Plasmid stability and kinetics of continuous production of glucoamylase by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in an airlift bioreactor

Production of glucoamylase by recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) in a continuous stirred tank bioreactor was studied at different dilution rates. Plasmid stability was found to be growth (dilution rate) dependent; it increased with the dilution rate. Bioreactor productivity and specific productivity also increased with the dilution rate. A kinetic equation was used to model the plasmid stability kinetics. The growth rate ratio between plasmid-carrying and plasmid-free cells decreased from 1.397 to 1.215, and segregational instability or probability of plasmid loss from each cell division decreased from 0.059 to 0.020 as the dilution rate increased from 0.10 to 0.37 1/h. The specific growth rates increased with dilution rate, while the growth rate difference between plasmid-carrying and plasmid-free cell populations was negligible. This was attributed to the low copy number of the hybrid plasmid pGAC9. Thus, the growth rate had no significant effect on plasmid instability. The proposed kinetics was consistent with experimental results, and the model simulated the experimental data well.     

Transference of a crystal protein gene from Bacillus thuringiensis and its expression in Bradyrhizobium sp. cells

The plasmid pHT409 that harbours the cryIA(a) gene for the production of a -endotoxin (crystal protein) from Bacillus thuringiensis was transferred into Bradyrhizobium sp. A conjugal transfer system aiming to introduce the plasmid into the Bradyrhizobium sp. host from colonies of an Escherichia coli donor strain (DH5::pHT409) has been developed. As a result exconjugants were obtained in which the transferred plasmid has been detected by both microbiological and electrophoresis techniques. The cryIA(a) gene when inside the new host had a low expression level which was detected by immunoblotting.     

Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae

The plasmid stability of a recombinant Saccharomyces cerevisiae strain, which expresses cloned -amylase, was increased when glucose or yeast extract was fed with multi-pulse mode in fed-batch culture. Using a novel strategy combining constant rate fed-batch culture and multi-pulse feeding of yeast extract, the plasmid stability was maintained over 90%, meanwhile, 36 g cells l^–1 and 208 units of cloned -amylase activity ml^–1 were obtained.     

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA

Summary The designation of the yeast 2 circle as a selfish DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%–3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Enhanced plasmid stability through post-segregational killing of plasmid-free cells

Summary In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, -galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10^–11 with full promoter induction under non-selective conditions.     

Silicon carbide fiber-mediated stable transformation of plant cells

Summary Maize (Zea mays, cv Black Mexican Sweet) (BMS) and tobacco (Nicotiana tabacum, cv Xanthi) tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA. Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding -glucuronidase (GUS). Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTII) and GUS from the respective plasmids. Thirty-four BASTA-resistant BMS colonies and 23 kanamycin-resistant tobacco colonies recovered following selection contained intact copies of the BAR gene and NPTII genes, respectively, as determined by Southern blot analysis. Sixty-five percent of the resistant BMS colonies and 50% of the resistant tobacco colonies also expressed GUS activity. Intact copies of the GUS gene were observed in Southern blots of all resistant BMS and tobacco colonies that expressed GUS activity. These results indicate that a simple, inexpensive DNA delivery procedure employing silicon carbide fibers can be used to reproducibly transform cells of both monocotyledonous and dicotyledonous plant species.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitableCooperative investigation of the Minnesota Agriculture Experiment Station and the US Department of Agriculture, Agricultural Research Service. Supported in part by grants from The Quaker Oats Company, and Midwest Plant Biotechnology Consortium, USDA Subgrant # 593-0009-04. Minnesota Agricultural Experiment Station Publication No. 19,226.     

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture

A recombinant strain of Saccharomyces cerevisiae, containing a 2-m-fragment-based plasmid (pYEa4) was grown under non-selective conditions in continuous culture. The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions. The difference in growth rate (µ) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique. Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures. Plasmid loss was µ-dominated in cultures grown on defined media whereas µ and R were co-dominant in cultures grown on complex medium. Loss rates increased with increasing dilution rate in complex glucose-limited cultures. The reverse was found in defined glucose-limited cultures. Plasmid retention and loss kinetics determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures. Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased µ indicated an alternative physiological effect of phosphate limitation on plasmid stability.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

Biodegradation of 2-Chlorobenzoate by Recombinant Burkholderia Cepacia Expressing Vitreoscilla Hemoglobin Under Variable Levels of Oxygen Availability

The influence of bacterial hemoglobin, VHb, on dechlorinationand degradation of 2-chlorobenzoate (2-CBA) by recombinantBurkholderia sp. under variable oxygen availability with an initial dissolved oxygenconcentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Abilityto express VHb was provided to recombinant Burkholderia by transformationwith the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded incultures with 85% and 70% of total volume as headspace air in closed reactorsby both wild type and recombinant Burkholderia. The recombinant cultures were able todechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 °Ccompared to more than 120 hours for wild type cultures. The rate and extent of CBAdegradation by recombinant cultures with 40% of total volume as headspace air was higher than thoseachieved by wild type cells at the end of the 168 hours of incubation period, 98and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40%of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinantstrains, whereas it was non-stoichiometric (molar ratio = 0.24)for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorinationand degradation pathway for recombinant cells under hypoxic conditions.The degradation and dechlorination ability of both types of cells was alsoinvestigated in continuous reactor studies by varying the dilution rate under hypoxicconditions. Regarding potential of the recombinant strain for 2-CBA degradation in eitheropen ecosystems or closed bioreactor bioremediation systems, the stability of the plasmidcontaining vgb in the recombinant cells was also studied; the plasmid was100% stable at 0.025 h^-1 dilution rate (1.7 d hydraulic retention time),even after one month.     

Differentiation betweenPhaeocystis pouchetii (Har.) Lagerheim andPhaeocystis globosa Scherffel

An Arctic clone ofPhaeocystis pouchetii LAGERHEIM was compared toPhaeocystis globosa SCHERFFEL isolated from the southern North Sea with regard to temperature tolerance and colony shapes. Already youngP.pouchetii colonies (<100 m) show the typical distribution of the cells in groups, separated from each other by wide zones of cell-free mucilage; the maximum colony size is ca 2 mm in diameter.P.pouchetii colonies form clouds with bubble-like vesicles, spherical colony-shapes are seldom found.P.globosa colonies are spherical up to a size of 2 mm; the cells are distributed homogeneously over the periphery of the colonies. A pouchetii-like distribution of cells never occurs either in the spherical young colonies or in the pear-shaped old colonies (size up to 8 mm). A development from the colony shape of the globosa-type to the pouchetii-type or vice versa was never found. Therefore the colony shape has to be considered a constant distinctive character. Single cells ofP.pouchetii andP.globosa cannot be separated from each other by using the light microscope; this also holds for the flagellates and the non-motile cells.P.pouchetii grows well between 0°C and 14°C,P.globosa between 4°C and 22°C, respectively. Because of the distinctive differences in the morphology of the colonies and the differences in temperature tolerances we propose thatPhaeocystis globosa should no longer be considered conspecific withPhaeocystis pouchetii.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

Population heterogeneity of plasmid-bearing and plasmid-freeBacillus subtilis strains under different environmental conditions

The population heterogeneity of recombinant and plasmid-freeBacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strainB. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strainB. subtilis 2335/105 (Km^rInf⁺) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates ofB. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0. I× M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 × M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

A dominant nuclear streptomycin resistance marker for plant cell transformation

Summary Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5 end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3 end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.     

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells

Summary A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.Correspondence to: T. Schweder