Charles Abbas

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

SUMMARY Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

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.

Biochemical Analysis of a β-d-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-beta-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 beta-D-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative beta-D-glucosidase has activity for pNP-beta-D-xylopyranoside, pNP-alpha-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 beta-D-xylosidase.

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

BackgroundThe thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°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.ResultsSite-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 Δxyl1 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 Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°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 × (L × h)-1 as compared to 4.2 mg × (L × h)-1 for the wild type strain.ConclusionRecombinant 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 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.

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.

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°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. Biotechnol. Bioeng. 2009; 104: 911–919.

Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate

ABSTRACT We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOSFA,Ac) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOSFA,Ac, a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose.

Chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses by dogs to select corn fibers.

The objective was to examine the chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses of fiber-rich corn coproducts resulting from corn wet milling. Native corn fibers, native corn fibers with fines, hydrolyzed corn fibers, and hydrolyzed extracted corn fibers were analyzed chemically and their capacity to produce short-chain fatty acids determined. Ash content was low (<1.2%), crude protein content varied little, but fat and fiber concentrations varied widely. Most fiber was in the insoluble form, with glucose being predominant followed by xylose. Total short-chain fatty acid production ranged from 211.6 to 699.52 micromol/g of dry matter, whereas branched-chain fatty acid production was low. Four corn fibers (native and processed) were included in a canine diet matrix at the 7% inclusion level. Nutrient digestibility, food intake, and fecal characteristics were not affected by corn fiber inclusion in canine diets, suggesting that they should be considered as potential dietary fiber sources in dog foods.

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. Copyright