YEAST MUTANTS EXCRETING VITAMIN B1 AND THEIR USE IN THE PRODUCTION OF THIAMINE RICH BEERS*,†

In certain strains of Saccharomyces cerevisiae and Saccharomyces uvarum (carlsbergensis), high doses of ultraviolet radiation induced stable mutants excreting thiamine from their living cells during the ethanol production. In S. cerevisiae, the first mutagenesis step yielded mutants with the production of 0·3–0·5 mg extracellular thiamine. HCL/litre minimum medium without thiamine and the second one increased the production up to 2·0–2·1 mg thiamine. HCL/litre. In S. uvarum (carlsbergensis), mutants producing up to 0·76 mg thiamine. HCL/litre 10% Plato hopped wort were obtained in the first mutagenesis step combined with mitotic recombinations. The increase up to 0·92 mg/litre was achieved here by repeated selections. Both laboratory and pilot plant fermentations in 10 and 4% Plato hopped worts showed the suitability of selected mutants for the production of thiamine rich beers which fulfilled all quality requirements and contained 0·67–0·80 and 0·22–0·33 mg thiamine. HCI/litre, respectively.     

枯草杆菌纤溶酶高产菌株的物理化学诱变

以枯草杆菌FM-S2作为出发菌株,经γ-射线和2.5%硫酸二乙酯复合诱变,结合牛奶蛋白平板从2341株存活菌中初筛得到200株诱变菌株,经摇瓶测定纤溶酶活性进行复筛得到7株生产性能较出发菌株显著提高的突变株,它们的纤溶酶活性超过2000UK.U/ml。其中突变株γ-DES36纤溶酶活性最高达到2719.89±242.51UK.U/ml,同时该菌株遗传性能稳定。     

烟草抗野火病细胞突变体筛选

烤烟品种红花大金元叶片组织悬浮培养细胞经过４～５代继代培养后，铺平板于含野火病粗毒的ＭＳ＋２，４－Ｄ２．０ｍｇ／Ｌ＋６－ＢＡ０．１ｍｇ／Ｌ（简称ＭＳＩ）培养基上进行筛选或置于含野火病粗毒的ＭＳＩ液体培养基上进行浅层筛选培养。存活愈伤组织转移至相同培养基进一步筛选，经两次筛选得到的愈伤组织在ＭＳ＋２，４－Ｄ０．１ｍｇ／Ｌ＋６－ＢＡ２．０ｍｇ／Ｌ培养基上诱导分化形成植株，通过上述方法筛选得到的抗性愈伤组织细胞比未经筛选进行继代培养的愈伤组织细胞对野火病粗毒表现出较强的抵抗力；对筛选得到的愈伤组织再生植株及其后代进行人工接种，从中筛选出一些抗病株系，研究结果表明，利用体细胞无性系变异，采用细胞离休筛选技术，可以获得抗野火病的烟草新材料。     

Morphological Changes in Autolytic Wine Yeast during Aging in Two Model Systems

ABSTRACT: Morphological changes produced in Saccharomyces cerevisiae IFI473 and 2 autolytic mutants derived from it (M1 and M2 mutants) were studied during yeast aging in 2 model systems (rich medium and model wine). Different conditions affecting autolysis, including temperature, culture media, nitrogen starvation, or phenyl-methylsulfonylfluoride addition, were analyzed. In rich medium, morphological changes mainly consisted in variation of cell size, presence of autophagic bodies inside the cytoplasm, detachment of the cytoplasm from the cell wall, spore formation, and loss of cytoplasmic material. Morphological changes were greater for mutant M2 than for the rest of the strains studied. In the wine medium, a decrease in cell size was the most relevant feature and the morphological changes observed were similar for all strains. Results obtained show morphological differences between autophagy and autolysis suggesting that yeast cells with accelerated autolysis could also present accelerated autophagy.     

In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants

The aggregation of alpha-synuclein (α-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson’s disease (PD). Ninety percent of α-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. α-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of α-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT α-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated α-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) α-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of α-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated α-SYN in vitro, as was shown previously for WT α-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of α-SYN.     

Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant

The photoproduction of H₂ was studied in a sulfur-deprived Chlamydomonas reinhardtii D1 mutant that carried a double amino acid substitution. The leucine residue L159 was replaced by isoleucine, and the asparagine N230 was replaced by tyrosine (L159I-N230Y). Phenotypic characterization of the mutant showed some interesting features compared to its wild type, namely: (1) a lower chlorophyll content; (2) a higher photosynthetic capacity and higher relative quantum yield of photosynthesis; (3) a higher respiration rate; (4) a very high conversion of violaxanthin to zeaxanthin during H₂ production; (5) a prolonged period of H₂ production. In standard conditions, the mutant produced more than 500 ml of H₂, that is, more than one order of magnitude greater than its wild type, and about 5-times greater than the CC124 strain that was used for comparison. The better performance of the mutant was mainly the result of a longer production period. Biogas produced contained up to 99.5% H₂.     

Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: Ultrastructural analysis of spo mutants

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation-deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild-type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18-h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore-like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore-wall precursors, instead of anucleate spore-like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.