Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background  

Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.     

Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis

The growth of two strains of Lactococcus lactis subsp. lactis from vegetable (NCDO 2118) and dairy origin (IL 1403) were compared on various culture media. Both strains grew more rapidly on a complex organic medium than on a defined synthetic medium. The best growth was obtained under nitrogen gas phase. The single omission technique was applied to each component of a non-optimized synthetic medium in order to determine the true nutritional requirements. Requirements for macro-elements, oligo-elements, bases and vitamins were identical for the two strains. As expected, the dairy strain (IL 1403) was seen to be auxotrophic for some amino acids, whereas the vegetable strain (NCDO 2118) was seen to be prototrophic for all amino acids when using the single omission technique. Growth was then characterized on progressively simplified media and the composition of the absolute minimal media for the growth of both strains was defined. Sustained growth of the vegetable strain was only possible in minimal media supplemented with six amino acids (Glu, Met, Ile, Leu, Val, Ser), indicating that the definition of prototrophy/auxotrophy is partly dependent upon the medium composition. The dairy strain showed a requirement for Arg, His and Thr in addition to the six amino acids necessary for growth of the vegetable strain. The removal of ammonium salt from the medium did not affect the growth, illustrating that the amino acids may satisfy the totality of the nitrogen requirement for biomass synthesis.     

Auxotrophic mutants in the selection of the rubomycin producer, Actinomyces coeruleorubidus

A total of 351 auxotrophic mutants with different antibiotic activity, including several mutants with activity higher than that of the parent prototrophic strains were obtained under the effect of gamma-rays from 3 prototrophic strains of Act. coeruleorubidus. It was shown that most of the auxotrophic mutants did not preserve the property of biochemical insufficiency on passages on complete media. A mutant strain 1059-32 with activity 2 times higher than that of the prototrophic strain 2-39 and the parent auxotrophic culture was obtained from the revertants. Requirements in 29 growth factors including 17 amino acids, 4 nitrous bases, 8 vitamins and coenzymes were determined in 46 stable auxotrophic mutants isolated. The effect of the specific and non-specific growth factors on the culture antibiotic production was studied.     

Commonly used Saccharomyces cerevisiae strains (e.g. BY4741, W303) are growth sensitive on synthetic complete medium due to poor leucine uptake

It is reported that some of the widely used laboratory strains of Saccharomyces cerevisiae (e.g. W303, BY4741) are sensitive to synthetic media containing all 20 amino acids [e.g. synthetic complete (SC) medium or supplemented minimal medium]. To determine the molecular basis for this unexpected sensitivity, a genomic library was screened and three genes were identified that, when overexpressed, rescue cells from this phenotype. Two of the 'rescuing' genes, BAP2 and TAT1, are related to transport of leucine, and one, LEU2, to synthesis of leucine, showing that sensitivity to SC medium is associated with the leu2 mutation. The sensitive strains seem incapable of transporting leucine when grown on synthetic complete media. This effect of the leu2 mutation should be taken into consideration when analyzing the results of genetic screens and other experiments performed with these strains.     

Availability of Amino Acids Extends Chronological Lifespan by Suppressing Hyper-Acidification of the Environment in Saccharomyces cerevisiae

The chronological lifespan of Saccharomyces cerevisiae represents the duration of cell survival in the postdiauxic and stationary phases. Using a prototrophic strain derived from the standard auxotrophic laboratory strain BY4742, we showed that supplementation of non-essential amino acids to a synthetic defined (SD) medium increases maximal cell growth and extends the chronological lifespan. The positive effects of amino acids can be reproduced by modulating the medium pH, indicating that amino acids contribute to chronological longevity in a cell-extrinsic manner by alleviating medium acidification. In addition, we showed that the amino acid-mediated effects on extension of chronological longevity are independent of those achieved through a reduction in the TORC1 pathway, which is mediated in a cell-intrinsic manner. Since previous studies showed that extracellular acidification causes mitochondrial dysfunction and leads to cell death, our results provide a path to premature chronological aging caused by differences in available nitrogen sources. Moreover, acidification of culture medium is generally associated with culture duration and cell density; thus, further studies are required on cell physiology of auxotrophic yeast strains during the stationary phase because an insufficient supply of essential amino acids may cause alterations in environmental conditions.     

Saccharomyces cerevisiae mitochondria are required for optimal attractiveness to Drosophila melanogaster

While screening a large collection of wild and laboratory yeast strains for their ability to attract Drosophila melanogaster adults, we noticed a large difference in fly preference for two nearly isogenic strains of Saccharomyces cerevisiae, BY4741 and BY4742. Using standard genetic analyses, we tracked the preference difference to the lack of mitochondria in the BY4742 strain used in the initial experiment. We used gas chromatography coupled with mass spectroscopy to examine the volatile compounds produced by BY4741 and the mitochondria-deficient BY4742, and found that they differed significantly. We observed that several ethyl esters are present at much higher levels in strains with mitochondria, even in fermentative conditions. We found that nitrogen levels in the substrate affect the production of these compounds, and that they are produced at the highest level by strains with mitochondria when fermenting natural fruit substrates. Collectively these observations demonstrate that core metabolic processes mediate the interaction between yeasts and insect vectors, and highlight the importance mitochondrial functions in yeast ecology.     

Construction and characterization of centromeric, episomal and GFP-containing vectors for Saccharomyces cerevisiae prototrophic strains

A set of shuttle yeast vectors containing the dominant selectable markers KanMX4 or HphMX4 cassettes, conferring resistance to geneticin and hygromycin B, respectively, was constructed. Dominant selectable markers are useful for genetic manipulation of natural, wine and industrial strains which do not contain any auxotrophic markers as well as of strains which cannot grow on synthetic mineral medium. Vectors were characterized by (i) copy number, (ii) mitotic stability both in selective and non-selective conditions, (iii) the efficiency and frequency of transformations, (iv) optimal adaptation times in non-selective media, (v) optimum conditions for transformation of various laboratory, commercial and wine strains, and (vi) expression level of an inserted gene. Furthermore we produced GFP-containing vectors that can be used for protein subcellular localization in prototrophic strains.     

