Obtainment, selection and characterization of a mutant strain of Kluyveromyces marxianus that displays improved production of 2-phenylethanol and enhanced DAHP synthase activity.

AIMS: Yeasts produce 2-phenylethanol (2-PE) from sugars via de novo synthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to select Kluyveromyces marxianus mutant strains with improved capacity to produce 2-PE from sugars. METHODS AND RESULTS: Kluyveromyces marxianus CCT 7735 mutant strains were selected from UV irradiation coupled with screening of p-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium without l-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced both ARO3 and ARO4 genes of Km_PFP41 mutant and identified mutations in ARO4 which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. CONCLUSIONS: The Km_PFP41 mutant strain presented improved 2-PE production via de novo synthesis and enhanced DAHP synthase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.

Exogenous fungal quorum sensing molecules inhibit planktonic cell growth and modulate filamentation and biofilm formation in the Sporothrix schenckii complex.

This study investigated the effect of the quorum sensing molecules (QSMs) farnesol, 2-phenylehtanol, tyrosol and tryptophol against planktonic cells, filamentation and biofilms of Sporothrix spp. The antifungal activity of QSMs was evaluated by broth microdilution. QSMs showed MICs in the ranges of 0.01-1 µM (farnesol), 1-8 mM (2-phenylehtanol and tyrosol), and >16 mM (tryptophol). Filamentous biofilm formation was inhibited by farnesol and 2-phenylehtanol and stimulated by tyrosol. Yeast biofilm formation was inhibited by 2-phenylehtanol and tyrosol. Tryptophol did not affect Sporothrix biofilm formation. QSMs showed MICs against mature biofilms of 8-32 µM (farnesol), 8-32 mM (2-phenylehtanol) and 64-128 mM (tyrosol). In conclusion, farnesol, 2-phenylethanol and tyrosol have antifungal activity against planktonic and sessile cells and modulate filamentation and biofilm formation in Sporothrix spp.

2-Phenylethanol and 2-phenylethylacetate production by nonconventional yeasts using tequila vinasses as a substrate.

Vinasses from the tequila industry are wastewaters with highly elevated organic loads. Therefore, to obtain value-added products by yeast fermentations, such as 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEA), could be interesting for industrial applications from tequila vinasses. In this study, four yeasts species (Wickerhamomyces anomalus, Candida glabrata, Candida utilis, and Candida parapsilosis) were evaluated with two different chemically defined media and tequila vinasses. Differences in the aroma compounds production were observed depending on the medium and yeast species used. In tequila vinasses, the highest concentration (65 mg/L) of 2-PEA was reached by C. glabrata, the inhibitory compounds decreased biomass production and synthesis of 2-PEA, and biochemical and chemical oxygen demands were reduced by more than 50 %. Tequila vinasses were suitable for the production of 2-phenylethylacetate by the shikimate pathway. A metabolic network was developed to obtain a guideline to improve 2-PE and 2-PEA production using flux balance analysis (FBA).

Effects of aroma quality on the biotransformation of natural 2-phenylethanol produced using ascorbic acid

Background Natural 2-phenylethanol (2-PE) is an important flavoring that emits the aroma of roses. During biotransformation, the aroma quality of natural 2-PE is affected by its main by-products, which include butanol, isobutyric acid, butyric acid, and isovaleric acid. Thus, controlling undesirable by-product formation can reduce the effect of odor on 2-PE aroma quality. Results 2-PE was produced through biotransformation using l-phenylalanine as a substrate and glucose as a carbon source. Ascorbic acid was added to the system to improve the redox reaction and suppress the generation of by-products. Principal component analysis of the aroma quality of 2-PE was performed using an electronic nose. Similarity analysis revealed that the effects of four by-products on 2-PE aroma quality may be ranked in the following order: isovaleric acid > butyric acid > isobutyric acid > butanol. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Conclusions 2-PE produced through the addition of ascorbic acid exhibited the closest aroma similarity to the standard 2-PE sample.

In vitro determination of volatile compound development during starter culture-controlled fermentation of Cucurbitaceae cotyledons.

The effects of Lactobacillus plantarum UFLA CH3, Pediococcus acidilactici UFLA BFFCX 27.1, and Torulaspora delbrueckii UFLA FFT2.4 inoculation on the volatile compound profile of fermentation of Cucumeropsis mannii cotyledons were investigated. Different microbial associations were used as starters. All associations displayed the ability to ferment the cotyledons as judged by lowering the pH from 6.4 to 4.4-5 within 24h and increasing organic acids such as lactate and acetate. The population of lactic acid bacteria (LAB) and yeasts increased during fermentation. In the fermentation performed without inoculation (control), the LAB and yeast populations were lower than those in inoculated assays at the beginning, but they reached similar populations after 48 h. The Enterobacteriaceae population decreased during the fermentation, and they were not detected at 48 h in the L. plantarum UFLA CH3 and P. acidilactici UFLA BFFCX 27.1 (LP+PA) and L. plantarum UFLA CH3, P. acidilactici UFLA BFFCX 27.1, and T. delbrueckii UFLA FFT2.4 (LP+PA+TD) samples. The assays inoculated with the yeast T. delbrueckii UFLA FFT2.4 exhibited the majority of volatile compounds (13 compounds) characterized by pleasant notes. The LP+PA+TD association seemed to be appropriate to ferment C. mannii cotyledons. It was able to control the Enterobacteriaceae population, and achieved high concentrations of esters and low concentrations of aldehydes and ketones.

Impact of different cocoa hybrids (Theobroma cacao L.) and S. cerevisiae UFLA CA11 inoculation on microbial communities and volatile compounds of cocoa fermentation.

The aim of this work was to study the microbial communities and volatile compounds profile of different fermentations: using four different cocoa hybrids and adding Saccharomyces cerevisiae UFLA CA11 as starter culture. Each hybrid showed particular characteristics: size, peel, seed and pulp. The temperature of the cocoa mass increased during fermentations (24°C to 47°C). The hybrid FA13 inoculated with S. cerevisiae showed the lowest temperatures (26 to 37°C). The pulp's compositions were different between the hybrids, mainly regarding citric acid (0.5 to 3.2g/kg). The carbohydrates were more rapidly (60h) metabolized in inoculated fermentations than in spontaneous fermentations (84h). Thirty-nine volatile compounds were identified by GC-FID for all fermentation processes. Esters (14 compounds) and alcohols (12) were the most important groups. Yeast communities were similar among the different processes while bacterial communities were dependent on the hybrid and process. The inoculation accelerated the fermentation and the hybrid characteristics influenced on the fermentation requiring particular management.