Reference - Detail
| RRC ID | 60583 |
|---|---|
| Author | Zhang JJ, Yamanaka K, Tang X, Moore BS. |
| Title | Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination. |
| Journal | Methods Enzymol |
| Abstract |
Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways. |
| Volume | 621 |
| Pages | 87-110 |
| Published | 2019-1-1 |
| DOI | 10.1016/bs.mie.2019.02.026 |
| PII | S0076-6879(19)30045-X |
| PMID | 31128791 |
| PMC | PMC6555405 |
| MeSH | Bacteria / genetics* Bacteria / metabolism Biological Products / metabolism Biosynthetic Pathways Cloning, Molecular / methods* Firmicutes / genetics Firmicutes / metabolism Genetic Engineering / methods Micromonosporaceae / genetics Micromonosporaceae / metabolism Multigene Family* Proteobacteria / genetics Proteobacteria / metabolism Recombination, Genetic* Saccharomyces cerevisiae / genetics* Saccharomyces cerevisiae / metabolism Streptomyces / genetics Streptomyces / metabolism |
| IF | 1.862 |
| Times Cited | 7 |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | X(Twitter) |
| Total number of mentions | 6 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| General Microbes | JCM20779 |