Enhanced deacidification activity in Schizosaccharomyces pombe by genome shuffling

Abstract

A problem frequently occurring in making some kinds of wines, particularly Vitis quinquangularis Rehd wine, is the presence of malic acid at high concentrations, which is detrimental to the quality of wines. Thus, there is a need of the ways for effectively reducing the malic acid levels in wine. This study aimed to generate shuffled fusants of Schizosaccharomyces pombe with enhanced deacidification activity for reducing the excessive malic acid content in wine. Sz. pombe CGMCC 2.1628 was used as the original strain. The starting mutant population was generated by UV treatment. The mutants with higher deacidification activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by using the indicator of malic acid concentration of fermentation supernatants on 96-well microtitre plates, measured with bromocresol green. After three rounds of genome shuffling, the best-performing fusant, named GS3-1, was obtained. Its deacidification activity (consumed 4.78 g/l malic acid within 10 days) was increased by 225.2% as compared to that of original strain. In the Vitis quinquangularis Rehd wine fermentation test, GS3-1 consumed 4.0 g/l malic acid during the whole cycle of fermentation, providing up to 185.7% improvement in malic acid consumption compared with that of the original strain. This study shows that GS3-1 has great potential for improving the quality of Vitis quinquangularis Rehd wine.

Flavor Formation in Chinese Rice Wine (Huangjiu): Impacts of the Flavor-Active Microorganisms, Raw Materials, and Fermentation Technology

“…Apart from traditional screening, genetic modification techniques are also capable of creating new yeast strains with specific metabolic profiles for alcoholic fermentation (Krogerus et al, 2017). Ding et al (2015) enhanced deacidification activity in Schizosaccharomyces pombe using genome shuffling, and Dong et al (2019) increased production of acetate ester in S. cerevisiae using a "self-cloning" integration strategy. Ohashi et al (2020) reported that the removal of feedback inhibition of NAGK activity resulted in the significantly higher production of ornithine in sake and sake cake by gene expression.…”

Selected Schizosaccharomyces pombe Strains Have Characteristics That Are Beneficial for Winemaking

“…Several research teams are studying the winemaking potential of non- Saccharomyces yeast strains [ 1 ]. For example, fission yeasts from the genus Schizosaccharomyces show more rapid malic acid deacidification, by converting malic acid to ethanol and CO 2 [ 2 – 4 ]. Schizosaccharomyces pombe possesses several remarkable metabolic properties that may be useful in modern quality winemaking [ 2 , 5 – 6 ], including a malic dehydrogenase activity, high autolytic polysaccharides release [ 7 ], ability of gluconic acid reduction [ 8 – 11 ], urease activity [ 12 , 13 ], elevated production of pyruvic acid and colour improvement [ 14 , 15 ], as well as low production of biogenic amines and ethyl carbamate [ 6 , 16 ].…”

EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains

“…Metabolic engineering undertakes a rational approach to redirect the metabolic fluxes towards the desired product by targeted manipulation of the cell’s genome involving insertion of heterologous pathways, overexpression and downregulation of multiple genes, construction of synthetic regulatory circuits, etc. [ 14 , 24 ]. Multiple rounds of genetic engineering are commonly required until the cell with desired properties is obtained.…”

“…A series of vectors with various auxotrophic markers, promoters, multiple cloning sites and integration sequences have been developed [ 9 , 15 , 38 , 47 ]. Recently, an EasyClone vector set was developed [ 17 ] enabling integration by double cross-over events into several intergenic regions, which have been validated for sufficient and reliable expression level and absence of growth impairment, on three different S. cerevisiae chromosomes [ 24 ]. The insertion sites are interspaced with essential genes voiding risk of chromosomal rearrangements even when the expression cassettes share long sequence homologies [ 26 ].…”