Mapping of Genomic Regions (Quantitative Trait Loci) Controlling Production and Quality in Industrial Cultures of the Edible Basidiomycete Pleurotus ostreatus

Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.     

Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.     

Continuous culture studies on the production of staphylocoagulase by Staphylococcus aureus

The production of staphylocoagulase was studied with continuous cultures of various S. aureus strains in a simple salts medium supplemented with mannitol, casein hydrolysate and three vitamins. Conditions of low oxygen availability and magnesium-limitation were required for optimal steady-state staphylocoagulase production. It was demonstrated that the specific rate of staphylocoagulase production was dependent on the growth rate.In two bovine strains, the production rate pattern was similar to that of an inducible enzyme sensitive to catabolite repression, although no specific inductor or repressor could be demonstrated. The human strain, on the other hand, produced staphylocoagulase constitutively. In all strains the specific rate of production of total extracellular protein was strictly proportional to the growth rate. The bovine strains produced 6 times more staphylocoagulase in chemostat culture as compared with batch cultures of the same organisms.It is likely that mannitol functioned as an energy source rather than as a carbon source because it was converted for a major part to acetate and for a minor part to lactate and not to new cell material. Repression of staphylocoagulase production by mannitol, acetate or lactate was not observed. The probable nature of the regulating mechanism(s) underlying staphylocoagulase production is discussed.     

Role of Surface Substances on Germinated Spores of Ceratosystis fimbriata,Black Rot Fungus,in Host-parasite Interaction

Surface substances were isolated by sonication from the germinated spores of various strains of Ceratocystis fimbriata and characterized in relation to host-parasite specificity. The substances from the sweet potato strain, compatible with sweet potato, potently inhibited the spore agglutination of various strains by spore-agglutinating factor from sweet potato roots, while the substances from incompatible strains, that is, coffee, taro, and almond strains, weakly inhibited this agglutination. The substances from the sweet potato strain increased ethylene production from sweet potato roots infected by all strains tested, sweet potato, coffee, taro, and almond strains, which was possibly an index of pathogenicity. On the other hand, the substances from incompatible strains, coffee, taro, and almond strains, suppressed the ethylene production from the tissue infected by all four strains except the substances from almond strains on almond strain. Heat and trypsin treatments inactivated the spore agglutination inhibitory activity of the surface substances. Coincidently, these treatments extinguished the effect of the surface substances on pathogenicity of C. fimbriata on sweet potato roots.     

The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori

 A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion. Received: 3 August 1995/Received revision: 20 December 1995/Accepted: 8 January 1996     

Production of S-Methylthioacetate by Brevibacterium linens

Volatile sulfur compounds production by eight strains of Brevibacterium linens isolated from cheeses was demonstrated: methanethiol, dimethyldisulfide, and 2,3,4-trithiapentane. Four of these strains also produced S-methylthioacetate, an important aroma component of smear-coated cheeses. It is the first demonstrated microbiological production of a thioester.     

The nature of the competitive ability of spontaneous staphylocoagulase-negative mutants of Staphylococcus aureus with respect to growth of the parent strains in continuous culture

During prolonged cultivation of S. aureus strains 104 and NCTC 8178 in continuous culture, staphylocoagulase-negative mutants arose and accumulated progressively in increasing proportions. The resulting loss of production of staphylocoagulase was accompanied by a simultaneous loss of production of -haemolysin and PV-leucocidin. Characterization of the strains revealed no further differences in biotype, exoenzymes, phage pattern and plasmid content.Cultivation in batch cultures showed that the maximal specific growth rates and specific oxygen-consumption rates of the mutant strains were slightly higher than those of the parent strains, whereas the production of total extracellular protein of the mutant strains had decreased significantly.From competition experiments between parent and mutant strains in chemostat cultures at different dilution rates and cultivation temperatures, it was concluded that the underlying mechanism of accumulation of staphylocoagulase-negative mutants in the chemostat is based on differences in affinity for the limiting substrate(s) rather than on differences in the production rates of total extracellular proteins. The complete repression of three exoenzymes, a partial repression of the total extracellular protein production, and an increased affinity for the limiting substrate(s) suggested that a mutation in a regulatory gene is involved. The possible role of a transposon in this mutation is discussed.     

Levels of Thermostable Direct Hemolysin Produced by Vibrio parahaemolyticus O3:K6 and Other Serovars Grown Anaerobically with the Presence of a Bile Acid

Twenty-three V. parahaemolyticus strains, including 12 pandemic O3:K6 strains, were examined for their growth and production of thermostable direct hemolysin (TDH) under an anaerobic culture condition with or without presence of a bile acid, taurocholic acid (TCA). Both bacterial growth and TDH production were markedly enhanced by TCA for a majority of the strains, but the scale of the TDH production was disproportionately greater than that of the corresponding growth for 14 strains. Such enhancement was, however, not specific to the pandemic strains. Received: 27 August 2001 / Accepted: 15 October 2001     

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures

Aims:  To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures.
Methods and Results:  Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus–Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus–Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms.
Conclusions:  The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma -mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes.
Significance and Impact of the Study:  Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.     

Invasive Salmonella Typhimurium ST313 with Naturally Attenuated Flagellin Elicits Reduced Inflammation and Replicates within Macrophages

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones

Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.     

