Optimized Heterologous Expression of the Polymorphic Human Glutathione Transferase M1-1 Based on Silent Mutations in the Corresponding cDNA

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression inEscherichia coliafforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5′ region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.     

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.     

A rapid, high-throughput method for quantitative determination of ethanol tolerance in Saccharomyces cerevisiae

There are a variety of methods available for determining ethanol tolerance phenotypes in Saccharomyces cerevisiae. Many of these methods are limited in through-put and/or application. We describe here a microtiter-plate, growth-based, liquid-culture, rapid ethanol tolerance assay (RETA) that overcomes these limitations. Determinations of ethanol tolerance gave results that were comparable to shake-flask cultures, and there was a high level of intra- and inter-plate reproducibility. We report the successful application of this assay to determining the segregation pattern of ethanol tolerance in backcrosses of two ethanol-tolerant mutants (SM and CM) to a non-tolerant parent (W303-1A); RETA was used to determine ethanol tolerance in numerous progeny resulting from these crosses. This assay, or variations thereof, will prove be of great value for anyone attempting to develop quantitative, high-throughput growth assays for yeast or other microorganisms.     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Experimental reduction of codon bias in the Drosophila alcohol dehydrogenase gene results in decreased ethanol tolerance of adult flies

The ethanol tolerance of adult transgenic flies of Drosophila containing between zero and ten unpreferred synonymous mutations that reduced codon bias in the alcohol dehydrogenase (Adh) gene was assayed. As the amino acid sequences of the ADH protein were identical in the four genotypes assayed, differences in ethanol tolerance were due to differences in the abundance of ADH protein, presumably driven by the effects of codon bias on translational efficiency. The ethanol tolerance of genotypes decreased with the number of unpreferred synonymous mutations, and a positive correlation between ADH protein abundance and ethanol tolerance was observed. This work confirms that the fitness effects of unpreferred synonymous mutations that reduce codon bias in a highly expressed gene are experimentally measurable in Drosophila melanogaster.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.     

Pathways of Genetic Adaptation: Multistep Origin of Mutants Under Selection Without Induced Mutagenesis in Salmonella enterica

In several bacterial systems, mutant cell populations plated on growth-restricting medium give rise to revertant colonies that accumulate over several days. One model suggests that nongrowing parent cells mutagenize their own genome and thereby create beneficial mutations (stress-induced mutagenesis). By this model, the first-order induction of new mutations in a nongrowing parent cell population leads to the delayed accumulation of visible colonies. In an alternative model (selection only), selective conditions allow preexisting small-effect mutants to initiate clones that grow and give rise to faster-growing mutants. By the selection-only model, the delay in appearance of revertant colonies reflects (1) the time required for initial clones to reach a size sufficient to allow the second mutation plus (2) the time required for growth of the improved subclone. We previously characterized a system in which revertant colonies accumulate slowly and contain cells with two mutations, one formed before plating and one after. This left open the question of whether mutation rates increase under selection. Here we measure the unselected formation rate and the growth contribution of each mutant type. When these parameters are used in a graphic model of revertant colony development, they demonstrate that no increase in mutation rate is required to explain the number and delayed appearance of two of the revertant types.     

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.     

Transport of acetate in mutants of Saccharomyces cerevisiae defective in monocarboxylate permeases

The strain Saccharomyces cerevisiae W303-1a, able to grow in a medium containing acetic acid as the sole carbon and energy source, was subjected to mutagenesis in order to obtain mutants deficient in monocarboxylate permeases. Two mutant clones exhibiting growth in ethanol, but unable to grow in a medium with acetic acid as the sole carbon and energy source, were isolated (mutants Ace12 and Ace8). In both mutants, the activity for the acetate carrier was strongly affected. The mutant Ace8 revealed not to be affected in the transport of lactate, while the mutant Ace12 did not display activity for that carrier. These results reinforced those previously found in the strain IGC 4072, where two distinct transport systems for monocarboxylates have been described, depending on the growth carbon source. It is tempting to postulate that the Ace8 mutant seems to be affected in the gene coding for an acetate permease. In contrast, the absence of activity for both monocarboxylate permeases in mutant Ace12 could be attributed to a mutation in a gene coding for a regulatory protein not detected before.     

Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2

We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S.?cerevisiae W303-1A and in the isogenic mutants jen1?, ady2? and jen1? ady2?, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells.     

Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Characterization of herpes simplex virus type 1 thymidine kinase mutants selected under a single round of high-dose brivudin

A broad variety of herpes simplex virus type 1 clones was selected under a single round of high-dose selection with brivudin. Mutations in the thymidine kinase (TK) genes consisted of 42% frameshift mutations within homopolymer repeats of G's and C's and single nucleotide substitutions (58%) that produced stop codons (Q261 and R281) or a new codon at the site of the substitution (A168T, R51W, G59W, G206R, R220H, Y239S, and T287 M). The A168T change, associated with an altered TK phenotype, proved to be the most commonly selected substitution. For the different mutants, a correlation between phenotype, genotype, and in vivo neurovirulence was observed.     

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.     

Engineering improved ethanol production in Escherichia coli with a genome-wide approach

A key challenge to the commercial production of commodity chemical and fuels is the toxicity of such molecules to the microbial host. While a number of studies have attempted to engineer improved tolerance for such compounds, the majority of these studies have been performed in wild-type strains and culturing conditions that differ considerably from production conditions. Here we applied the multiscalar analysis of library enrichments (SCALEs) method and performed a growth selection in an ethanol production system to quantitatively map in parallel all genes in the genome onto ethanol tolerance and production. In order to perform the selection in an ethanol-producing system, we used a previously engineered Escherichia coli ethanol production strain (LW06; ATCC BAA-2466) (Woodruff et al., in press), as the host strain for the multiscalar genomic library analysis (>10⁶ clones for each library of 1, 2, or 4 kb overlapping genomic fragments). By testing individually selected clones, we confirmed that growth selections enriched for clones with both improved ethanol tolerance and production phenotypes. We performed combinatorial testing of the top genes identified (uspC, otsA, otsB) to investigate their ability to confer improved ethanol tolerance or ethanol production. We determined that overexpression of otsA was required for improved tolerance and productivity phenotypes, with the best performing strains showing up to 75% improvement relative to the parent production strain.     

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。