Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.     

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.     

The cellular location of nitrilase and amidase enzymes of Brevibacterium R312

Abstract Growth of Brevibacterium R312 on acetonitrile results in the appearance of acetate and ammonia in the medium, whereas incubation of harvested bacteria with acetonitrile results in appearance of acetamide, acetate and ammonia in the medium. Acetonitrilase and acetamidase activities were found to be located in the cytosolic fraction of the cell, suggesting that acetonitrile and acetamide readily enter and leave the bacterium. This was confirmed by the inability of these compounds to osmotically stabilise protoplasts, whereas acetate does so. The small size and neutral non-ionic structure of acetonitrile and acetamide suggest they probably permeate by simple diffusion.     

Unusual flocculation characteristics of the yeast Candida famata (Debaryomyces hansenii)

Candida famata NCYC 576 cells aggregated throughout growth in YEPD. Aggregates were dispersed by Pronase E, EDTA or specific sugars. EDTA-dispersed cells reaggregated after calcium ion addition. Unlike Saccharomyces cerevisiae, C. famata cells lost the ability to flocculate with repeated EDTA washings. These cells regained flocculation when resuspended in the first washing solution after calcium addition. Candida famata NCYC 576 aggregation is consistent with lectin-mediated yeast flocculation, where lectins are not surface-anchored, as in S. cerevisiae but attached to cells only by lectin action.     

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.     

Nitrile-metabolizing potential of Bacillus cereus strain FA12; Nitrilase production,purification, and characterization

Nitrile-hydrolyzing bacteria have the potential to perform useful biotransformations such as the production of industrially useful acids and amides. In this study, we report a nitrile-degrading bacterium with significant nitrile metabolism. Molecular characterization of 16S rDNA gene characterized this strain as Bacillus cereus. Medium optimization of B. cereus FA12 showed that biomass and nitrilase production was strongly supported by glucose (10 gL^{? 1}) and yeast extract (10 gL^{? 1}). Enzymatic production improved slightly in the pH range from 6.0 to 7.0. The addition of Mg⁺², Fe⁺², and Na⁺ supported biomass and nitrilase production; however, other metal ions, Co⁺² and Cu⁺², inhibited production. The apparent molecular mass of the puri?ed FA12 nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was about 45 kDa. Nitrilase FA12 shows relatively high activity and stability at pH 7.0 and 40°C. Nitrilase FA12 was marginally inhibited with Ca^{+ 2} and Co⁺², whereas inhibition in the presence of dithiothreitol or DTT was 80%. The pseudo K_m (mM) values of resting cells (i.e., treating whole cells as if they were an enzyme) for acetonitrile and acetamide were determined to be 2.36 and 1.81, respectively. Under optimum situations, B. cereus FA12 resting cells produced 83 and 58 (U/mg) acetonitrile/acetamide degrading activity, respectively. Ammonia production from acetamide and acetonitrile by the B. cereus FA12 was maximum after 5 and 7 h of incubation, respectively. These results indicate that B. cereus FA12 resting cells may be used in nitrile biotransformations to produce commercially useful compounds.     

Metabolism of acetonitrile and propionitrile by Nocardia rhodochrous LL100-21.

Six nitrile compounds and two amide derivatives were degraded by Nocardia rhodochrous LL100-21. Acetonitrile, hydroacrylonitrile, and propionitrile were the best sources of carbon and nitrogen for growth, whereas butenenitrile, succinonitrile, and acetamide supported less growth. Acrylonitrile and acrylamide supported growth but only as a source of nitrogen. Gas chromatography of the culture medium revealed a decrease in acetonitrile with the sequential formation of acetamide and acetic acid. Ammonia was also detected by colorimetric procedures. The enzyme system responsible for the hydrolysis of acetonitrile was shown to be intracellular and inducible. The breakdown of acetonitrile by a crude bacterial extract was a two-step enzymatic hydrolysis with acetamide as the intermediate product and acetic acid and ammonia as the final products. Product formation was stoichiometric with substrate disappearance. When propionitrile was the growth substrate, there was complete conversion of the nitrile to propionic acid and ammonia as the major products. The enzymatic breakdown of the propionitrile, although slower than acetonitrile, yielded the corresponding carboxylic acid and ammonia. Propionamide was produced in very small amounts as an intermediate product.     

