Selection of transcriptional parts as starting point for efficient expression of a synthetic spider silk protein construct in Bacillus subtilis

Spider silk exhibits outstanding mechanical properties often compared to steel and Kevlar. These properties make spider silk proteins, also known as spidroins, highly attractive for developing both medical and technical materials. The exceptional mechanical properties originate from their long length and the modular and repetitive protein structure. However, producing these proteins through heterologous expression yielded varying degrees of success. One of the challenges encountered is the construction of expression vectors, because of the size and repetitive nature of the coding sequence. To facilitate assembly of expression constructs with repetitive DNA sequences, a special cloning strategy is important. Here we show the adaptation of the Modular Cloning (MoClo) system and choice of suitable transcriptional parts to build an expression system for efficient production of a synthetic spidroin construct in Bacillus subtilis. Using a reportergene encoding superfolder GFP, we evaluated the effects of different promotors, and ribosomal binding sites on transcription and translation efficiency in B. subtilis. The results from testing individual and paired parts allows the selection of best-suited transcriptional elements as an effective starting point for optimizing recombinant spidroin production in B. subtilis. Future work will focus on assessing and comparing the heterologous production of the synthetic spidroin construct in B. subtilis, exploring both single- and multi-copy integration strategies. Notably, B. subtilis is an ideal host to produce synthetic spidroins on an industrial scale as it possesses an excellent protein secretion machinery, is highly amenable to genetic manipulation and can be easily cultivated on inexpensive substrates.