yeast were examined. XI‐displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

The effect of xylose reductase genes on xylitol production by industrial Saccharomyces cerevisiae in fermentation of glucose and xylose

The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol co-production from straw feedstocks.     

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

Continuous alcoholic fermentation of glucose/xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae

Viable Saccharomyces cerevisiae and Candida shehatae cells were co-immobilized in a composite agar layer/microporous membrane structure. This immobilized-cell structure was placed in a vertical position between the two halves of a double-chambered, stainless-steel bioreactor of original design and applied to the continuous alcoholic fermentation of a mixture of glucose (35 g dm⁻³) and xylose (15 g dm⁻³). Various dilution rates and initial cell loadings of the gel layer were tested. Simultaneous consumption of the two sugars was always observed. The best fermentation performance was obtained at low dilution rate (0.02 h⁻¹) with an excess of C. shehatae over S. cerevisiae in the initial cell loading of the gel (5.0 mg dry weight and 0.65 mg dry weight cm⁻³ gel respectively): 100% of glucose and 73% of xylose were consumed with an ethanol yield coefficient of 0.48 g g total sugars⁻¹. In these conditions, however, the ethanol production rate per unit volume of gel remained low (0.37 g h⁻¹ dm⁻³). Viable cell counts in gel samples after incubation highlighted significant heterogeneities in the spatial distribution of the two yeast species in both the vertical and the transverse directions. In particular, the overall cell number decreased from the bottom to the top of the agar sheet, which may explain the low ethanol productivity relative to the total gel volume. Received: 26 February 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998     

Thermus thermophilus HB8葡萄糖异构酶在大肠杆菌中表达

为了增加高热稳定性的葡萄糖异构酶的得率,采用PCR技术扩增得到Thermus thermophilusHB8葡萄糖异构酶基因xylA,连接到表达载体pET-22b( )上,获得重组质粒pET-22b( )-xylA。将重组质粒转化到大肠杆菌Rosetta(DE3)中,经IPTG诱导后,通过半胱氨酸-咔唑法测葡萄糖异构酶酶活。重组菌经诱导培养,SDS-PAGE电泳结果显示出明显的分子量约为44 kD特异性蛋白质条带,比酶活约为18.562 U/mg,比野生型菌株提高了2倍。     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus

A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.     

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose–xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L⁻¹ acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L⁻¹) in all cultures. Xylose fermentation in acetic –acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.     

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.     

Regulation of the urea active transporter gene (DUR3) in Saccharomyces cerevisiae.

The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined. The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins. Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function. Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway. High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes. Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product. DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.     

Effect of xylose incubation on the glucose transport system in Saccharomyces cerevisiae

Incubation of Saccharomyces cerevisiae with xylose and ethanol for 16 hours leads to a decrease of hexokinase (and glucokinase) activity in the cells. It does not alter the levels of polyphosphate, orthophosphate and ATP. The transport of the glucose derivative 2-deoxy-D-glucose, a sugar that can be phosphorylated, is inhibited after this treatment, whereas transport of 6-deoxy-D-glucose, which has a blocked phosphorylation site, is not inhibited. Even though, both deoxyglucoses use the same transport system. The decrease in initial velocity of 2-deoxy-D-glucose transport is most pronounced under anaerobic conditions. Incubation of the cells with antimycin A, a treatment which has a similar effect as anaerobiosis, shows, that the inhibition of the transport of 2-deoxy-D-glucose is presumably the result of an increase in the Km of the carrier transport. Transport of glucose is probably regulated by kinase enzymes.