Very early acetaldehyde production by industrial Saccharomyces cerevisiae strains: a new intrinsic character

During a general survey of the acetaldehyde-producing properties of commercially available wine yeast strains, we discovered that, although final acetaldehyde production cannot be used as a discriminating factor between yeast strains, initial specific acetaldehyde production rates were of highly interest for classifying yeast strains. This parameter is very closely related to the growth- and fermentation-lag phase durations. We also found that this acetaldehyde early production occurs with very different extent between commercial active dry yeast strains during the rehydration phase and could partially explain the known variable resistance of yeast strains to sulfites. Acetaldehyde production appeared, therefore, as very precocious, strain-dependent, and biomass-independent character. These various findings suggest that this new intrinsic characteristic of industrial fermenting yeast may be likely considered as an early marker of the general fermenting activity of industrial fermenting yeasts. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by Saccharomyces cerevisiae.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Genome-wide identification of the targets for genetic manipulation to improve l-lactate production by Saccharomyces cerevisiae by using a single-gene deletion strain collection

To identify genome-wide targets for gene manipulation for increasing l-lactate production in recombinant Saccharomyces cerevisiae strains, we transformed all available single-gene deletion strains of S. cerevisiae with a plasmid carrying the human l-lactate dehydrogenase gene, and examined l-lactate production in the obtained transformants. The thresholds of increased or decreased l-lactate production were determined based on l-lactate production by the standard strain in repetitive experiments. l-lactate production data for 4802 deletion strains were obtained, and deletion strains with increased or decreased l-lactate production were identified. Functional category analysis of genes whose deletion increased l-lactate production revealed that ribosome biogenesis-related genes were overrepresented. Most deletion strains for genes related to ribosome biogenesis exhibited increased l-lactate production in 200-ml batch cultures. We deleted the genes related to ribosome biogenesis in a recombinant strain of S. cerevisiae with a genetic background different from that of the above deletion strains, and examined the effect of target gene deletion on l-lactate production. We observed that deletion of genes related to ribosome biogenesis leads to increased l-lactate production by recombinant S. cerevisiae strains, and the single-gene deletion strain collection could be utilized in identifying target genes for improving l-lactate production in S. cerevisiae recombinant strains.     

Formation of biogenic amines as criteria for the selection of wine yeasts

This work deals with biogenic amine production by yeast strains isolated from grapes and wines. A total of 50 strains were tested for their capacity to produce biogenic amines in wine. In general, all the species produced very low or non-detectable amounts of histamine, whereas methylamine and agmatine were formed by all the species considered. The highest concentration of total biogenic amines was formed by Brettanomyces bruxellensis, with an average value of 15 mg/l, followed by Saccharomyces cerevisiae with an average of 12.14 mg/l. The other species formed less than 10 mg of total biogenic amines per litre. Wines fermented with the most fermentative strains of S. cerevisiae species had the highest contents of ethanolamine, from 2.3 to 16 mg/l, and of agmatine, from 3.1 to 7.5 mg/l. The strains of the other species, which exhibited a low fermentative ability, Kloeckera apiculata, B. bruxellensis and Metschnikowia pulcherrima, varied in the production of agmatine and phenylethylamine. A significant variability in the production of cadaverine was characteristic of Candida stellata strains, which varied also in ethanolamine production. Our results emphasize the importance of using selected strains of S. cerevisiae, not only for the expression of desirable technological traits, but also to avoid potentially negative effects on human health. Therefore, the characterization of strains of S. cerevisiae for the 'production of biogenic amines' becomes of applicative interest.     

Overexpression of the glucoamylase-encoding STA1 gene of Saccharomyces cerevisiae var. diastaticus in laboratory and industrial strains of Saccharomyces

Production of glucoamylase encoded by the Saccharomyces cerevisiae (var. diastaticus) STA1 gene has been assayed in laboratory S. cerevisiae strains of different ploidy and in different industrial Saccharomyces strains, in which STA1 was expressed under control of an inducible promoter. Highest enzyme activity was achieved with a tetraploid strain constructed by crossing preselected parental strains. Maximal glucoamylase production correlated with heterogeneity in enzyme mass, likely due to incomplete glycosylation, suggesting that the secretion-glycosylation process is the limiting step in the production of the STA-encoded glucoamylase by Saccharomyces. Industrial strains showed quite different capacity to produce glucoamylase. High production was achieved with a S. pastorianus brewer’s strain. Overall, our results allowed the selection of strains capable of yielding a high level of glucoamylase and suggest specific approaches for further enhancing this capability.     

Quantitative gas chromatography of Bacteroides species under different growth conditions

From 56 strains of strictly anaerobic gram-negative rods isolated from stool and purulent lesions the fermentation products in the presence and absence of hemin were determined by quantitative gas-solid chromatography, using a simple and more rapid chromatographic procedure. With hemin the fermentation products were propionic, acetic, lactic and succinic acid. Without hemin no or little succinic acid was formed and the main products were lactic and acetic acid. In both groups the distribution of subspecies was determined and the production of fatty acids measured quantitatively.Fourteen strains of the lesion group showed a higher metabolic activity, resulting in an increased total acid production caused by an excessive production of acetic and lactic acid. This characteristic is probably a virulence factor in these strains.All strains were protoporphyrin- and oxgall-dependent. It is postulated that these substances are used for the production of cytochromes which permits the formation of succinic acid by a fumarate reductase resulting in an increased growth rate and growth yield.     

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A–NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.