Minor structural consequences of alternative CUG codon usage (Ser for Leu) in Candida albicans exoglucanase

In some species of Candida the CUG codon is encoded as serine and not leucine. In the case of the exo-beta-1,3-glucanase from the pathogenic fungus C. albicans there are two such translational events, one in the prepro-leader sequence and the other at residue 64. Overexpression of active mature enzyme in a yeast host indicated that these two positions are tolerant to substitution. By comparing the crystal structure of the recombinant protein with that of the native (presented here), it is seen how either serine or leucine can be accommodated at position 64. Examination of the relatively few solved protein structures from C. albicans indicates that other CUG encoded serines are also found at non-essential surface sites. However such codon usage is rare in C. albicans, in contrast to C. rugosa, with direct implications for respective recombinant protein production.     

A site-directed integration system for the nonuniversal CUGSer codon usage species Pichia farinosa by electroporation

Halotolerant yeast, Pichia farinosa, is a valuable yeast strain in fermentation industry because it produces high yield of glycerol and xylitol, and can tolerate both contamination and high-density growth during fermentation. However, the lack of genetic manipulation tools makes it less popular as a gene engineering strain. Expression systems commonly used in other yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris cannot be used in P. farinosa because it translates universal Leu codon CUG as Ser. Here we reported a modified expression vector and a transformation system with enhanced efficiency in P. farinosa. The results showed that cells of OD₆₀₀ 0.8–1.0 with DTT treatment can obtain high transformation efficiency. The optimized electroporation condition was 900 V, 25 μF, and 200 Ω. The DNA concentration did not influence the transformation. Our system provides the potential not only for applying P. farinosa as an industrial strain of gene engineering, but also for studying gene function in its native host.     

Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae

Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.     

Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.     

Drosophila melanogaster tRNA(Ser) suppressor genes function with strict codon specificity when introduced into Saccharomyces cerevisiae

The anticodon of the wild-type tRNA(7Ser) gene of Drosophila melanogaster was mutated using oligodeoxyribonucleotide-directed, site-specific mutagenesis, and all three nonsense suppressor derivatives of the gene were constructed. These constructs were cloned into an Escherichia coli-yeast shuttle vector (YRp7), and used to transform a Saccharomyces cerevisiae strain [JG 369-3B(alpha)] containing an array of nonsense alleles. When tested on appropriate omission media, the D. melanogaster suppressor genes were found to function in the yeast with strict codon specificity. Subsequent Northern hybridization analyses revealed that the D. melanogaster suppressor genes were transcribed and processed well, when in S. cerevisiae.     

Physiological conditions affecting the sensitivity of Saccharomyces cerevisiae to a Pichia kluyveri killer toxin and energy requirement for toxin action

The interaction between the killer toxin of Pichia kluyveri 1002 and cells of Saccharomyces cerevisiae SCF 1717 is strongly affected by the physiological state of sensitive cells. The killing effect is maximal for cells in the lag and early exponential phase of growth, whereas stationary cells are completely resistant. Furthermore, sensitivity is markedly enhanced by a rise of the pH (from 3.2 to 6.8) at which cells are cultured.Three successive stages can be distinguished in the killing process: (I) binding of the toxin to the primary binding site; (II) transmission of the toxin to its reactive site in the plasma membrane; (III) occurrence of functional damage (K⁺-leakage; decrease of intracellular pH). The transition from stage I to II is prevented in the absence of metabolic energy or at low temperature (below 10°C). Sensitive cells in stage I can be rescued from toxin-induced killing by a short incubation at pH 7.0, which treatment is not effective for cells in stage II. Cells in stage II are able to resume growth when plated in a rich medium containing suitable concentrations of potassium and hydrogen ions. Rescue was not observed for cells in stage III of the killing process.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

Prionization of the Pichia methanolica SUP35 gene product in the yeast Saccharomyces cerevisiae

Induction of the prionlike form of the SUP35 gene of Pichia methanolica, the [PSIP+] factor, was shown in the transgenic yeast Saccharomyces cerevisiae containing the P. methanolica SUP35 gene located in the chromosome instead of the indigenous SUP35 gene. Either the induction of the [PSIP+] factor in the transgenic yeast, unlike that of the classical [PSI+] factor, does not depend on the presence of the [PIN+] determinant in the cell or the substitution of the S. cerevisiae SUP35 gene for the P. methanolica SUP35 gene changes the PIN status of the strain. The [PSIP+] factor is unstable in mitosis and meiosis and is not effectively eliminated upon over-production of the chaperone protein Hsp104p of S. cerevisiae. The existence of an interspecific barrier during transmission of the prionlike state from S. cerevisiae Sup35p to P. methanolica Sup35p was shown.     

