• Journal of Life Science
• /
• v.12 no.2
• /
• pp.53-56
• /
• 2002

The baculovirus expression vector system (BEVS) is one of the powerful heterologous protein expression systems using insect cells. As a result this has become a hot issue in the fleld of biotechnology. The advantage of the BEVS is that the large-scale production of heterologous proteins, which undergo posttranslational modification in the endoplasmic reticulum (ER), can be accomplished. Altrough posttranslational modification of heterologous proteins in insect cells is more similar to mammalian cells than yeast, it is not always identical. Therefore, aggregation and degradation can sometimes occur in the ER. To produce a high level of bioactive heterologous proteins using BEVS in insect cells, the prerequisite is to completely understand the posttranslational conditions that determine how newly synthesized polypeptides are folded and assembling with ER chaperones in the ER lumen. Here, we provide information on current BEVS problems and the possibility of successful heterologous protein production from mammalian cells.

• KSBB Journal
• /
• v.16 no.1
• /
• pp.15-23
• /
• 2001

The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.12
• /
• pp.1931-1937
• /
• 2019

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.

• Asian-Australasian Journal of Animal Sciences
• /
• v.13 no.7
• /
• pp.894-896
• /
• 2000

In vitro fertilization can be used for salvaging superior buffalo germplasm which otherwise goes waste after the slaughter of animals. This technology has also increased our basic understanding of growth of germ cells and embryos. The requirement of growing embryos is peculiar and stage specific. In the present study the cleavage and development of buffalo embryos were studied with homologous (buffalo) and heterologous (goat) oviductal cell co-culture systems. The cleavage rate improved significantly (p<0.01) in both homologous and heterologous co-culture as compared to control (55.3, 46.8 and 11.4%). The morula formation using homologous and heterologous oviductal cells also increased significantly as compared to control group (43.6, 21.9 & 1.9%). There was no blastula formation in control group, but addition of oviductal cells either from homologous or heterologous species significantly increased the blastula formation (9.5, 12.5%). The cleavage rate and embryo development was slightly better (non significant) in homologous as compared to heterologous oviductal cell culture. It was concluded that the use of oviductal cell co-culture (homologous and heterologous species) have significantly improved cleavage and development of buffalo embryos in vitro.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.11
• /
• pp.1465-1480
• /
• 2021

Violacein, a purple pigment first isolated from a gram-negative coccobacillus Chromobacterium violaceum, has gained extensive research interest in recent years due to its huge potential in the pharmaceutic area and industry. In this review, we summarize the latest research advances concerning this pigment, which include (1) fundamental studies of its biosynthetic pathway, (2) production of violacein by native producers, apart from C. violaceum, (3) metabolic engineering for improved production in heterologous hosts such as Escherichia coli, Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica, (4) biological/pharmaceutical and industrial properties, (5) and applications in synthetic biology. Due to the intrinsic properties of violacein and the intermediates during its biosynthesis, the prospective research has huge potential to move this pigment into real clinical and industrial applications.

• Molecules and Cells
• /
• v.27 no.5
• /
• pp.601-608
• /
• 2009

The 2-deoxystreptamine and paromamine are two key intermediates in kanamycin biosynthesis. In the present study, pSK-2 and pSK-7 recombinant plasmids were constructed with two combinations of genes: kanABK, and kanABKF and kacA respectively from kanamycin producer Streptomyces kanamyceticus ATCC12853. These plasmids were heterologously expressed into Streptomyces lividans TK24 independently and generated two recombinant strains named S. lividans SK-2/SL and S. lividans SK-7/SL, respectively. ESI/ MS and ESI-LC/MS analysis of the metabolite from S. lividans SK-2/SL showed that the compound had a molecular mass of 163 $[M+H]^+$, which corresponds to that of 2-deoxystreptamine. ESI/MS and MS/MS analysis of metabolites from S. lividans SK-7/SL demonstrated the production of paromamine with a molecular mass of $324[M+H]^+$. In this study, we report the production of paromamine in a heterologous host for the first time. This study will evoke to explore complete biosynthetic pathways of kanamycin and related aminoglycoside antibiotics.

• Journal of Microbiology
• /
• v.37 no.2
• /
• pp.102-110
• /
• 1999

Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.1
• /
• pp.135-137
• /
• 2008

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. celluloslim containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.9
• /
• pp.1295-1299
• /
• 2010

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone $3{\beta}$-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

• Molecules and Cells
• /
• v.28 no.4
• /
• pp.369-373
• /
• 2009

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and ${\beta}$-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an ${\alpha}$-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and ${\beta}$-glucosidase was able to produce ethanol from ${\beta}$-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.