The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli.

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-alpha2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-alpha2b expression remained poor even when fused to a signal sequence, and an alternative IFN-alpha2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

[Comparative analysis of in vitro antifungal activity and in vivo acute toxicity of the nystatin analogue S44HP produced via genetic engineering].

New polyene macrolide S44HP was purified from the culture of recombinant Streptomyces noursei strain with engineered nystatin polyketide synthase. S44HP, nystatin (NYS), and amphotericin B (Amph-B) were tested against 19 clinical fungal isolates in agar diffusion assay, which demonstrated clear differences in antifungal activities of these antibiotics. Sodium deoxycholate suspensions of all three antibiotics were subjected to acute toxicity studies in vivo upon intravenous administration in mice. NYS exhibited the lowest acute toxicity in mice in these experiments, while both Amph-B and S44HP were shown to be 4 times more toxic as judged from the LD50 values. While the acute toxicity of S44HP was higher than that of Amph-B, the data analysis revealed a significantly increased LD10 to LD50 dose interval for S44HP compared to Amph-B. The data revealed structural features of polyene macrolides, which might have an impact on both the activity and toxicity profiles of these antibiotics. These results represent the first example of preclinical evaluation of an "engineered" polyene macrolide, and can be valuable for rational design of novel antifungal drugs with improved pharmacological properties.

Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli.

In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.

High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4.

Streptomyces lividans 1,326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of a-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes.

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway.

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.

Molecular cloning and analysis of a pleiotropic regulatory gene locus from the nystatin producer Streptomyces noursei ATCC11455.

A regulatory gene locus from Streptomyces noursei ATCC14455, the producer of the antifungal antibiotic nystatin, was cloned in Streptomyces lividans based on its ability to activate actinorhodin (Act) production in this host. Deletion and DNA sequencing analyses showed that a small gene, designated ssmA, located downstream of an afsR homologue (a known pleiotropic regulator) was responsible for the Act overproduction in S. lividans. Database searches for the ssmA gene product revealed its limited similarity to the AfsR2 regulatory protein from S. lividans and CREA catabolite repressor from Aspergillus nidulans. To study the effect of ssmA on nystatin production, this gene was either deleted from S. noursei genome, or placed under control of PermE* promoter and introduced in S. noursei. The properties of the corresponding strains indicate that ssmA is involved in regulation of growth and antibiotic production only in the media with certain carbon sources.