Pdc Genes Research Articles

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes

Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5kb) and pRK2 (5.4kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae

The production of higher alcohols by engineered bacteria has received significant attention. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis. Initially, we overexpressed 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in S. cerevisiae to enhance the endogenous activity of the Ehrlich pathway. We then overexpressed Ilv2, which catalyzes the first step in the valine synthetic pathway, and deleted the PDC1 gene encoding a major pyruvate decarboxylase with the intent of altering the abundant ethanol flux via pyruvate. Through these engineering steps, along with modification of culture conditions, the isobutanol titer of S. cerevisiae was elevated 13-fold, from 11 mg/l to 143 mg/l, and the yield was 6.6 mg/g glucose, which is higher than any previously reported value for S. cerevisiae.

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

BackgroundThe use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.ResultsThe presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations.ConclusionIn mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxilase.

Improvement of Ethanol Yield from Glycerol via Conversion of Pyruvate to Ethanol in Metabolically Engineered Saccharomyces cerevisiae

The conversion of low-priced glycerol to higher value products has been proposed as a way to improve the economic viability of the biofuels industry. In a previous study, the conversion of glycerol to ethanol in a metabolically engineered strain of Saccharomyces cerevisiae was accomplished by minimizing the synthesis of glycerol, the main by-product in ethanol fermentation processing. To further improve ethanol production, overexpression of the native genes involved in conversion of pyruvate to ethanol in S. cerevisiae was successfully accomplished. The overexpression of an alcohol dehydrogenase (adh1) and a pyruvate decarboxylase (pdc1) caused an increase in growth rate and glycerol consumption under fermentative conditions, which led to a slight increase of the final ethanol yield. The overall expression of the adh1 and pdc1 genes in the modified strains, combined with the lack of the fps1 and gpd2 genes, resulted in a 1.4-fold increase (about 5.4g/L ethanol produced) in fps1Δgpd2Δ (pGcyaDak, pGupCas) (about 4.0g/L ethanol produced). In summary, it is possible to improve the ethanol yield by overexpression of the genes involved in the conversion of pyruvate to ethanol in engineered S. cerevisiae using glycerol as substrate.

Enhancement of ethanol production from glycerol in a Klebsiella pneumoniae mutant strain by the inactivation of lactate dehydrogenase

Previously, using γ-irradiation treatment, we isolated a mutant strain of Klebsiella pneumoniae (named GEM167) that showed high-level ethanol production from glycerol. In the present study, in an effort to enhance ethanol production, we used a deletion of the lactate dehydrogenase gene to engineer a mutant strain incapable of lactate synthesis. In the Δ ldhA mutant of GEM167, the production of ethanol was significantly increased from 21.5 g/l to 28.9 g/l and from 0.93 g/(l h) to 1.2 g/(l h). Introduction of the Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase and aldehyde dehydrogenase, respectively, further improved the ethanol production level from glycerol to 31.0 g/l; this is the highest level reported to date.

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.

Inactivation of Snt2, a BAH/PHD‐containing transcription factor, impairs pathogenicity and increases autophagosome abundance in Fusarium oxysporum

The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskmelon wilt disease. The current study focused on the most virulent race of FOM-race 1,2. The tagged mutant D122, generated by Agrobacterium tumefaciens-mediated transformation, caused the delayed appearance of initial wilt disease symptoms, as well as a 75% reduction in pathogenicity. D122 was impaired in the gene product homologous to the Snt2-like transcription factor of Schizosaccharomyces pombe. Involvement of snt2 in the early stage of FOM pathogenesis and its requirement for host colonization were confirmed by targeted disruption followed by quantitative reverse transcription-polymerase chain reaction analysis of snt2 expression in planta. Δsnt2 mutants of FOM and Neurospora crassa exhibited similar morphological abnormalities, including a reduction in conidia production and biomass accumulation, slower vegetative growth and frequent hyphal septation. In N. crassa, snt-2 is required for sexual development, as Δsnt-2 mutants were unable to produce mature perithecia. Suppressive subtraction hybridization analysis of the D122 mutant versus wild-type isolate detected four genes (idi4, pdc, msf1, eEF1G) that were found previously in association with the target of rapamycin (TOR) kinase pathway. Expression of the autophagy-related idi4 and pdc genes was found to be up-regulated in the Δsnt2 FOM mutant. In N. crassa, disruption of snt-2 also conferred a significant over-expression of idi4.

