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.     

Inducible Secretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis

A host-vector system for inducible secretion during the logarithmic growth phase in Bacillus subtilis has been developed. The B. subtilis levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of Clostridium thermocellum was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of B. subtilis showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing B. subtilis sacU(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.     

Enhanced production of antibody fragment via SRP pathway engineering in Escherichia coli

In Escherichi coli, Sec-dependent pathway is the major pathway for protein secretion into periplasm, and it has been widely used for the production of antibody fragment. However, in many cases, the production yields of antibody fragments were not satisfactory due to inefficient secretion and low solubility. Here, we have developed the host-vector system for the secretory production of single chain Fv (scFv) via signal recognition particle (SRP)-dependent pathway instead of Sec-dependent pathway. Use of DsbA signal peptide for SRP-dependent pathway allowed more efficient production of scFv compared with Secdependent pathway. To further improve the production yield and solubility of scFv via SRP pathway, the effect of several factors which are closely related to SRP pathway were examined. Among those factors, the co-expression of YidC could significantly improve the solubility of scFv with high expression level. For the large-scale production, fed-batch cultivations with engineered host-vector system were performed and, two different nutrient feeding solutions (complex vs. defined) were examined. When defined feeding solution was supplied, higher production yield (90 mg/L of scFv) could be obtained than complex feeding solution.     

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.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory-scale fermentations

Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28 degrees C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37 degrees C, and low dissolved oxygen tension, i.e., <5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein. (c) 1993 John Wiley & Sons, Inc.     

Modeling Relapsing Disease Dynamics in a Host-Vector Community

Vector-borne diseases represent a threat to human and wildlife populations and mathematical models provide a means to understand and control epidemics involved in complex host-vector systems. The disease model studied here is a host-vector system with a relapsing class of host individuals, used to investigate tick-borne relapsing fever (TBRF). Equilibrium analysis is performed for models with increasing numbers of relapses and multiple hosts and the disease reproduction number, R₀, is generalized to establish relationships with parameters that would result in the elimination of the disease. We show that host relapses in a single competent host-vector system is needed to maintain an endemic state. We show that the addition of an incompetent second host with no relapses increases the number of relapses needed for maintaining the pathogen in the first competent host system. Further, coupling of the system with hosts of differing competencies will always reduce R₀, making it more difficult for the system to reach an endemic state.     

thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA_St and thyA_Lb, were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA_St or thyA_Lb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA_Lb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

Large-scale production of foreign proteins via the novel plant transient expression system in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factor-single chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agro-mediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

Analysis of the Functional Interaction of Arabidopsis Starch Synthase and Branching Enzyme Isoforms Reveals that the Cooperative Action of SSI and BEs Results in Glucans with Polymodal Chain Length Distribution Similar to Amylopectin

Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.     

Glass bead cultivation of fungi: Combining the best of liquid and agar media

Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors.     

Strategies for improving plasmid stability in genetically modified bacteria in bioreactors.

Exploitation of recombinant organisms for the large-scale, commercial production of foreign proteins is often hampered by the problem of plasmid instability. A wide range of strategies have been reported for improving the stability of recombinant organisms. A combination of manipulating both the genetic design of recombinants and the conditions of culturing the organisms may be used to achieve stable host-vector associations during culture of recombinant organisms in bioreactors.     

Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.     

Inducible expression of Norwalk virus capsid protein gene in plant cell suspension cultures

Plant cell suspension cultures can be used to make safe vaccines at a lower cost than conventional procedures. An inducible gene expression system provides an opportunity to optimize the conditions of vaccine production in a plant system. In this investigation, a dexamethasone-inducible Norwalk virus capsid protein (NVCP) gene expression system has been developed in cell suspension cultures for four different plant species: tobacco (Nicotiana tabacum), rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.) via Agrobacterium-mediated transformation. Resulting transgenic cell lines were confirmed by Southern blot analyses and NVCP gene expression was confirmed by Northern blot analysis. NVCP gene expression was observed in all 24 cell lines tested, but there were minor differences in transgene expression among the transgenic cell lines. The highest level of NVCP gene expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone (10 mg/l), for all transgenic cell lines derived from four different plant species. This investigation demonstrated that expression of NVCP in different transgenic cell lines and in different species was tightly controlled by the inducer, and the inducible gene expression system could be useful in controlling expression of NVCP or similar proteins for production of vaccines in cultured plant cells.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.