Screening of a Saccharomyces cerevisiae nonessential gene deletion collection for altered susceptibility to a killer peptide

Approximately 4,800 Saccharomyces cerevisiae mutants deleted for nonessential genes were screened for alterations in susceptibility to a synthetic killer peptide (KP). None of the tested strains, including mutants resistant to conventional antifungal drugs, showed increased or decreased susceptibility to KP in comparison with the parental strain. The results may reflect the peculiar mechanism of action of KP and claim the possible avoidance of vital resistant mutants.     

Synonymous codon usage in Thermosynechococcus elongatus (cyanobacteria) identifies the factors shaping codon usage variation

Analysis of synonymous codon usage pattern in the genome of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 using multivariate statistical analysis revealed a single major explanatory axis accounting for codon usage variation in the organism. This axis is correlated with the GC content at third base of synonymous codons (GC3s) in correspondence analysis taking T. elongatus genes. A negative correlation was observed between effective number of codons i.e. Nc and GC3s. Results suggested a mutational bias as the major factor in shaping codon usage in this cyanobacterium. In comparison to the lowly expressed genes, highly expressed genes of this organism possess significantly higher proportion of pyrimidine-ending codons suggesting that besides, mutational bias, translational selection also influenced codon usage variation in T. elongatus. Correspondence analysis of relative synonymous codon usage (RSCU) with A, T, G, C at third positions (A3s, T3s, G3s, C3s, respectively) also supported this fact and expression levels of genes and gene length also influenced codon usage. A role of translational accuracy was identified in dictating the codon usage variation of this genome. Results indicated that although mutational bias is the major factor in shaping codon usage in T. elongatus, factors like translational selection, translational accuracy and gene expression level also influenced codon usage variation.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiae

Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified. These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate. Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media. However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed. Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p. Moreover the available nitrogen source regulates the expression of SER genes. However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2. Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose. Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.     

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.     

Translation initiation: a regulatory role for poly(A) tracts in front of the AUG codon in Saccharomyces cerevisiae

The 5'-UTR serves as the loading dock for ribosomes during translation initiation and is the key site for translation regulation. Many genes in the yeast Saccharomyces cerevisiae contain poly(A) tracts in their 5'-UTRs. We studied these pre-AUG poly(A) tracts in a set of 3274 recently identified 5'-UTRs in the yeast to characterize their effect on in vivo protein abundance, ribosomal density, and protein synthesis rate in the yeast. The protein abundance and the protein synthesis rate increase with the length of the poly(A), but exhibit a dramatic decrease when the poly(A) length is ≥12. The ribosomal density also reaches the lowest level when the poly(A) length is ≥12. This supports the hypothesis that a pre-AUG poly(A) tract can bind to translation initiation factors to enhance translation initiation, but a long (≥12) pre-AUG poly(A) tract will bind to Pab1p, whose binding size is 12 consecutive A residues in yeast, resulting in repression of translation. The hypothesis explains why a long pre-AUG poly(A) leads to more efficient translation initiation than a short one when PABP is absent, and why pre-AUG poly(A) is short in the early genes but long in the late genes of vaccinia virus.     

Evolution of codon usage patterns: the extent and nature of divergence between Candida albicans and Saccharomyces cerevisiae.

Codon usage in a sample of 28 genes from the pathogenic yeast Candida albicans has been analysed using multivariate statistical analysis. A major trend among genes, correlated with gene expression level, was identified. We have focussed on the extent and nature of divergence between C.albicans and the closely related yeast Saccharomyces cerevisiae. It was recently suggested that significant differences exist between the subsets of preferred codons in these two species [Brown et al. (1991) Nucleic Acids Res. 19, 4293]. Overall, the genes of C.albicans are more A + T-rich, reflecting the lower genomic G + C content of that species, and presumably resulting from a different pattern of mutational bias. However, in both species highly expressed genes preferentially use the same subset of 'optimal' codons. A suggestion that the low frequency of NCG codons in both yeast species results from selection against the presence of codons that are potentially highly mutable is discounted. Codon usage in C.albicans, as in other unicellular species, can be interpreted as the result of a balance between the processes of mutational bias and translational selection. Codon usage in two related Candida species, C.maltosa and C.tropicalis, is briefly discussed.     

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.