Genome‐scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis

Bioethanol has been recognized as a potential alternative energy source. Among various ethanol-producing microbes, Zymomonas mobilis has acquired special attention due to its higher ethanol yield and tolerance. However, cellular metabolism in Z. mobilis remains unclear, hindering its practical application for bioethanol production. To elucidate such physiological characteristics, we reconstructed and validated a genome-scale metabolic network (iZM363) of Z. mobilis ATCC31821 (ZM4) based on its annotated genome and biochemical information. The phenotypic behaviors and metabolic states predicted by our genome-scale model were highly consistent with the experimental observations of Z. mobilis ZM4 strain growing on glucose as well as NMR-measured intracellular fluxes of an engineered strain utilizing glucose, fructose, and xylose. Subsequent comparative analysis with Escherichia coli and Saccharomyces cerevisiae as well as gene essentiality and flux coupling analyses have also confirmed the functional role of pdc and adh genes in the ethanologenic activity of Z. mobilis, thus leading to better understanding of this natural ethanol producer. In future, the current model could be employed to identify potential cell engineering targets, thereby enhancing the productivity of ethanol in Z. mobilis.

Increased ethanol resistance in Ethanolic Escherichia coli by Insertion of heat-shock genes BEM1 and SOD2 from Saccharomyces cerevisiae

Ethanol is generally toxic to microorganisms, and intracellular and extracellular accumulation of ethanol inhibits cell growth and metabolism. In this study, pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) were cloned into pET-32a vector and then introduced into E. coli BL21 to produce ethanol. Heat shock genes (BEM1 and SOD2) from Saccharomyces cerevisiae were inserted into recombinant ethanolic E. coli using pET28_a vector to improve ethanol shock resistance. Three different strains were constructed: Ethanolic E. coli (adhB and pdc genes inserted using pET32_a vector), BEM1 gene-inserted E. coli (BEM1 inserted using pET_28a), and SOD2-inserted E. coli (SOD2 inserted using pET28_a). Construction of these three different strains allowed comparison of the functions of these heat shock genes as well as their roles in ethanol tolerance. The toxicity of ethanol in recombinant ethanolic E. coli was tested by measuring cell growth in response to various ethanol concentrations. The results show that SOD2-inserted E. coli showed higher ethanol resistance than ethanolic E. coli.

Expression Patterns of Germ Cell-specific Phosducin-like 2 during Testicular and Ovarian Development in Chickens

Phosducin (PDC) is a photoreceptor cell-specific protein that is phosphorylated by cyclic nucleotide-dependent protein kinase. PDC and PDC-like proteins (PDCL, PDCL2, and PDCL3) are members of a conserved family of small thioredoxin-like proteins that modulate the β- and γ-subunits of G-proteins. In mammals, Pdc, Pdcl, and Pdcl3 genes show ubiquitous expression; however, Pdcl2 gene expression is limited to the testis and ovary. The aim of the present study was to examine the expression patterns of chicken Pdcl2 (cPdcl2) during testicular and ovarian development. Protein sequence comparisons performed using the CLUSTAL X program revealed that the amino acid sequences and potential phosphorylation sites of cPDCL2 and mammalian PDCL2 proteins were highly conserved. Quantitative real-time PCR analysis revealed that cPdcl2 was differentially expressed in the testis and ovary. Specifically, cPdcl2 expression was detected at low levels in the ovary at all time points. In the testis, cPdcl2 expression was detected at low levels until 5 weeks of age. At 8 weeks of age, however, cPdcl2 showed increased expression levels in the testis. Using in situ hybridization, we detected high levels of cPdcl2 expression in the testis, particularly in the spermatocytes and round spermatids. In summary, our data describe expression patterns of germ cell-specific Pdcl2 during testicular and ovarian development in chickens.

