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Yeast flocculation is the reversible formation of multicellular complexes mediated by lectin-like cell wall proteins binding to neighbouring cells. Strong flocculation can improve the inhibitor tolerance and fermentation performance of yeast cells in second generation bioethanol production. The strength of flocculation increases with the size of the flocculation protein and is strain dependent. However, the large number of internal repeats in the sequence of FLO1 from Saccharomyces cerevisiae S288c makes it difficult to recombinantly express the gene to its full length. In the search for novel flocculation genes resulting in strong flocculation, we discovered a DNA sequence, FLONF, that gives NewFlo phenotype flocculation in S. cerevisiae CEN.PK 113-7D. The nucleotide sequence of the internal repeats of FLONF differed from those of FLO1. We hypothesized that a chimaeric flocculation gene made up of a FLO1 variant derived from S. cerevisiae S288c and additional repeats from FLONF from S. cerevisiae CCUG 53310 would be more stable and easier to amplify by PCR. The constructed gene, FLOw, had 22 internal repeats compared to 18 in FLO1. Expression of FLOw in otherwise non-flocculating strains led to strong flocculation. Despite the length of the gene, the cassette containing FLOw could be easily amplified and transformed into yeast strains of different genetic background, leading to strong flocculation in all cases tested. The developed gene can be used as a self-immobilization technique or to obtain rapidly sedimenting cells for application in e.g. sequential batches without need for centrifugation.
Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw.
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