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ACS Synth Biol. 2018 Jul 20;7(7):1702-1708. doi: 10.1021/acssynbio.8b00151. Epub 2018 Jul 3.

Direct Pathway Cloning Combined with Sequence- and Ligation-Independent Cloning for Fast Biosynthetic Gene Cluster Refactoring and Heterologous Expression.

Author information

1
Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM) , Technical University of Munich , Lichtenbergstraße 4 , 85748 Garching bei München , Germany.

Abstract

The need  for new pharmacological lead structures, especially against drug resistances, has led to a surge in natural product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural product pathways have thus been developed. Direct pathway cloning (DiPaC) is an emerging synthetic biology strategy that utilizes long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture. As a proof of principle, the majority of the cyanobacterial hapalosin gene cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression. The complementation of DiPaC with SLIC depicts a time and cost-efficient method for simple capture and expression of new natural product pathways.

KEYWORDS:

direct pathway cloning (DiPaC); hapalosin; heterologous expression; sequence- and ligation-independent cloning (SLIC); synthetic biology

PMID:
29940102
DOI:
10.1021/acssynbio.8b00151

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