Production of 4‐ethylphenol from 4‐hydroxycinnamic acid byLactobacillussp. isolated from a swine waste lagoon

To determine the source material and the responsible micro-organisms of 4-ethylphenol production in swine lagoon sediment slurries. Swine lagoon sediment was blended and incubated with tryptone-yeast extract broth containing 10 mmol l(-1) each of 4-hydroxycinnamic as well as other phenolics. 4-Ethylphenol was only produced from 4-hydroxycinnamic acid. Denaturing gradient gel electrophoresis analysis indicated that the microbial community was substantially altered from inclusion of 4-hydroxycinnamic acid. Serial dilutions and selective plating were employed to isolate a culture capable of 4-hydroxycinnamic acid conversion to 4-ethylphenol. Morphological and ribosomal gene analysis indicated the isolate to be a Lactobacillus sp., Lactobacillus sp. pep8 converted 4-vinylphenol, but not 4-hydroxy-3-phenylpropionate, to 4-ethylphenol and did not convert 4-hydroxy-3-methoxycinnamic acid to 4-ethyl-2-methoxyphenol. A p-coumaric acid decarboxylase (pdc) gene identical to Lact. plantarum pdc genes was cloned from Lactobacillus sp. pep8. Swine lagoon sediments produce 4-ethylphenol from 4-hydroxycinnamic acid due, in part, to the activity of Lactobacillus spp. 4-Ethylphenol, a malodourant of swine and beef wastes, is generated from plant materials by indigenous lactobacilli suggesting that altering the amount dietary of plant material may influence levels of 4-ethylphenol in the wastes.

Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l(-1)) in the engineered yeast compared with the parent strain (0.83 g l(-1)). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l(-1)) in ethanol yield.

Control of sympathetic activity--new insights; new therapeutic targets?

Recently, a large international group of 25 investigators identified a candidate gene for hypertension which codes for phosducin (Pdc) as a novel player in the sympathetic nervous system [1]. In their study, a wide spectrum of findings documented its so-far unrecognized and apparently important role in experimental and human stress-induced hypertension. This conclusion is based on data ranging from detailed analyses of the sympathetic system in genetically manipulated Pdc �/� (knock-out) mice to the demonstration of its relevance in humans: in two separate cohorts, French Canadian and Afro-American individuals, respectively, a correlation was documented between singlenucleotide polymorphisms (SNPs) in the Pdc gene and stress-dependent blood pressure reactions. The target gene identified by the investigators codes for the G-protein regulator phosducin. In mice, targeted Pdc deletion increased blood pressure, increased catecholamine turnover in the peripheral sympathetic system and prolonged action potential firing. This finding suggests that Pdc is a hypotensive principle, explaining why reduced function of Pdc amplifies the hypertensive reaction to stressors. In other words: Pdc puts a brake on sympathetic activity, and conversely Pdc malfunction will lead to hypertension. What is the site of action of Pdc? The Pdc gene and the Pdc protein are expressed in sympathetic ganglia, although to a much lower extent than in the retina and pineal gland, but not in heart, blood vessels and kidney, important players in blood pressure control. To identify the site of action, experimental manipulations sequentially excluded changes in heart function, vascular function or function of the central nervous system as candidates. Early in life, cardiac morphology was not altered in Pdc�/�mice,butleftventricularcontractilitywasincreased despite no change of sensitivity to adrenoceptor agonists (norepinephrine, dobutamine)—consistent with increased sympathetic nerve traffic. In older animals (>4 months), however, significant hypertrophy was seen with expression of fetal genes as evidence of target organ injury.

Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice

Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual's lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc-/- mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc-/- mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc-/- mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

Efficient production of L‐lactic acid by Crabtree‐negative yeast Candida boidinii

Industrial production of L-lactic acid, which in polymerized form as poly-lactic acid is widely used as a biodegradable plastic, has been attracting world-wide attention. By genetic engineering we constructed a strain of the Crabtree-negative yeast Candida boidinii that efficiently produced a large amount of L-lactic acid. The alcohol fermentation pathway of C. boidinii was altered by disruption of the PDC1 gene encoding pyruvate decarboxylase, resulting in an ethanol production that was reduced to 17% of the wild-type strain. The alcohol fermentation pathway of the PDC1 deletion strain was then successfully utilized for the synthesis of L-lactic acid by placing the bovine L-lactate dehydrogenase-encoding gene under the control of the PDC1 promoter by targeted integration. Optimizing the conditions for batch culture in a 5 l jar-fermenter resulted in an L-lactic acid production reaching 85.9 g/l within 48 h. This productivity (1.79 g/l/h) is the highest thus far reported for L-lactic acid-producing yeasts.

PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum

Plant defensin corn 1 ( Pdc1) gene was amplified from corn genomic DNA with the primers designed from a corn EST sequence homologous to a barley defensin gene. The cloned gene contains two exons and an intron. The deduced 9 KDa PDC1 peptide has a sequence that is identical to corn γ2-zeathionin and has the typical features of a plant defensin, including a signal sequence of 35 amino acids, followed by a characteristic defensin domain of 47 amino acids containing 8 cysteines. The defensin protein was expressed from the cloned cDNA introduced into two different expression systems; prokaryotic, Escherichia coli and eukaryotic, yeast ( Pichia pastoris). The PDC1 protein was purified with a nickel resin column and was tested for its antifungal activities using the pathogen Fusarium graminearum. The protein expressed in both E. coli and P. pastoris had antifungal activity, however the protein expressed in P. pastoris was more efficient in inhibiting growth of F. graminearum. FTIR analysis of PDC1 protein expressed in the two expression systems showed that expression in P. pastoris gave a product with more β-sheets and less random unordered structure than when it was expressed in E. coli. In addition, removal of the His-tag used for purification increased the fungicidal activity of the PDC1 protein. The data presented here suggest that the defensin PDC1 peptide of corn could be effectively used to restrict the disease caused by F. graminearum.

A novel assay for rapid in vivo determination of phenotypic stability of recombinant ethanol-producing microorganisms

A rapid empirical assay is presented for assessing the phenotypic stability of continuous cultures of recombinant bacteria containing transposed pdc and adh genes for ethanol production. The method measures spectrophotometrically the rate of colour formation when cells oxidize added ethanol to acetaldehyde in the presence of Schiff’s reagent. During chemostat cultures of the recombinant ethanologen Escherichia coli KO11 on 20 g/l glucose, assay activities were stable and high at ca 8 × 10 −4 ΔOD 540/(s.OD 550), reflecting the high, stable ethanol yield ( ca 95%). On 20 g/l and 50 g/l xylose, ethanol yields declined rapidly to about 60% and this was closely mirrored by the assay activities which fell to ca 1.5 ΔOD 540/(s.OD 550), only slightly higher than those measured for the parent strain. Typically taking only about an hour to perform, the assay provides a faster means of gauging the phenotypic stability of ethanol production than is possible by conventional methods.

Molecular Screening of Wine Lactic Acid Bacteria Degrading Hydroxycinnamic Acids

The potential to produce volatile phenols from hydroxycinnamic acids was investigated for lactic acid bacteria (LAB) isolated from Spanish grape must and wine. A PCR assay was developed for the detection of LAB that potentially produce volatile phenols. Synthetic degenerate oligonucleotides for the specific detection of the pdc gene encoding a phenolic acid decarboxylase were designed. The pdc PCR assay amplifies a 321 bp DNA fragment from phenolic acid decarboxylase. The pdc PCR method was applied to 85 strains belonging to the 6 main wine LAB species. Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus pentosaceus strains produce a positive response in the pdc PCR assay, whereas Oenococcus oeni, Lactobacillus hilgardii, and Leuconostoc mesenteroides strains did not produce the expected PCR product. The production of vinyl and ethyl derivatives from hydroxycinnamic acids in culture media was determined by high-performance liquid chromatography. A relationship was found between pdc PCR amplification and volatile phenol production, so that the LAB strains that gave a positive pdc PCR response produce volatile phenols, whereas strains that did not produce a PCR amplicon did not produce volatile phenols. The proposed method could be useful for a preliminary identification of LAB strains able to produce volatile phenols in wine.

Genome-wide transcriptional responses to sulfite in Saccharomyces cerevisiae

Sulfite is a commonly used preservative in foods, beverages, and Pharmaceuticals because it is toxic to many microorganisms. In order to understand the global response of Saccharomyces cerevisiae to sulfite, genome-wide transcript profiling following sulfite exposure was obtained. The transcription levels of 21 genes were increased more than 2-fold, while those of 37 genes decreased to a similar extent. Genes involved in carbohydrate metabolism represented the highest proportion of induced genes, which may account for the easily acquired resistance to sulfite. Most of down-regulated genes are involved in transcription, protein biosynthesis, and cell growth. The down-regulation of these genes is thought to reflect growth arrest which occurs during sulfite treatment, allowing cells to save energy. Cells treated with sulfite generated more than 70% of acetaldehyde than untreated cells, suggesting that the increased acetaldehyde production is correlated with the induction of PDC1 gene encoding pyruvate decarboxylase.

Overexpression of pyruvate decarboxylase in the yeastHansenula polymorpharesults in increased ethanol yield in high-temperature fermentation of xylose

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.