Investigating the biosynthetic potential of VHPO-containing Actinobacteria using a Cas12a Direct Cloning approach

Gabriel Lefebre

McKinnie Lab, UCSC

Introduction

Vanadium-dependent haloperoxidases (VHPOs) are a family of enzymes that use a Vanadate prosthetic group and Hydrogen peroxide to catalyze the oxidation of Halide ions (Cl, Br, I). This allows the addition of halogen groups to chemical substrates, often improving the bioactivity and reactivity of the formed products. VHPOs have been found in Macroalgal species, where Vanadium is abundant in marine environments, and Actinobacteria, a family of high-GC, Gram-positive bacteria.

Biosynthetic Pathway of the mcl gene cluster, from the synthesis of the THN backbone to the site-specific chlorination, dearomatization and cyclization of the modified product to yield multiple bioactive products

Mcl24 as an example of site-specific VHPO

Enzymes within the mcl gene cluster of Streptomyces spp. CNH-189, namely Mcl 24 is shown to perform site-specific chlorination of a THN followed by a complex cyclization to generate final products with potent antibiotic activity in the range of 2-4 ug/mL MIC.

While a small number of site-specific VHPOs have been characterized in high-GC Gram-positive bacteria (Actinobacteria) and represent an enticing avenue for enzyme engineering, there is little known about this family of enzymes. One reason is that Actinobacteria are often difficult to cultivate in laboratory settings due to strict environmental requirements. Additionally, the expression of gene clusters may be impacted or silenced in certain bacterial strains. To investigate the biosynthetic potential of these putative VHPO-containing gene clusters, alternative methods must be applied.

Method

Bioinformatic searches of gene clusters through software such as antiSMASH have yielded several VHPO-containing gene clusters with homology to previously characterized enzymes. Once boundaries were properly defined, guide RNA could target the boundary regions of target gene clusters for excision.

Thanks to bioinformatic prediction of protein homology and function, direct cloning or capture of gene clusters offers a novel method for characterizing gene clusters, circumventing the need for random screening or library construction.

Cas12a CAPTURE method. Cas12a along with guide RNA is used to site-specifically excise the gene cluster. The fragment is is hybridized with generated DNA receivers. The DNA fragment is then circularized using a Cre-lox system present on the DNA fragments and is ready for replication and integration into a suitable host

In this particular method of direct cloning, the gene cluster of interest is removed from bacterial genomic DNA using CRISPR Cas12a, a nuclease that leaves 4-5 base pair overhangs at directed cut sites on either end of the gene cluster. The fragment is then "captured" using premade DNA receiver components hybridized with the overhangs generated from the Cas12a cut sites to form a linear fragment. This linear DNA is introduced into a bacteria possessing specialized genes called Cre recombinases that recognize sites on the DNA fragment and generate a circular plasmid containing the gene cluster. Once this has been completed, the biosynthetic gene cluster of interest can be conjugated into a suitable host, where it is integrated into the genome and investigated for metabolomic changes.

Results

For initial capture, a VHPO-containing gene cluster from Actinopolyspora mzabensis (amz) was chosen for its compact, 10-gene size and possession of necessary genes for the biosynthesis of merochlorin-like compounds. Following PCR confirmation of the construction of a suitable plasmid, the gene cluster was conjugated or transferred from a specialized E. coli donor species to our model Actinobacterial strain Streptomyces albidoflavus J1074. The gene cluster was then integrated into the genome of the bacteria and confirmed for presence of the genes through additional PCR screening.

Actinopolyspora mzabensis gene cluser (amz) possessing 10 genes, including THN synthase (black), prenyltransferases (blue), and a VHPO homolog (red)

Evaluation of metabolomics and Follow-up

LCMS, NMR, and HPLC were used to examine the differences among metabolomes of wild-type bacteria and conjugated strains, revealing stark differences in the metabolomes of the respective strains.

Major products were evident in extracts from conjugated strains, differing by growth conditions. Additional work is required to isolate these new compounds as they appear prone to degradation

Overall, the Actinopolyspora mzabensis gene cluster possessing a putative VHPO was successfully captured and conjugated into a host bacterial strain, yielding novel metabolomes and opening the door for further investigation into the identities of these newly produced compounds.

Additional strains have been found to contain VHPO-containing gene clusters and present as appealing next targets for further direct cloning studies.

Acknowledgements

McKinnie Lab

Shaun McKinnie

Austin Hopiavuori

Jackson Baumgartner

Jennifer Cordoza

Radcliff Huffman

Lia Salazar

Mel Jones

Lukas Varga

Anandi Radhakrishnan

Manasi Natu

Meron Mesgun

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