Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.

BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. RESULTS: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1 h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). CONCLUSIONS: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.

Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production.

Comprehension of viscous morphology--evaluation of fractal and conventional parameters for rheological characterization of Aspergillus niger culture broth.

The filamentous fungus Aspergillus niger is a widely used host in industrial processes from food, chemical to pharmaceutical industry. The most prominent feature of this filamentous microorganism in submerged cultivation is its complex morphology which comprises dense spherical pellets as well as viscous elongated filaments. Depending on culture conditions, the exhibited morphology has tremendous effect on the overall process, making a precise understanding of fungal growth and morphology indispensable. Morphology, however, is only industrially relevant as long as it can be linked to important cultivation characteristics of filamentous microorganisms such as culture broth flow behavior. In the present study, different conventional and fractal morphological parameters gained from automatic image analysis were tested for their eligibility to predict culture broth rheology from morphologic appearance. The introduced biomass independent rheological parameters K(BDW) and n(BDW) obtained by power law relationship were successfully estimated from morphology related fractal and conventional parameters. For improved characterization of morphologic appearance of filamentous fungi newly introduced fractal quotient and lacunarity were compared to conventional particle shape parameters in form of the earlier established Morphology number (MN).

Customization of Aspergillus niger morphology through addition of talc micro particles.

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology. It ranges from dense spherical pellets to viscous mycelia. Various process parameters and ingredients are known to influence fungal morphology. Since optimal productivity correlates strongly with a specific morphological form, the fungal morphology often represents the bottleneck of productivity in industrial production. A straight forward and elegant approach to precisely control morphological shape is the addition of inorganic insoluble micro particles (like hydrous magnesium silicate, aluminum oxide or titanium silicate oxide) to the culture medium contributing to increased enzyme production. Since there is an obvious correlation between micro particle dependent morphology and enzyme production it is desirable to mathematically link productivity and morphological appearance. Therefore a quantitative precise and holistic morphological description is targeted. Thus, we present a method to generate and characterize micro particle dependent morphological structures and to correlate fungal morphology with productivity which possibly contributes to a better understanding of the morphogenesis of filamentous microorganisms. The recombinant strain A. niger SKAn1015 is cultivated for 72 h in a 3 L stirred tank bioreactor. By addition of talc micro particles in concentrations of 1 g/L, 3 g/L and 10 g/L prior to inoculation a variety of morphological structures is reproducibly generated. Sterile samples are taken after 24, 48 and 72 hours for determination of growth progress and activity of the produced enzyme. The formed product is the high-value enzyme ß-fructofuranosidase, an important biocatalyst for neo-sugar formation in food or pharmaceutical industry, which catalyzes among others the reaction of sucrose to glucose. Therefore, the quantification of glucose after adding sucrose implies the amount of produced ß-fructofuranosidase. Glucose quantification is made by a GOD/POD-Assay, which is modified for high-throughput analysis in 96-well micro titer plates. Fungal morphology after 72 hours is examined by microscope and characterized by digital image analysis. In doing so, particle shape factors for fungal macro morphology like Feret's diameter, projected area, perimeter, circularity, aspect ratio, roundness und solidity are calculated with the open source image processing program ImageJ. Relevant parameters are combined to a dimensionless Morphology number (Mn), which enables a comprehensive characterization of fungal morphology. The close correlation of the Morphology number and productivity are highlighted by mathematical regression.