New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution.

The production of yeast oil from lignocellulosic biomasses is impaired by inhibitors formed during the pretreatment step, mainly acetic acid. Herein, we applied Adaptive Laboratory Evolution (ALE) to select three Acetic acid Tolerant Strains (ATS) of P. laurentii UFV-1. Different phenotypes emerged alongside evolution. The ATS II presented trade-offs in the absence of acetic acid, suggesting that it displays a specialized phenotype of tolerance to growth on organic acids. On the other hand, ATS I and ATS III presented phenotypes associated with the behavior of generalists. ATS I was considered the most promising evolved strain as it displayed the oleaginous phenotype in all conditions tested. Thus, we applied whole-genome sequencing to detect the mutations that emerged in this strain during the ALE. We found alterations in genes encoding proteins involved in different cellular functions, including multidrug resistance (MDR) transporters, energy metabolism, detoxification, coenzyme recycling, and cell envelope remodeling. To evaluate acetic acid stress responses, both parental and ATS I strains were cultivated in chemostat mode in the absence and presence of acetic acid. In contrast to ATS I, the parental strain presented alterations in the cell envelope and cell size under acetic acid stress conditions. Furthermore, the parental strain and the ATS I presented differences regarding acetic acid assimilation. Contrary to the parental strain, the ATS I displayed an increase in unsaturated fatty acid content irrespective of acetic acid stress, which might be related to improved tolerance to acetic acid. Altogether, these results provided insights into the mechanisms involved with the acetic acid tolerance displayed by ATS I and the responses of P. laurentii to this stressful condition.

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.

Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6.

Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 ± 0.38 mM and a maximum velocity (V max) of 0.96 ± 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae.

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate.

AIMS: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on beta-galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. METHODS AND RESULTS: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum beta-galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0.06, 0.09, 0.12, 0.18 to 0.24 h(-1). The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum beta-gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum beta-galactosidase activity at 0.24 h(-1) was observed. Following that stage beta-gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. CONCLUSION: The maximum beta-galactosidase activity per unit of cell mass was found to be 341.18 mmol ONP min(-1) g(-1) at a dilution rate of 0.24 h(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in beta-gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.