Nonutilizing Strains Research Articles

Mutagenesis, breeding, and characterization of sake yeast strains with low production of dimethyl trisulfide precursor

Dimethyl trisulfide (DMTS) is one of the main components responsible for hineka, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5'-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.

Yarrowia lipolytica cells mutant for the PEX24 gene encoding a peroxisomal membrane peroxin mislocalize peroxisomal proteins and accumulate membrane structures containing both peroxisomal matrix and membrane proteins.

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. Functional complementation of the oleic acid-nonutilizing strain mut1-1 of the yeast Yarrowia lipolytica has identified the novel gene, PEX24. PEX24 encodes Pex24p, a protein of 550 amino acids (61,100 Da). Pex24p is an integral membrane protein of peroxisomes that exhibits high sequence homology to two hypothetical proteins encoded by the open reading frames YHR150W and YDR479C of the Saccharomyces cerevisiae genome. Pex24p is detectable in wild-type cells grown in glucose-containing medium, and its levels are significantly increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex24 mutants are compromised in the targeting of both matrix and membrane proteins to peroxisomes. Although pex24 mutants fail to assemble functional peroxisomes, they do harbor membrane structures that contain subsets of peroxisomal proteins.

Selection and Evaluation of Microorganisms for Biocontrol of Fusarium Head Blight of Wheat Incited by Gibberella zeae.

Gibberella zeae incites Fusarium head blight (FHB), a devastating disease that causes extensive yield and quality losses to wheat and barley. Of over 700 microbial strains obtained from wheat anthers, 54 were able to utilize tartaric acid as a carbon source when the compound was supplied as choline bitartrate in liquid culture. Four tartaric acid-utilizing and three nonutilizing strains reduced FHB in initial tests and were selected for further assays. Antagonists were effective against three different isolates of G. zeae when single wheat florets were inoculated with pathogen and antagonist inoculum. All seven antagonists increased 100-kernel weight when applied simultaneously with G. zeae isolate Z3639 (P ≤ 0.05). Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strain OH 182.9 reduced disease severity by >77, 93, and 56%, respectively. Five antagonists increased 100-kernel weight of plants inoculated with G. zeae isolate DAOM 180378. All antagonists except one increased 100-kernel weight, and four of seven antagonists reduced disease severity (P ≤ 0.05) when tested against G. zeae isolate Fg-9-96. In spray-inoculation experiments, Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strains OH 71.4 and OH 182.9 reduced disease severity, regardless of the sequence, timing, and concentration of inoculum application (P ≤ 0.05), though 100-kernel weight did not always increase when antagonists were applied 4 h after inoculum of G. zeae. Overall, 4 of 54 isolates that utilized tartaric acid in vitro were effective against G. zeae versus only 3 of 170 isolates tested that did not utilize tartaric acid (P ≤ 0.05, χ-square test of goodness of fit), demonstrating the potential benefit of prescreening candidate antagonists of FHB for their ability to utilize tartaric acid. Biological control shows promise as part of an integrated pest management program for managing FHB.

Trans-dominant mutations in the GPR1 gene cause high sensitivity to acetic acid and ethanol in the yeast Yarrowia lipolytica.

Acetate non-utilizing strains harbouring trans-dominant mutations in the GPR1 gene (GPR1(d)) of the dimorphic yeast Yarrowia lipolytica have been selected and characterized. These mutants are highly sensitive to low concentrations of acetic acid and ethanol, even in presence of glucose. The toxic effect of acetic acid is pH-dependent and has the strongest effect at low pH. In contrast, the action of ethanol is pH-independent. One GPR1(d) mutant has been detected that was highly sensitive to acetic acid but could still grow on ethanol, which indicates putative differences in the function of the GPR1 gene product in the sensitivity to acetic acid and ethanol. The GPR1(d) mutants exhibit a complex pleiotropic phenotype. The mutations cause changed colony morphology as well as dimorphism of cells, and induce early cell death during growth on glucose, even without the presence of dicarbon compounds. Composition of intracellular membranes, as well as morphology of vacuole and mitochondria, were strongly changed. Back-crosses with wild-type strains and analysis of recombinant strains have shown that the expression of the pleiotropic phenotype depends on the site of mutation in the GPR1 gene, as well as on the genetic background of the strain harbouring the responsive mutation. Our data suggest that Gpr1p is involved in a general response of cells to the toxic action of dicarbon compounds like acetic acid and ethanol.