Characterisation of the nitrile hydratase gene clusters of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strains AJ270 and AJ300 and <Emphasis Type="Italic">Microbacterium</Emphasis> sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.     

Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

Utilization of nitriles by yeasts isolated from a Brazilian gold mine

Yeast strains from the genera Candida, Debaryomyces, Aureobasidium, Geotrichum, Pichia, Rhodotorula, Tremella, Hanseniaspora, and Cryptococcus were isolated from samples of a gold mine from liquid extraction circuit. These strains were tested for their ability to utilize acetonitrile at 12 mM as the sole nitrogen source. The yeasts that grew using acetonitrile at 12 mM were tested in the presence of acetonitrile, isobutyronitrile, methacrylnitrile, and propionitrile at concentrations of 12, 24, 48, 97, and 120 mM. One strain was selected for each nitrile and the concentration of nitrile in which the best growth occurred. Cryptococcus sp. strain UFMG-Y28 had a better growth on 120 mM propionitrile and 97 mM acetonitrile, Rhodotorula glutinis strain UFMG-Y5 on 48 mM methacrylnitrile, and Cryptococcus flavus strain UFMG-Y61 on 120 mM isobutyronitrile. The utilization of different nitriles and amides by yeast strains involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.     

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.     

Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

Utilization of acrylonitrile by bacteria isolated from petrochemical waste waters

A bacterium, utilising acrylonitrile as a sole source of carbon and nitrogen, was isolated from Indian Petrochemical Corporation Limited (IPCL) waste waters and identified as Arthrobacter sp. This strain could also utilize acetonitrile, acetamide and acrylamide individually as a source of carbon and nitrogen. The metabolic studies with the whole cells indicated the sequential conversion of the nitrile to the respective amide and then to the respective acid and ammonia. The rate of nitrile hydrolysis was slower than the corresponding amide hydrolysis. Acrylic acid, the end product of acrylonitrile breakdown, did not support the growth when provided as a carbon source.     

Subcellular location of the enzymes of purine breakdown in the yeast Candida famata grown on uric acid

Abstract The subcellular location of the enzymes of purine breakdown in the yeast Candida famata , which grows on uric acid as sole carbon and nitrogen source, has been examined by subcellular fractionation methods. Uricase was confirmed as being peroxisomal, but the other three enzymes, allantoinase, allantoicase and ureidoglycollate lyase were shown to be cytosolic. In addition the peroxisomes harboured catalase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase.     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

Factors affecting killer activity of some yeast species occurring in rokpol cheese

Fourteen strains of Candida famata and 7 strains of C. sphaerica originating from blue-veined Rokpol cheese were studied for their ability to produce killer toxins against 3 strains of Yarrowia lipolytica selected as potential starter cultures for cheesemaking. All tested strains revealed killer activity in the presence of salt, at 14 degrees C and pH 4.6. Only one Y. lipolytica strain was resistant to C. famata and C. sphaerica killer toxins.     

Studies on Osmophilic Yeasts

In a medium containing high concentration of yeast extract was observed aerobic formation of a large amount of ethanol by various yeasts of non-fermenting or extremely poor fermenting type such as Torulopsis famata, Candida polymorpha and Pichia membranaefaciens etc. Acetaldehyde formed as a metabolic intermediate leading to ethanol formation was trapped by the addition of sulfite and was identified as 2,4-dinitrophenyl-hydrazone.     

Induction and Repression of Amidase Enzymes in Aspergillus nidulans

Aspergillus nidulans can grow on acetamide as both a carbon and nitrogen source and can also grow on formamide as a nitrogen source. Two distinct enzymes, an acetamidase and a formamidase, are produced. The control of the synthesis of these two enzymes in a wild-type strain was investigated. The formamidase is induced by acetamide and formamide and repressed by ammonia. The acetamidase is induced by formamide and acetamide, repressed by carbon metabolites derived from glucose and acetate, and repressed by ammonia. Repression of the acetamidase by ammonia depends on the carbon source; growth on glucose but not on acetate or acetamide allows repression to occur. The pattern of acetamidase repression is compared with that of histidine catabolic enzymes in various bacteria.