Identification and functional study of a new FLO10-derivative gene from the industrial flocculating yeast SPSC01
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- He, L., Zhao, X., Ge, X. et al. J Ind Microbiol Biotechnol (2012) 39: 1135. doi:10.1007/s10295-012-1121-1
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Yeast flocculation is an important property for the brewing industry as well as for ethanol fermentation to facilitate biomass recovery by sedimentation from the fermentation broth, which is cost-effective. In this study, a new flocculating gene FLO10spsc of 4,221 bp homologous to FLO10 was identified in the industrial flocculating yeast SPSC01. Sequence analysis indicated that the N- and C-terminus of the deduced protein of this new FLO gene are 99 % identical to that of FLO10, but more intragenic repeats are included. The study on the function of FLO10spsc by its integrative expression in the non-flocculating industrial yeast indicated severe inhibition in the flocculation of the transformant by mannose and maltose, moderate inhibition by sucrose and glucose and no inhibition by xylose and galactose, and thus the NewFlo type was established. Meanwhile, the flocculation of the transformant was stable when the temperature was below 50 °C and the pH was in the range of 4.0–6.0. Furthermore, the medium containing 250 g/l glucose was completely fermented within 48 h by the transformant, with about 110 g/l ethanol and 5.5 g(DCW)/l biomass produced, and no significant difference in ethanol fermentation performance was observed compared to its wide-type strain. Therefore, the FLO gene and corresponding transformation strategy provide a platform for engineering yeast strains with the flocculation phenotype to facilitate biomass recovery.
KeywordsYeast flocculationBiomass recoveryFLO10spscIntegrative expressionEthanol fermentation
The flocculation of Saccharomyces cerevisiae is important for the brewing industry, through which yeast cells aggregate and form flocs that can be recovered conveniently from the fermentation broth by cost-effective sedimentation . Although yeast flocculation has been explored for a long time, the mechanism underlying this phenomenon is still not fully understood due to its intrinsic complexity, making its control in the brewing industry dependent to a large extent on expertise. Both medium composition and fermentation conditions affect yeast flocculation [2, 11], but the genetic background of yeast strains ultimately determines their flocculation phenotype, which was highlighted by the expression of the single gene FLO1 in the non-flocculating laboratory yeast S.cerevisiae S288C for its flocculation .
Several FLO genes, including FLO1, Lg-FLO1, FLO5, FLO9, FLO10, and FLO11 have been identified in S. cerevisiae and reviewed recently [10, 20]. While Flo1p, Lg-Flo1p, Flo5p, Flo9p, and Flo10p allow yeast cells to form macroscopic flocs [5, 14], Flo11p confers various phenotypes in S. cerevisiae, including adhesive and invasive growth, pseudohyphal formation and the formation of biofilm [3, 7, 19]. In addition to the above-mentioned flocculation genes, FLO8 encodes a transcription factor that is capable of activating the transcription of FLO1 and FLO11 in S. cerevisiae [1, 4, 6]. Moreover, FLO genes contain conserved intragenic tandem repeats, which is responsible for the flocculating strength .
Compared to the unstable flocculation of yeast cells triggered by the depletion of fermentable sugars in the brewing industry, the flocculation of the yeast SPSC01 is constitutive and stable , which presents a valuable reservoir for exploring new FLO genes and their functions. In this study, FLO10spsc, a homologue of FLO10 but with more intragenic repeats, was identified, and its function was explored by its integrative expression in a non-flocculating S. cerevisiae for the flocculation phenotype.
Materials and methods
Strains and plasmids
The self-flocculating yeast SPSC01, a protoplast fusant developed for ethanol production from the non-flocculating yeast S. cerevisiae K2 and the flocculating yeast Schizosaccharomyces pombe and deposited at Chinese General Microbiological Culture Collection Center (CGMCC 0587), was used to identify FLO10spsc. The non-flocculating yeast S. cerevisiae 6525 was used to study the function of the FLO10spsc. The integrative expression vector pHO10, derived from the sequence of HO-poly-KanMX4-HO , was constructed in this study.
E. coli DH5α was used as cloning host and grown at 37 °C and 200 rpm in LB medium containing 10 g/l tryptone, 5 g/l yeast extract, and 10 g/l NaCl supplemented with 50 μg/ml ampicillin. Yeast strains were cultured at 30 °C and 150 rpm in YPD medium containing 10 g/l yeast extract, 20 g/l peptone and 20 g/l glucose, and the resistant strains were selected on the YPD agar plate supplemented with 1 M sorbitol and 300 μg/ml G418, incubating for 48 h at 30 °C after electroporation transformation. The LB and YPD media were solidified by adding 1.5 % agar.
