Yeasts strains with the ability to flocculate are very useful in industrial applications, as the flocculent phenotype allows for an easier, and cheaper, way of separating the yeast culture from the final, economically-important, fermentation product (Ratledge and Kristiansen, [2001
]); and also because of the higher production rate and cellular densities obtained with these strains (Teixeira et al. [1990
]). As a result of this, flocculent strains are ideally suited for use in continuous fermentative processes (Domingues et al. [2000b
]). However, the flocculent phenotype negatively affects the yeast growth (Zhao et al. [2009
]), therefore an inducible flocculent phenotype would be most desirable to use in fermentation procedures (Govender et al. [2010
The present work describes the successful construction of a novel recombinant K marxianus
CECT 11769 yeast strain with an inducible flocculating phenotype, for the industrial production of EPG and ethanol. This was achieved by engineering a yeast expression vector containing the FLO5 gene, from S. cerevisiae,
under the control of the EPG1 promoter. This promoter originates from the CECT 11769 strain and has the advantages of not being repressed by glucose (even in high concentrations, such as 100
g/L; Serrat et al. [2002
]), as well as being repressed under aerobic conditions. These two properties make this promoter ideally suited for the modulation of yeast flocculent phenotypes in industrial fermentations. In this way, we could use this novel inducible flocculating K. marxianus
strain in two-steps fermentations. In the first fermentation step, the yeasts are grown, under aerobic conditions, to high cell densities (the aerobic conditions would prevent flocculation, hence favoring cell growth). On the other hand, the second fermentation step involves the production of ethanol and EPG, under anaerobic conditions (producing yeast flocculation at just the right time). This second step could be carried out continuously.
Analyses of the recombinant K. marxianus
clones obtained revealed that those generated by a processes favoring homologous recombination (when the plasmid DNA was linearized with a restriction enzyme with a cleavage site inside the EPG1 promoter region) resulted in strains displaying the flocculent phenotype in a constitutive manner. On the other hand, the clones generated by a process favoring random recombination (plasmid linearized with a restriction enzyme with a cleavage site located before the start of the promoter) produced strains with inducible flocculent phenotypes. The latter also produced a far higher number of recombinant colonies than the former, in agreement with the results obtained by Nonklang et al. ([2008
]) indicating that this yeast species has a strong preference for random over homologous recombination processes. DNA sequencing of the promoter regions from the recombinant yeast revealed that all the clones constitutively expressing FLO5 contained a deletion (4 to 7
bp in length) at position -273 to -279 upstream of the ATG, in a DNA region encompassing a novel putative repressor site (Figure ), next to the TATA box. This deletion did not appear in the inducible clones, with promoter sequences identical to that of the wild-type strain. This upstream repressor site is closely related to the oxygen repression mechanism that regulates the EPG1 promoter and this could explain why the flocculent phenotype is not repressed under aerobic conditions. Some of these upstream repressor sites involved in oxygen regulation are well characterized in S. cerevisiae
(Kwast et al. [1998
]) and the deletion reported here could be due to a mistake in the impaired homologous recombination mechanisms of this yeast species. Both flocculating phenotypes were stable at 42°C, this is contrary to the data reported by Nonklang et al. ([2009
]), indicating that the flocculation phenotype generated by expression of the FLO5 gene is sensitive to temperatures above 40°C.
As shown in Table , the constitutive flocculent phenotype displayed slower growth, as indicated by its lower biomass production, than both the inducible and the wild-type strains. This is not surprising, as it had been previously shown by Zhao et al. ([2009
]) that expression of the flocculent phenotype interferes with the ability of the yeast to grow. This is the reason why our aim centered on the development of a K. marxianus
CECT 11769 strain displaying an inducible flocculent phenotype.
EPG is an enzyme produced by K. marxianus
under stress conditions. This type of enzyme is used by pectinolytic microorganisms to soften plant tissues and get access to the nutrients located inside the plant cells (Blanco et al. [1999
]). The constitutive phenotype produced high amounts of EPG, at the two temperatures assayed (Figure A), and this could be a result of the higher stress conditions within the floc, as compared to cells growing planktonically.
The inducible flocculating phenotype yielded higher ethanol production that the constitutive strain (Figure B and C), but neither of the flocculent phenotypes could match the level of ethanol production by the wild-type when the yeast were grown at 30°C. On the other hand, the differences in yield were greatly reduced when the yeasts were grown at 42°C, with the inducible yeast strain producing a very similar amount of ethanol as the wild-type strain. These results indicate that the inducible strain could be successfully, and advantageously, used in fermentative processes taking place at high temperatures, which are of industrial interest because the use of higher temperatures results in the reduction of both the cost and the risk of microbial contamination (Abdel-Banat et al. [2010b
In conclusion, the present work resulted in the generation of two novel K marxianus CECT 11769 flocculent phenotypes, one constitutive and the other inducible, that successfully produce ethanol and EPG. Apart from the advantage that flocculation represents in industrial fermentations, the constitutive phenotype produces more EPG enzymatic activity than the wild-type strain, whereas the inducible phenotype produces similar amounts of ethanol as the wild-type.