Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

Abstract

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to alpha-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 muM alpha-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 muM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.

Conversion of lignocellulosic agave residues into liquid biofuels using an AFEX™-based biorefinery

“…We did not measure saponin hydrolysis during AFEX pretreatment and more study is needed to understand the nature of the inhibitors in A. salmiana leaf hydrolysates. Some fungi have apparently been developed that produce detoxifying enzymes [ 71 ], and S. cerevisiae might also potentially be engineered to resist saponins [ 73 ].…”

Development of Agave as a dedicated biomass source: production of biofuels from whole plants

“…It has been noted that some Agave carbohydrate extracts are known to be unfermentable [ 33 ]. The impact of inclusion of inulinase in Agave hydrolysis used for yeast fermentation is surprising yet beneficial as the unfermentable extracts from about a dozen Agave species can now be used for production of biofuels and other chemicals using both the abundant readily solubilized and structural carbohydrates.…”

“…However, Kluyveromyces marxianus successfully fermented the juice [ 31 ], possibly due to its inulinase production along with other enzymes [ 34 ]. Indeed, Agave is known to produce anti-fungal glycosylated triterpenoids called saponins that have strong anti-fungal and anti-yeast activity [ 33 , 35 – 37 ]. Their action on fungi has been shown to be membrane permeabilization [ 38 ].…”

“…During this work, it was noticed that during yeast fermentation of Agave hydrolysates in the absence of solids, the lack of inulinase resulted in a clearing of the visible yeast turbidity, and no viable yeast based upon direct Petri plate tests, indicating possible cell lysis consistent with membrane disruption. Saponins can be inactivated enzymatically as shown in the pioneering work by Cira et al [ 33 ] where a common tomato saponin, α-tomatidine, can be inactivated by a Fusarium tomatinase after cloning this gene into S. cerevisiae . When this genetically modified yeast was tested on the must (extract) of A. tequilana and A. salmiana , the fermentations produced ethanol while the wild-type yeast did not.…”

Hydrolysis of Agave fourcroydes Lemaire (henequen) leaf juice and fermentation with Kluyveromyces marxianusfor ethanol production

“…It is well documented that agave plants produce saponins with inhibitory activity [16,17]. To corroborate this, Cira et al [18] produced a modified strain of S. cerevisiae which resists the saponins present in the musts of Agave tequilana Weber var. azul and Agave salmiana , and succeeded in fermenting them into ethanol.…”