[A CASE OF SUSPECTED ALLERGIC BRONCHOPULMONARY MYCOSIS CAUSED BY SCEDOSPORIUM APIOSPERMUM THAT SHOWED CLINICAL IMPROVEMENT AFTER BRONCHOSCOPIC REMOVAL OF A MUCUS PLUG].

A 69-year-old woman presented with a persistent cough and high fever. Thoracic computed tomography revealed atelectasis and high-attenuation mucus. The blood test results showed eosinophils at 18.2%, an absolute eosinophil count of 980 cells/µL, and a total serum immunoglobulin E of 1980IU/mL. Bronchoscopy revealed a mucous plug, which upon photomicrograph examination, showed eosinophils. A culture study of the mucus yielded Scedosporium apiospermum, leading to the suspicion of allergic bronchopulmonary mycosis (ABPM) caused by the fungus. After the bronchoscopic removal of the mucous plug, her symptoms quickly diminished. She was successfully treated without medication, and ABPM has not recurred for 2 years. To our knowledge, ABPM caused by Scedosporium apiospermum is rare, and close follow-up was effective without the administration of systemic steroids or antifungal drugs.

Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast.

BACKGROUND: During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed. RESULTS: We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance. CONCLUSIONS: In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.

Overexpression of the transcription activator Msn2 enhances the fermentation ability of industrial baker's yeast in frozen dough.

We constructed a self-cloning diploid baker's yeast strain that overexpressed the transcription activator Msn2. It showed higher tolerance to freeze-thaw stress and higher intracellular trehalose level than observed in the wild-type strain. Overexpression of Msn2 also enhanced the fermentation ability of baker's yeast cells in frozen dough. Hence, Msn2-overexpressing baker's yeast should be useful in frozen-dough baking.

Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance.

The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase (crtI product) and bifunctional phytoene synthase/lycopene cyclase (crtYB product) resulted in the accumulation of a small amount of beta-carotene in S. cerevisiae. Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from S. cerevisiae (the BTS1 gene product) increased the intracellular beta-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the X. dendrorhous crtS gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with X. dendrorhous CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the beta-carotene-producing yeast cells transformed by the bacterial genes crtW and crtZ, encoding beta-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on S. cerevisiae cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.

Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses.

During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1P247S and PRO1E415K gene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses.

Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.

Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor.

Improvement of fermentation ability under baking-associated stress conditions by altering the POG1 gene expression in baker's yeast.

During the bread-making process, yeast cells are exposed to many types of baking-associated stress. There is thus a demand within the baking industry for yeast strains with high fermentation abilities under these stress conditions. The POG1 gene, encoding a putative transcription factor involved in cell cycle regulation, is a multicopy suppressor of the yeast Saccharomyces cerevisiae E3 ubiquitin ligase Rsp5 mutant. The pog1 mutant is sensitive to various stresses. Our results suggested that the POG1 gene is involved in stress tolerance in yeast cells. In this study, we showed that overexpression of the POG1 gene in baker's yeast conferred increased fermentation ability in high-sucrose-containing dough, which is used for sweet dough baking. Furthermore, deletion of the POG1 gene drastically increased the fermentation ability in bread dough after freeze-thaw stress, which would be a useful characteristic for frozen dough baking. Thus, the engineering of yeast strains to control the POG1 gene expression level would be a novel method for molecular breeding of baker's yeast.

Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough.

Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking.