Stable multicopy integration of vector sequences inHansenula polymorpha

Plasmids without an origin of replication, but bearing theURA3 gene ofSaccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously inHansenula polymorpha, indicating that parts of theS. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function inH. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued inE. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into theH. polymorpha genome. The integration event usually occurred in high copy number (approx. 30–50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. It contrast, the use of homologousURA3 gene under similar conditions led to low-copy-number transformants.     

Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to the edible straw mushroom, Volvariella volvacea. Mycelium pellets were transformed to cold stress resistance using the afp gene as both a selective marker and a reporter gene, under the control of a heterologous Lentinula edodes gpd promoter. The efficiency of transformation is over 100 times higher than that previously reported in V. volvacea. Stable integration of the afp gene with 1-4 copy numbers was confirmed in all 10 randomly selected transgenic events by Southern blot analysis. The mitotic stability of the transformants was demonstrated after five successive transfers on PDA medium without selection pressure and the PCR analysis of basidiospores harvested from transformants.     

Bioluminescent monitoring enables observation of intracellular events in real time without cell and tissue destruction

Secreted reporter proteins provide monitoring of intracellular events in real time without cell destruction. To create human melanoma cell lines that enables noninvasive bioluminescent monitoring of metabolic activity, a comparison of the efficiency of isoforms and mutant variants of luciferase from the Metridia longa as secreted reporter proteins in the cells of human melanoma lines Mel IL was conducted. The MLM3 deletion mutant had the highest activity in the medium of two studied isoforms and two deletion mutants of secreted M. longa luciferase during the Mel IL melanoma cell transfection. It was established that optimization of the gene structure of the selected MLM3 variant for expression in human cells increases the level of bioluminescent activity in the Mel IL cells by almost an order of magnitude. A stable Mel IL melanoma cell line with constitutive expression of the humanized hMLM3 reporter gene was obtained and characterized. The linear range of identification of living cells by the hMLM3 reporter activity was more than three orders of magnitude with a sensitivity of detection of 10 cells.

     

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.     

Stability of the pBR322 plasmid derivative pBB210 in Escherichia coli TG1 under non-selective and selective conditions

The stability behaviour of the pBR322 plasmid derivative pBB210 with β-lactamase gene and human interferon-α1 gene in Escherichia coli TG1 was studied in chemostat cultures under non-selective (medium without antibiotics), selective (medium with β-lactam antibiotic ampicillin) and modified selective (medium with ampicillin and the β-lactamase inhibitor sulbactam) conditions. Under non-selective conditions, a behaviour typical of unstable systems was found. Under selective conditions, the behaviour predicted by the models was obtained – the fraction of plasmid-bearing cells in the population approached a constant value which was dependent on the ampicillin concentration in the feeding and on the cell concentration in the chemostat. Under modified selective conditions, the higher the concentration of sulbactam in the medium was, the higher the fraction of plasmid-bearing cells was in steady state conditions.     

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.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid. Received: 27 March 1997 / Received revision: 13 August 1997 / Accepted: 25 August 1997     

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-1β, 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.     

Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured. (c) 1993 Wiley & Sons, Inc.     

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     

Improved production and stability ofE. coli recombinants expressing transketolase for large scale biotransformation

Summary TheE.coli tkt gene has been subcloned into high copy number vectors. In fed batch fermentations up to 4gL^–1 of soluble intracellular transketolase was produced representing 43% of the total cell protein. Increased plasmid stability during fed-batch fermentations was obtained by using kanamycin resistant pBGS vectors rather than the ampicillin resistant pUC vectors. Plasmid stability was maintained throughout growth in a complex medium without any selective pressure by incorporating thecer region fromColE1 into the expression construct.     

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.     

Plasmid maintenance and growth of recombinantSaccharomyces cerevisiae producing hepatitis B virus surface antigen

Summary The effects of host strains (Saccharomyces cerevisiae) and medium composition on the plasmid stability and expression level of hepatitis B virus surface antigen were investigated. Specific growth rates of cells carrying a plasmid, pMHBS, was found to be slower than those without the plasmid. It was also found that the plasmid was maintained in a more stable manner in the selective medium. The nonselective complex medium, however, was greatly favored for the growth of recombinant hosts as well as the HBsAg production.     

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.     

Maximizing the expression of recombinant proteins in Escheria coli by manipulation of culture conditions

The expression of hybrid proteins β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain A (constitutive), and β-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.     

Host-plasmid interactions in Saccharomyces cerevisiae: effect of host ploidy on plasmid stability and copy number

The segregational stability of two chimaeric plasmids has been examined in an isogenic series of haploid, diploid and tetraploid strains of Saccharomyces cerevisiae, constructed by transformation-associated spheroplast fusion. For the highly unstable, ARS-based plasmid YRp7M, a significant increase in its segregational stability was observed with increasing ploidy, while the relatively stable, 2 microns-based plasmid pMA3a showed only a small increase in stability in strains of higher ploidy. The copy number of both pMA3a and the endogenous 2 microns plasmid increased in proportion with the host cell ploidy, while the copy number of TRp7M was increased in the higher ploidy strains but did not correlate with ploidy. These results suggest that the copy numbers of both the 2 microns plasmid and a plasmid derived from it are controlled by a nuclear gene and that, in addition, there are 2 microns sequences, other than those required for the FLP-mediated recombination system, that play a role in maintaining copy number.