Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.     

Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

A novel gene amplification system in yeast based on double rolling-circle replication

Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication. This system produced three types of amplification products. Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.     

A DEX gene conferring production of extracellular amyloglucosidase on yeast

A DEX gene from Saccharomyces diastaticus (strain BRG536 alpha DEX1) has been cloned in the hybrid vector pJDB207. The gene is included within a 3.6-kb fragment and confers production of extracellular amylo-alpha-1,4-glucosidase (AMG) and, thereby the ability to hydrolyse starch or dextrins on Dex- strains of Saccharomyces cerevisiae. The cloned gene hybridizes to three fragments produced by ClaI digestion of DNA from BRG536; one of these (11 kb) cosegregates in crosses with DEX1, while another (10 kb) is present in all Dex+ and Dex- strains examined. Accumulation of extracellular AMG by Dex+ transformants is up to five-fold that of BRG536, and escapes regulation by the CDX1 gene under conditions of excess glucose. The enzyme produced by Dex+ transformants resembles that of BRG536 with respect to Mr (approx. 150 X 10(3)) and effects of temperature and pH. The cloned DEX gene can be used as a selectable marker for introducing recombinant plasmids into wild-type strains of S. cerevisiae.     

Analysis of threonine uptake in Escherichia coli threonine production strains

Threonine uptake in Escherichia coli wild-type and in threonine-producing strains decreased throughout threonine production. In contrast to previously published results, the SstT uptake system is not the sole serine/threonine permease in E. coli, since a novel transport system was detected in an sstT deletion strain.     

Plasmid maintenance and threonine production by recombinantEschcrichia coli strains

Summary E. coli NRRL 12100-a recombinant strain obtained by genetic manipulationwas used forL-threonine production. When cultured in a rich medium without antibiotics three types of colonies were isolated (Ap^sTc^s, Ap^rTc^s, and Ap^rTc^r). The Ap^rTc^r clones were best threonine producers (9 to 12 g/1) and the plasmid was maintained, in 65 to 93% of the host cells. When inocula were grown under selective pressure we obtained about 10 g/1 of threonine and a plasmid maintenance of 83%. When growth of inocula was done without antibiotic, threonine yields dropped to 5 g/1 and 64% of the cells have lost the plasmid. Batch culture experiments were performed with 3, 4 and 6% of glucose, added at the initial stage or in a discontinuous feed. Threonine yields and plasmid stability were not affected. The elimination of the maximum level of threonine produced (from 13.8 to 6.7 g/1) and on the plasmid maintenance (from 94 to 4% of the cells) while growth of the strain was not affected.     

Genome mapping by arbitrary amplification of yeast artificial chromosomes

Several methods have been described for using the polymerase chain reaction (PCR) to isolate fragments of DNA for genome mapping. We have developed an approach for isolating discrete fragments by amplifying DNA with single oligonucleotides (10-mers) with arbitrarity selected sequences. The method is rapid and technically simple. We isolated fragments from a contig of three yeast artificial chromosomes (YACs) from the human Xq28 chromosomal region. We purified YACs yWXD 37, yWXD348, and yWXD705 from a preparative pulsed field gel. Amplifications of each YAC were performed with single 10-mers as the PCR primers and the products were visualized on agarose gels. These fragments have been successfully used as hybridization probes against Southern blots containing the YACs and against blots containing human genomic DNA and somatic cell hybrids containing Xq28 as their only human constituent. The results have been concordant with the known order of the YACs. We have also successfully combined 10-mers with primers derived from vector arm sequences to isolate YAC ends. We discuss several uses of this method in comparative mapping and in filling in gaps in physical and genetic maps.     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

Effect of production method and gene amplification on the glycosylation pattern of a secreted reporter protein in CHO cells

We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

Genetic analyses of yeast protein serine/threonine phosphatases

Abstract Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threoninc phosphatases in yeast.     

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.     

利用酵母菌生产有机酸的研究进展

有机酸是含有一种或多种低分子量酸性基团(如羧基、磺酸基)的可生物合成的有机化合物,广泛应用于食品、农业、医药、生物基材料工业等领域。酵母菌具有生物安全、抗逆性强、底物谱广泛、方便遗传改造,以及大规模培养技术成熟等独特优点,因此利用酵母菌生产有机酸的研究日益受到国内外学者的关注。目前利用酵母生产有机酸还存在浓度低、副产物多,以及发酵效率低等缺陷。随着酵母菌代谢工程和合成生物学技术的发展,利用酵母菌生产有机酸取得了快速进展。本文总结了利用酵母合成11种有机酸的研究,包括内源和异源合成的大宗羧酸和高价值有机酸,并对该领域的未来研究方向进行了展望。     

Enzymatic breakdown of threonine by threonine aldolase

1. The enzyme which splits threonine to acetaldehyde and glycine has been partially purified from rat liver (five- to sixfold purification) and the name threonine aldolase proposed for it. 2. The general properties of threonine aldolase have been studied. The enzyme is unstable to a pH below 5. The pH optimum of the enzyme reaction is at 7.5-7.7. The initial rate of production of acetaldehyde is proportional to the enzyme concentration, and when the enzyme concentration is constant, the production of acetaldehyde is proportional to the time, provided that the substrate is in excess. The enzyme is inhibited by the carbonyl group reagent, hydroxylamine. Attempts to demonstrate that pyridoxal phosphate is a cofactor were unsuccessful. 3. The enzyme splits only L-allothreonine and L-threonine and is inactive against the D-forms of these amino acids. 4. The enzyme reaction on DL-allothreonine follows first order kinetics. From the first order velocity constants and the initial rates of the rates of the reaction at various substrate concentrations the Michaelis constant, Ks, for this substrate has been evaluated. Michaelis constants have also been determined for threonine. 5. The optimum temperature for the enzymatic breakdown of DL-allothreonine at pH 7.65 was found to be 50 degrees C. in phosphate buffer and 48 degrees C. in tris-maleate buffer. The rate of thermal inactivation of the enzyme threonine aldolase obeys a first order reaction. The heat of thermal inactivation was calculated by the aid of the van't Hoff-Arrhenius equation to be 43,000 cal. per mole for the temperature range 41.2-46.6 degrees C. 6. Equivalent amounts of acetaldehyde and glycine were formed from DL-allothreonine and the enzymatic breakdown of DL-allothreonine was found to be irreversible.