Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7.

The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.

CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis.

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.

Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT).

Branched chain aminotransferase (BCAT) is one of the enzymatic tools of choice for the production of chiral amines or amino acids; especially, non-natural amino acids are of interest as building blocks for the pharmaceutical industry. The expression and subsequent secretion of BCAT counteracts limited cell permeability of target substrates and facilitates downstream processing. Since Pichia pastoris secretes a negligible amount of native proteins and was previously shown to efficiently secrete recombinant proteins, it was chosen as the expression host. We examined different promoters and glycosylation states and also engineered the host strain by disrupting genes encoding proteins related to cell wall assembly (Scw10, Cwp1) and glycosylation (Och1). Finally, we were able not only to increase the extracellular BCAT production, but also to achieve a more homogenous product in terms of glycosylation and identified a deletion strain, which counteracts typical cell clustering in the Δoch1 strain.

Meta4: a web application for sharing and annotating metagenomic gene predictions using web services.

Whole-genome shotgun metagenomics experiments produce DNA sequence data from entire ecosystems, and provide a huge amount of novel information. Gene discovery projects require up-to-date information about sequence homology and domain structure for millions of predicted proteins to be presented in a simple, easy-to-use system. There is a lack of simple, open, flexible tools that allow the rapid sharing of metagenomics datasets with collaborators in a format they can easily interrogate. We present Meta4, a flexible and extensible web application that can be used to share and annotate metagenomic gene predictions. Proteins and predicted domains are stored in a simple relational database, with a dynamic front-end which displays the results in an internet browser. Web services are used to provide up-to-date information about the proteins from homology searches against public databases. Information about Meta4 can be found on the project website, code is available on Github, a cloud image is available, and an example implementation can be seen at.

Further investigation on the turnover of Escherichia coli biotin synthase with dethiobiotin and 9-mercaptodethiobiotin as substrates.

Biotin synthase, a member of the "radical-SAM" family, produces biotin by inserting a sulfur atom between C-6 and C-9 of dethiobiotin. Each of the two saturated carbon atoms is activated through homolytic cleavage of a C-H bond by a deoxyadenosyl radical, issued from the monoelectronic reduction of S-adenosylmethionine (SAM or AdoMet). An important unexplained observation is that the enzyme produces only 1 mol of biotin per enzyme monomer. Some possible reasons for this absence of multiple turnovers are considered here, in connection with the postulated mechanisms. There is a general agreement among several groups that the active form of biotin synthase contains one (4Fe-4S)(2+,1+) center, which mediates the electron transfer to AdoMet, and one (2Fe-2S)(2+) center, which is considered the sulfur source [Ugulava, N. B., Sacanell, C. J., and Jarrett, J. T. (2001) Biochemistry 40, 8352-8358; Tse Sum Bui, B., Benda, R., Schunemann, V., Florentin, D., Trautwein, A. X., and Marquet, A. (2003) Biochemistry 42, 8791-8798; Jameson, G. N. L., Cosper, M. M., Hernandez, H. L., Johnson, M. K., and Huynh, B. H. (2004) Biochemistry 43, 2022-2031]. An alternative hypothesis considers that biotin synthase has a pyridoxal phosphate (PLP)-dependent cysteine desulfurase activity, producing a persulfide which could be the sulfur donor. The absence of turnover was explained by the inhibition due to deoxyadenosine, an end product of the reaction [Ollagnier-de Choudens, S., Mulliez, E., and Fontecave, M. (2002) FEBS Lett. 535, 465-468]. In this work, we show that our purified enzyme has no cysteine desulfurase activity and the required sulfide has to be added as Na(2)S. It cannot be replaced by cysteine, and consistently, PLP has no effect. We observed that deoxyadenosine does not inhibit the reaction either. On the other hand, if the (2Fe-2S)(2+) center is the sulfur source, its depletion after reaction could explain the absence of turnover. We found that after addition of fresh cofactors, including Fe(2+) and S(2)(-), either to the assay when one turn is completed or after purification of the reacted enzyme by different techniques, only a small amount of biotin (0.3-0.4 equiv/monomer) is further produced. This proves that an active enzyme cannot be fully reconstituted after one turn. When 9-mercaptodethiobiotin, which already contains the sulfur atom of biotin, is used as the substrate, the same turnover of one is observed, with similar reaction rates. We postulate that the same intermediate involving the (2Fe-2S) cluster is formed from both substrates, with a rate-determining step following the formation of this intermediate.