Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan.

The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding alpha-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for beta-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 degrees C.

Production of flavin mononucleotide by metabolically engineered yeast Candida famata.

Recombinant strains of the flavinogenic yeast Candida famata able to overproduce flavin mononucleotide (FMN) that contain FMN1 gene encoding riboflavin (RF) kinase driven by the strong constitutive promoter TEF1 (translation elongation factor 1alpha) were constructed. Transformation of these strains with the additional plasmid containing the FMN1 gene under the TEF1 promoter resulted in the 200-fold increase in the riboflavin kinase activity and 100-fold increase in FMN production as compared to the wild-type strain (last feature was found only in iron-deficient medium). Overexpression of the FMN1 gene in the mutant that has deregulated riboflavin biosynthesis pathway and high level of riboflavin production in iron-sufficient medium led to the 30-fold increase in the riboflavin kinase activity and 400-fold increase in FMN production of the resulted transformants. The obtained C. famata recombinant strains can be used for the further construction of improved FMN overproducers.

Construction of Hansenula polymorpha strains with improved thermotolerance.

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high-temperature resistance and high-temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat-shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12-fold increased tolerance to heat-shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8-fold improvement of ethanol production from xylose at 50 degrees C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L(-1), our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high-temperature ethanol producers from this pentose.

Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha.

BACKGROUND: The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 - 48 degrees C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved. RESULTS: Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Deltaxyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341 --> R N343 --> D), while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Deltaxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48 degrees C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg x (L x h)-1 as compared to 4.2 mg x (L x h)-1 for the wild type strain. CONCLUSION: Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha.

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results 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.

Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation.

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Deltaxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.

Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha.

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.

Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis.

Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.