Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae

Abstract The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1 . Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11 . The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3 ) that, however, was unable to suppress other FLO1 genes in other flocculent strains.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

The identification, characterization, and mapping of a gene for flocculation in Saccharomyces sp.

Genetic studies of a flocculent haploid strain of Saccharomyces have revealed the flocculation to be dominant and controlled at a single gene locus. The flocculation character of both hybrids and haploids derived from such hybrids appears to be influenced by the repression or derepression status of the culture. Mapping studies of this flocculation gene have revealed that it is linked to ade 1 and therefore located on Chromosome I. Consequently, this is a different gene to the three flocculation genes studied by other laboratories because they have found such genes to be unlinked to ade 1. The flocculation gene being discussed in this paper has been designated FLO4. FLO4 has been located 32-33 cM from the Chromosome I centromere and 37 cM from ade 1 (i.e., FLO4 is on the opposite side of the centromere to ade 1).     

Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain

A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment. The shortest DNA fragment (XbaI-XbaI) able to confer the flocculating phenotype was 3.1 kb. Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I. Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII. The gene was detected in a variety of S. cerevisiae strains regardless of their being phenotypically flocculating. This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors (fsu3) that do not affect other FLO genes.     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。     

高温高浓发酵工业啤酒酵母菌种的构建

高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。     

Identification and functional study of a new FLO10-derivative gene from the industrial flocculating yeast SPSC01

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 FLO10 (spsc) 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 FLO10 (spsc) 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.     

Flocculation of Saccharomyces cerevisiae tup1 mutants.

Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

絮凝性强的优良面包酵母菌株的选育

通过初筛、单倍体分离、DES诱变、絮凝基因的克隆表达及杂交等育种技术成功构建了高生物量、耐高糖、强絮凝的优良面包酵母菌株(Saccharomyces cerevisiae) ZLTH58(MATa/α,leu,FLO1)。菌株ZLTH58具有双亲的优良性状,遗传性状稳定。对其生物量、耐高糖能力、絮凝特性进行了检测,结果表明,菌株ZLTH58的生物量是原始亲株BL56的1.21倍;耐高糖能力优于原始亲株BL61;絮凝性能明显优于原始亲株BL56和BL61。对其培养条件进行了优化,在优化的培养条件下,生物量可以达到83.06g/L,为初始培养条件下的1.35倍。     

絮凝基因的克隆和在工业啤酒酵母菌株中表达

The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E.coli shuttle plasmid YCp50 as vector.Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation,designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15.Six transformants PJ208-5-15-1(pCF1)～PJ208-5-15-6(pCF1)possessing strong flocculation ability were obtained.Th…     

Flocculation in Saccharomyces cerevisiae: a review

The present work reviews and critically discusses the aspects that influence yeast flocculation, namely the chemical characteristics of the medium (pH and the presence of bivalent ions), fermentation conditions (oxygen, sugars, growth temperature and ethanol concentration) and the expression of specific genes such as FLO1, Lg‐FLO1, FLO5, FLO8, FLO9 and FLO10. In addition, the metabolic control of loss and onset of flocculation is reviewed and updated. Flocculation has been traditionally used in brewing production as an easy and off‐cost cell‐broth separation process. The advantages of using flocculent yeast strains in the production of other alcoholic beverages (wine, cachaça and sparkling wine), in the production of renewal fuels (bio‐ethanol), in modern biotechnology (production of heterologous proteins) and in environmental applications (bioremediation of heavy metals) are highlighted. Finally, the possibility of aggregation of yeast cells in flocs, as an example of social behaviour (a communitarian strategy for long‐time survival or a means of protection against negative environmental conditions), is discussed.     

絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。     

絮凝基因(FLOIG)的序列测定及分析

虽然酵母细胞絮凝的确切机制至今尚无定论,但已克隆了多个与絮凝相关的基因,如FLOI、FLO5、FLO11等[1-3].这些基因的表达可以赋予非絮凝酵母细胞以絮凝能力.酵母细胞的絮凝特性在酿造工业、固定化酶、精细化工和物生制药等领域具有广泛的应用价值[4,5].从一株强絮凝酿酒酵母菌株中克隆到一个约4.3kb的NDA片段,酵母转化实验证明该DNA片段能够赋予非絮凝酵母菌株以絮凝能力[6].本文简要报道对该DNA片段进行序列测定和分析的结果.