Construction of the integrative expression vector and yeast transformation
Primers used in this work
Nucleotide sequence (5′ → 3′)
Impact of medium composition on the flocculation of the transformant
The selected transformant was grown in a flask with the YPD medium for 24 h at 30 °C and 150 rpm, harvested through sedimentation and washed two times with the de-flocculating buffer (0.1 M sodium citrate, pH4.5) to de-flocculate the yeast flocs so that biomass concentration could be controlled accurately for further study.
The deflocculated yeast cells were adjusted to the OD620 of 2.0, and 20 ml suspension was sampled and centrifuged to collect cell pellets, which were washed two times with the flocculating buffer (50 mM sodium acetate, 0.1 % CaCl2, pH 4.5) and suspended to 20 ml for yeast cells to re-flocculate. The impact of temperature, pH, sugar concentrations (0–0.6 M) including mannose, glucose, sucrose, maltose, galactose, and xylose and Ca2+ on the flocculation was evaluated with triplicate experiments.
The yeast was incubated in flask with the YPD medium for overnight at 30 °C, 150 rpm, and yeast flocs were collected and washed two times with the de-flocculating buffer. Then, 1 ml de-flocculated yeast cell suspension was adjusted to the OD620 of 1.0 and inoculated into a 250-ml flask containing 100 ml of fermentation medium composed of 5 g/l yeast extract, 10 g/l peptone, and 250 g/l glucose. The ethanol fermentation was carried out at 30 °C and 150 rpm.
Glucose and ethanol were analyzed by the Biosensor (SBA-40C, China), and biomass (dry cell weight) was evaluated at the end of fermentation by collecting all yeast cells, washing three times with deionized water, and drying to a constant weight at 85 °C.
Results and discussion
Sequence analysis of FLO10spsc
Introduction of the FLO10spsc into the industrial yeast S. cerevisiae 6525
Effect of temperature, pH, sugars, and Ca2+ on the flocculation phenotype
The flocculation of the transformant was inhibited by sugars with the following sequence: mannose > maltose > sucrose > glucose > xylose > galactose, which confers different sensitivity of sugar inhibition: severe inhibition by mannose and maltose, moderate inhibition by sucrose and glucose, and no inhibition by xylose and galactose (Fig. 5c). When the sequence of the deduced product of FLO10spsc was analyzed, it was found that the N-terminus sugar recognition region is the same with that of FLO10 . However, the two sequences differ largely in the number of the repeated units. According to the difference in sugar sensitivity, yeast flocculation is classified into two main groups: Flo1 type is inhibited by mannose and presented in most laboratory strains; NewFlo type is inhibited by mannose and glucose, and is found in many brewing industrial strains [12, 13]. The experimental data showed that the transformant is the NewFlo phenotype.
Ca2+ is necessary for maintaining the active conformation of the flocculating protein , and the flocculation of the transformant was improved with the increase of Ca2+ concentration, but the saturation phenomenon was observed when Ca2+ was increased to about 10 mM under the designated biomass concentration (Fig. 5d).
Ethanol fermentation performance of the transformants
Ethanol fermentation performance of the transformants and their wild-type strain
Residual glucose (g/l)
Ethanol yield (%)
109.3 ± 2.3
0.48 ± 0.20
5.53 ± 0.22
43.82 ± 0.93
111.3 ± 3.1
0.45 ± 0.18
5.49 ± 0.18
44.61 ± 1.22
110.0 ± 2.0
0.52 ± 0.13
5.50 ± 0.24
44.09 ± 0.80
The new functional FLO gene was identified from the industrial flocculating yeast SPSC01, and the sequence analysis indicates that it is a derived form of FLO10 with a full length of 4,221 bp encoding a protein of 1,406 amino acids. The transformant developed by the integrative expression of FLO10spsc into the non-flocculating industrial yeast displays the NewFlo flocculation phonotype, which is thermo-stable when the temperature is in the range of 20–50 °C, and not affected significantly by the pH change from 4.0 to 6.0. Moreover, the transformant exhibits similar growth and ethanol fermentation performance to its host strain, indicating that the FLO gene and corresponding transformation strategy could be a platform for engineering yeast strains with the flocculation phenotype to facilitate biomass recovery.
Funding for this research was provided in part from the National Natural Science Foundation of China with a grant number of 20806014.