Effect of Temperature and Dew Period on Infection of Northern Jointvetch by Wild-Type and Mutant Strains ofColletotrichum gloeosporioidesf. sp.aeschynomene1

Abstract Colletotrichum gloeosporioides f. sp. aeschynomene causes an anthracnose on Aeschynomene virginica, northern jointvetch. Infectivity, measured as the number of lesions per plant caused by three strains of the fungus including a wild-type strain (3-1-3), a benomyl-resistant strain (B21), and a nitrate nonutilizing strain (Nit A), was measured on seedling plants. Infectivity of each strain was measured for nine combinations of three dew temperatures (20, 26, and 32°C) and incubation temperatures (20, 28, and 32°C). Generally, results show that 3-1-3 and B21 caused significantly more lesions than did Nit A at all dew and incubation temperature combinations. However, strain 3-1-3 caused significantly more lesions than did B21 when given 20°C dew at 28°C incubation temperature treatments. Infection of northern jointvetch by the mutant strains was reduced at the highest and lowest temperatures studied while infection by 3-1-3 was reduced only at 32°C. Thus, the wild-type strain appeared to be more tolerant to temperature changes than did the mutant strains. In addition, the length of dew period required for infection of northern jointvetch by each strain was studied at each dew temperature. The wild-type strain appeared to require shorter dew periods than did the mutant strains to cause a similar number of lesions.

Infection Components of Wild-Type and Mutant Strains of Colletotrichum gloeosporioides f. sp. aeschynomene on Northern Jointvetch.

Colletotrichum gloeosporioides f. sp. aeschynomene causes an anthracnose of northern jointvetch, Aeschynomene virginica. Infection components, including lesion number, latent period, lesion expansion rate, and sporulation, were measured in experiments conducted in controlled environments. Two wild-type strains (3-1-3 and CLA 5A), four benomyl-resistant strains (B13, B15, B18 and B21), and four nitrate nonutilizing mutant strains (Nit A, Nit R, Nit L, and Nit T) of the pathogen were tested. Nitrate nonutilizing strains caused significantly fewer lesions on northern jointvetch than did wild-type and benomyl-resistant strains. Latent periods were significantly shorter for the wild-type strain CLA 5A than for most other strains. Lesion expansion rates of all benomyl-resistant strains were significantly slower than those of the wild- type strains. Large variations in sporulation were observed for most strains, and no differences in sporulation were found between wild-type and mutant strains. The usefulness of infection component analysis for the identification of competitiveness of strains of fungal pathogens for biological control of weeds is discussed.

Isolation and characterization of a Saccharomycopsis lipolytica mutant showing increased production of citric acid from canola oil

A process for the production of citric acid from canola (rapeseed) oil using the yeast Saccharomycopsis lipolytica was examined. A citrate nonutilizing strain, designated NTG9, which had an improved citric to isocitric acid ratio, was isolated after mutagenesis of S. lipolytica ATCC 20228 with nitrosoguanidine. Although the mutant grew well on canola oil, unlike the parent strain or a spontaneous revertant (JF2), it did not grow on glycolytic intermediates below glycerate-3-phosphate, amino acids, hexadecane, or yellow kerosene. The mutant was shown to be impaired in the gluconeogenic pathway because of a loss of phosphoenolpyruvate carboxykinase activity. A preliminary study of the effect of micronutrients on citric acid production by S. lipolytica NTG9 showed that manganese had a stimulatory effect on the process whereas zinc and iron were inhibitory. A revised growth medium was tested and found to increase citric acid production while decreasing that of isocitric acid.

Characterization of glycerol nonutilizing and protoperithecial mutants of Neurospora.

Mutants defective in polyol metabolism and/or in protoperithecial development were selected in Neurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed into N. crassa to facilitate genetic analysis. One, glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, between ad-1 and rib-1; the other, glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal to ad-9. Another mutant, gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal to ad-9.

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a β-1, 4-glucosidase or -cellulase.

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria1

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.

Rate of Isolated Hemicellulose Degradation and Utilization by Pure Cultures of Rumen Bacteria

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens , of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly. Images Fig. 6 Fig. 7

Rate of Isolated Hemicellulose Degradation and Utilization by Pure Cultures of Rumen Bacteria1

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.

Studies of the genetics and physiology of a nitrate non-utilizing strain of Neurospora

Studies of the genetics and physiology of a nitrate non-utilizing strain of Neurospora

Neurospora lactase. I: Properties of lactase preparations from a lactose utilizing and a lactose non-utilizing strain

Mutant strains of Neurospora which utilize lactose poorly as a carbon source are all found to form an enzyme with β- d-galactosidase activity. Purification and properties of β- d-galactosidase from a normal strain of Neurospora is described, and these properties are then compared with the properties of the β- d-galactosidase of a mutant strain which cannot utilize lactose as a carbon source. No differences between the two enzyme preparations were observed.