Background

For decades, non-standard amino acids (nsAAs) have been used to introduce chemistries that are not ordinarily found in biological systems through genetically encoded site-specific incorporation within target proteins in live cells. When incorporated within proteins, nsAAs have been used for bioconjugation (Chin et al., 2002b; Seitchik et al., 2012), biocontainment (Mandell et al., 2015; Rovner et al., 2015), photo-crosslinking (Chin et al., 2002a, 2002b), fluorescence (Wang et al., 2006), and biomaterial production (Israeli et al., 2020). Akin to the ribosomally mediated process of translation for standard amino acids, nsAAs require a unique aminoacyl-tRNA synthetase and tRNA pair that exhibits minimal cross talk with other cellular components for codon recognition and addition of the nsAA to the growing polypeptide chain. The systems used to enable this for nsAAs are referred to as orthogonal translation systems (OTS), and they usually contain an aminoacyl-tRNA synthetase (AARS) and tRNA from an evolutionarily distant microbial species, such as the archaea Methanocaldococcus jannaschii or Methanomethylophilus alvus (Dumas et al., 2015; Beranek et al., 2019). Most commonly, the OTS is engineered to have a CUA anticodon for incorporation of the nsAA at the amber stop codon, UAG. This technology has been developed primarily in Escherichia coli and mammalian cell lines, thus limiting its use to a handful of organisms (Dumas et al., 2015). Extensive characterization of nsAAs technology in these species has led to discoveries of specific enzyme mechanisms, protein–protein interactions, and protein structures (Xie et al., 2004; Ai et al., 2011; Zhao et al., 2020).

Bacillus subtilis is a prime target for expansion of nsAA incorporation technology due to its broad utility as a Gram-positive rhizobacterium model in applied and fundamental research. B. subtilis has been used to study a variety of biological phenomena, including asymmetric cell division, biofilm formation, and sporulation (Losick et al., 1986; Kearns et al., 2005; McKenney et al., 2013; Bisson-Filho et al., 2017), which could be further investigated through incorporation of functional nsAAs such as those for post-translational modifications or photo-crosslinking. Having been conferred the status Generally Regarded As Safe (GRAS) by the US FDA, B. subtilis has also been used as a probiotic and vaccine vector for plants, animals, and humans (Cutting, 2011; Oh et al., 2020). Such applications could benefit from the site-specific incorporation of nsAAs within proteins for augmented capabilities or for the implementation of safeguards such as synthetic auxotrophy. B. subtilis is commercially used to produce antibiotics, cosmetic small molecules, and proteins (Westers et al., 2004; Stein, 2005; Su et al., 2020; Park et al., 2021). Given the variety of potential applications for B. subtilis in fundamental and applied science, we must continue to advance the tools available to scientists working with this organism.

Recent work from our groups demonstrated the potential application of site-specific nsAA incorporation within proteins in B. subtilis, including validation of theoretical protein–protein interactions, and tuning of cell wall biosynthesis with nsAA titration (Stork et al., 2021). Our study also provided preliminary evidence of the portability of this technology from E. coli to B. subtilis. Thus, the method presented here describes the application of these technologies, but it also establishes a fundamental base upon which the community can continue to build new technologies.

Existing technology for nsAAs incorporation in Bacilli species has been limited to a specific, single nsAA for a specific purpose (Scheidler et al., 2020; Tian et al., 2020). Our method presents the first demonstration of incorporation of a broad spectrum of nsAAs within proteins in B. subtilis for a variety of functions, including bioconjugation, photo-crosslinking, and fine-tuned control of protein expression. We demonstrated the incorporation of 20 unique nsAAs with six different orthogonal translation systems. In particular, we were able to incorporate 13 unique nsAAs with a single OTS system providing a platform strain for a diverse range of applications. This method also demonstrates the best overall nsAA incorporation with an upwards of 60% of native protein produced, thus far the best reported in B. subtilis. Some existing limitations to this system include background incorporation of the nsAA at natural stop codon sites and apparent limitations of the incorporation level in rich media. Despite those, this method provides an opportunity to introduce new tools to chemical biology and B. subtilis communities.