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Article Abstract
Here, we developed a direct cloning method that aims to capture superlarge biosynthetic gene clusters (BGCs) (e.g., >150 kb), namely, ACQUIRE (dvanced as12a nuclease assisted precise and ck digestion of in-gel prepared genomic DNA in combination with the transformation-associated combination in yeast). Following the ACQUIRE method, we successfully cloned three BGCs with the size from 94 kb to 180 kb, including the rifamycin BGC (, ∼94 kb, GC 73%) with an efficiency of 12.8% from the actinomycete U32 and one superlarge polyketide synthase BGC (∼180 kb, GC 73%, with efficiency of ∼1.6%) from Y0289. Subsequently, we introduced the BGC into U32 and generated high rifamycin producers. Therefore, the ACQUIRE method developed here greatly complements the current direct cloning toolbox and has an advantage to capture superlarge BGCs that can be harnessed for enhancement of antibiotic production or for genome mining of new natural products.
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| http://dx.doi.org/10.1021/acssynbio.5c00281 | DOI Listing |
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