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Ackerley, David F.

Brown, Alistair S.

Calcott, Mark J.

Francis, Jenni J.B.

Lee-Harwood, Hannah R.

Linton-de Boer, Alexandra J.

McGuinniety, Edward M.

Messenger, Sarah R.

Mumm, Jeff S.

Rich, Michelle H.

Sharrock, Abigail V.

Skurr, Thomas W., V

English

Engineering Conferences International

ACCESSING BACTERIAL DARK MATTER FOR IMPROVED ENZYME DISCOVERY AND ENGINEERING  David F. Ackerley, Victoria University of Wellington david.ackerley@vuw.ac.nz Michelle H. Rich, Victoria University of Wellington Hannah R. Lee-Harwood, Victoria University of Wellington Sarah R. Messenger, Victoria University of Wellington Jenni J.B. Francis, Victoria University of Wellington Thomas W. Skurr, Victoria University of Wellington Edward M. McGuinniety, Victoria University of Wellington Alexandra J. Linton-de Boer, Victoria University of Wellington Jeff S. Mumm, Johns Hopkins University Abigail V. Sharrock, Victoria University of Wellington Alistair S. Brown, Victoria University of Wellington Mark J. Calcott, Victoria University of Wellington   Key Words: New technologies for enzyme engineering, Enzyme discovery, Antibiotic resistance, Nitroreductases, Non-ribosomal peptide synthetases.  Functional screening of environmental DNA libraries is a powerful strategy to recover enzymatic ‘unknown unknowns’ from the dark web of bacteria that occupy complex environments such as soil. However, it can also be heavily biased toward the tiny subset of genes that are preferentially transcribed and translated by the screening strain. To overcome this bias, we have developed generally applicable methods to precision clone captured genes downstream of a strong host promoter and ribosome-binding sequence. Our approach greatly improves screening for weak phenotypes and also permits direct recovery of the corresponding enzymes, providing rapid access to a broad diversity of previously unexplored biocatalysts. We have also shown that environmental DNA can be a rich source of enzymatic domains or sub-domains to boost the throughput of non-rational engineering strategies.   

Accessing bacterial dark matter for improved enzyme discovery and engineering

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1030&context=enzyme_xxvii&type=additional

Accessing bacterial dark matter for improved enzyme discovery and engineering

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Engineering Conferences International