Defined minimal media for the growth of prototrophic and auxotrophic strains of Bacillus stearothermophilus

Minimal chemically defined media for Bacillus stearothermophilus were developed at 60°C and quantitative requirements for each nutrient were determined. A prototrophic strain of B. stearothermophilus was grown in medium containing only glucose and mineral salts whereas auxotrophic strains in addition required biotin, thiamine, nicotinic acid and DL-methionine. Metabolic interaction between L-valine and L-leucine was observed with auxotrophic organisms. The presence of L-leucine in minimal medium necessitated the addition of L-valine. Growth took place in the absence of both amino acids.     

CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建

谷胱甘肽(Glutathione,GSH)是具有多种生理功能的非蛋白质类巯基化合物,已广泛应用于药品、食品等行业,且市场需求量逐年增加。遗传工程育种是提高细胞内GSH含量的重要策略,但在遗传操作过程中使用到的营养缺陷型遗传标记可能会影响菌株的正常生长,且不利于高密度发酵的进行。为回复工程菌株的营养缺陷型,利用g RNA转录表达框和靶基因同源DNA片段直接共转化酵母细胞,由细胞内表达的Ⅱ型CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9)介导的基因组编辑技术将营养缺陷型GSH工程菌株W303-1b/FGP回复为原养型菌株。结果显示,与营养缺陷型菌株相比,原养型菌株生长周期缩短,且可以利用简单的合成培养基进行培养,方便菌株的大规模培养。     

Polyphosphates as an energy source for growth of Saccharomyces cerevisiae

Cells of the yeast Saccharomyces cerevisiae with a low content of polyphosphates (polyP) are characterized by disturbance of growth in medium with 0.5% glucose. The parent strain with polyP level reduced by phosphate starvation had a longer lag phase. The growth rate of strains with genetically determined low content of polyP due to their enhanced hydrolysis (CRN/pMB1_PPN1 Sc is a superproducer of exopolyphosphatase PPN1) or reduced synthesis (the BY4741 vma2Δ mutant with impaired vacuolar membrane energization) was lower in the exponential phase. The growth of cells with high content of polyP was accompanied by polyP consumption. In cells of strains with low content of polyP, CRN/pMB1_PPN1 Sc and BY4741 vma2Δ, their consumption was insignificant. These findings provide more evidence indicating the use of polyP as an extra energy source for maintaining high growth rate.     

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.     

Induced parasexual processes in Claviceps sp. strain SD58.

A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.     

Induced parasexual processes in Claviceps sp. strain SD58

A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.     

Prototrophic hybrids of bakers yeast. I. Selection of haploids and diploids of prototrophic yeast

153 haploids, including 8 prototrophs, 12 biotin prototrophs and 4 biotin auxotrophs were isolated from Y, F and FB strains of the baker's yeast Saccharomyces cerevisiae. Remaining haploids were dependent on vitamins of B group other than biotin. Obtained haploids were characterized by large cell sizes, a or α mating type, the ability to fermentation of sugars and the assimilation of non-fermented carbon sources. Haploids obtained from the yeast of Y and FB strains a good growth in the synthetic medium. Prototrophic haploids, prototrophic haploids with biotin auxotrophs and biotin prototrophs with biotin auxotrophs were crossed. 89 prototrophic hybrids capable of growth in synthetic medium without vitamins were selected out of 178 hybrids obtained. Hybrids are characterized by features typical for baker's yeast; however, not all of them are capable of sporulation. As result of selection, prototrophic hybrids and 16 hybrids characterized by a good increase of biomass in molasses medium were chosen. The efficiency of the biomass of hybrids designated as YY 3040/2, YY 3040/3 and FFB 1910/2 is considerably higher than one obtained from the cultivation of the industrial strain French Mautner (Mf). All hybrids possess adequate enzymatic activity in anaerobic metabolism of saccharose and maltose. Selected prototrophic hybrids were sent out to two yeast factories in the country for experimental propagation.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Anhydrobiosis in yeast: role of cortical endoplasmic reticulum protein Ist2 in Saccharomyces cerevisiae cells during dehydration and subsequent rehydration

Antonie van Leeuwenhoek - Two Saccharomyces cerevisiae strains, BY4741 and BY4741-derived strain lacking the IST2 gene (ist2Δ), were used to characterise the possible role of cortical...     

Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.     

Strain improvement of Claviceps purpurea by protoplast fusion without introducing auxotrophic markers

Protoplasts of morphologically and biochemically different Claviceps purpurea strains producing ergotoxins were fused without introducing selective auxotrophic markers. Fused strains thus obtained differed significantly in biosynthetic activity and morphology from the prototrophic isolates obtained after fusion of the same parent strains marked by auxotrophy. Comparison of the two types of fused strains showed about tenfold higher alkaloid production in fusants obtained from prototrophic strains. Selected stable prototrophic isolates also showed a significant productivity improvement in comparison with the original parent strains. Correspondence to: M. Didek-Brumec     

Minimal Growth Requirements for Clostridium perfringens and Isolation of Auxotrophic Mutants

The minimal growth requirements for two strains of Clostridium perfringens were defined, and both synthetic and semisynthetic plating media were developed. Plate counts of the wild-type strains on both of these minimal media were equivalent to those on complex media. A number of auxotrophic mutants of each strain were isolated, and their phenotypes were defined.