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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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-017-0625-9) contains supplementary material, which is available to authorized users.

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