Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Some aspects of xylose isomerase constitutive biosynthesis in <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis>

The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product of xylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M_r of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA _Kp 5 upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5 upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.     

Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae

Summary The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity. A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used. Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping. Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences. Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7. The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis. The coding region includes a 2.05 kb EcoRI fragment common to all four inserts. A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus. Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus. This showed that the PGI1 gene had been isolated. Finally, and in contrast to the results of Kempe et al. (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis.     

Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis sp. PCC 6803 and mammalian glucose transporters

Synechocystis sp. PCC 6803 is capable of facultative photoheterotrophy with glucose as the sole carbon source. Eight mutants that were unable to take up glucose were transformed with plasmids from pooled gene banks of wild-type Synechocystis DNA prepared in an Escherichia coli vector that does not replicate in Synechocystis. One mutant (EG216) could be complemented with all gene banks to restore ability for photoheterotrophic growth. One of the gene banks was fractionated into single clones and plasmid DNA from each clone used to complement EG216. This yielded a 1.5 kb DNA fragment that was sequenced. It contained one complete open reading frame (gtr) whose putative gene product displayed high sequence conservation with the xylose transporter of E. coli and the mammalian glucose transporters. Further, the isolated gtr gene interrupted in vitro by a kanamycin resistance cassette could be used to construct mutants from wild-type Synechocystis sp. PCC 6803 that lacked a functional glucose transporter, thus confirming the identity of the gtr gene with the glucose transporter gene. This is the first prokaryotic glucose transporter known to share a sequence relationship with mammalian glucose transporters and the first sugar transporter from a cyanobacterium characterized at the sequence level.     

Construction of a new strain of Streptomyces violaceoniger,having strong,constitutive and stable glucose-isomerase activity

Summary A newly isolated strong Streptomyces promoter (P1) has been cloned in front of the xylA gene of Streptomyces violaceoniger. This led to a strong and constitutive expression. To avoid instability of plasmid and glucose isomerase activity, the P1-xylA gene has been integrated into the chromosome using the integrative vector pTS55. The resultant CBS1 strain has about seven times higher glucose-isomerase activity in absence of xylose compared to that of wild type strain fully induced by xylose. In addition, glucose isomerase specific activity of the CBS1 strain increases in the secondary growth phase, in contrast to wild type strain.     

Thermal inducible expression of xylose isomerase and its performance in a hollow fiber bioreactor

Summary TheEscherichia coli xylose isomerase (EC 5.3.1.5) has been expressed under the control of a thermal inverting promotor system (att-nutL-p-att-N block) and its performance in a hollow fiber bioreactor measured. The conversion of xylose to xylulose was inversely proportional to the flow rate and the system operated up to 60°C. The maximum conversion efficiency observed was 19.05% at 55°C.     

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Xylose and Glucose Utilization by Bacteroides xylanolyticus X5-1 Cells Grown in Batch and Continuous Culture

During cultivation on a mixture of xylose and glucose, Bacteroides xylanolyticus X5-1 showed neither diauxic growth nor a substrate preference. Xylose-limited continuous-culture cells were able to consume xylose and glucose both as single substrates and as mixed substrates without any lag phase. When glucose was the growth-limiting substrate, the microorganism was unable to consume xylose. However, in the presence of a small amount of glucose or pyruvate, xylose was utilized after a short lag phase. In glucose-limited cells, xylose isomerase was present at low activity but xylulose kinase activity could not be detected. On addition of a mixture of xylose and glucose, xylose isomerase was induced immediately and xylulose kinase was induced after about 30 min. The induction of the two enzymes was sensitive to chloramphenicol, showing de novo synthesis. Xylose uptake in glucose-grown cells was very low, but the uptake rate could be increased when incubated with a xylose-glucose mixture. The increase in the uptake rate was not affected by chloramphenicol, indicating that a constitutive uptake system had to be activated. The inability of B. xylanolyticus X5-1 cells undergoing glucose-limited continuous culture to induce the xylose catabolic pathway after the addition of only xylose probably was caused by energy limitation.     

Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a V_max of 0.142 U/mg purified rXI, and a K_M for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol K_I values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low K_M of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca²⁺ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher V_max at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016     

The plasmids of Acetobacter xylinum and their interaction with the host chromosome

Summary Acetobacter xylinum contains a complex system of plasmid DNA molecules. Plasmids of molecular weights or copy numbers different from the original wild-type, are found in different types of mutants. Restriction endonuclease digestion and DNA/DNA hybridization analysis, showed that the plasmids often contained partly, but not completely the same DNA sequences. Two of these plasmid classes were analysed in more detail, and could be shown to differ in size by about 5 kb. Hybridization analysis using cloned DNA fragments as probes, showed that sequences lacking in the smallest plasmid were still present in a DNA fraction co-migrating with linearized chromosomal DNA. In addition, at least part of the DNA in the smallest plasmid was present both in the plasmid and chromosomal DNA fraction. Analysis of a particular strain containing an insertion of transposon Tn1, also indicated the existence of complex interactions between plasmids and chromosomal DNA. Together with experiments on conjugative transfer and curing of the plasmids, the results indicate that at least part of the genetic system of A. xylinum is unusual when compared to that of other genetically characterized bacteria.     

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion.     

Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae

Summary The structural gene PG11 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PG11 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PG11 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 strains did not grow in glucose as sole carbon source. On the other hand pgi1/pgi1 diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that (a) the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, (b) glucose-6-P is essential in yeasts, and (c) the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.     

High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli

Summary A thermophilic Bacillus sp. producing xylose (glucose) isomerase has been isolated. Its xy/A gene when cloned in Escherichia coli and expressed gave 37.5 and 12.8 units/ mg protein respectively for xylose and glucose isomerase activities at 85°C. A single heat treatment of the crude extract purified the enzyme further yielding the highest ever recorded activities of 150 and 49.02 units /mg protein.     

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.     

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL^CS displayed significantly increased anaerobic growth rate (0.203±0.006 h^?1) and xylose consumption rate (1.866 g g^?1 h^?1) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.