Construction of a MoClo-compatible vector for efficient protein production and secretion in Bacillus subtilis

Heterologous protein production requires a suitable vector system to ensure adequate expression rates, correct protein folding and, where desired, efficient secretion. With a Modular Cloning (MoClo) approach, the construction of standardised parts allows the creation of complex genetic constructs using type II restriction endonucleases. These enzymes cut outside of their recognition sequence, allowing the design of individual four base pair overhangs to assemble multiple parts in a defined order within a one-pot reaction. MoClo can then be used to build genetic circuits for the production of complex proteins, such as spider silk proteins, spidroins. These proteins exhibit incredible chemo-mechanical properties, but researchers still face problems with low production rates due to their high molecular weight and repetitive nature. Here we show the construction of three MoClo-compatible E. coli - B. subtilis shuttle vectors, based on the parent vector pBSMuL1. This vector was constructed for the overproduction and secretion of heterologous proteins in B. subtilis [1]. Our MoClo-compatible vector system allows the exchange of the coding sequence or even the replacement of the entire transcription unit. This makes an individual change of the coding sequence and regulatory elements possible, allowing easy exchange of constitutive and inducible promoters, different ribosomal binding sites and transcription terminators. It is also possible to change signal sequences upstream of the coding sequence for protein secretion and add purification tags downstream for protein purification. In each construct, a constitutively expressed mRFP1 cassette, which leads to red colouration of E. coli colonies, provides a visible and economical selection for successful insertion during cloning. The functionality of the MoClo-compatible pBSMuL1 shuttle vector will be tested for the production of synthetic spider silk proteins in the Gram-positive host B. subtilis. Spidroins are known as high-performance materials because they combine strength, toughness and elasticity. This makes them an environmentally friendly component for building materials and, in the form of long fibres, ideal for use in the textile industry. [1] Brockmeier, U., Wendorff, M., & Eggert, T. (2006). Versatile expression and secretion vectors for Bacillus subtilis. Current microbiology, 52(2), 143–148. doi.org/10.1007/s00284-005-0231-7