Heterologous expression of gene clusters
Biosynthetic gene clusters (BGCs) of interest can be cloned either by making genome libraries in Supercos1 or phage artificial chromosomes such as pESAC. Companies such as BIO S&T will construct and probe custom made libraries and provide clones of interest. BGCs that have been cloned from their native host can then be expressed heterologously in a different host organism. This can help improve production of known products, or aid the discovery of novel natural products.
Cloned gene clusters can be introduced into a host strain via intergenic conjugation.
Useful host strains:
- Streptomyces albus [1]
This species has a naturally minimised genome and fast growth, which can help improve production of a target gene cluster.
Streptomyces superhosts: [2]
- Streptomyces coelicolor M1146
Δact Δred Δcpk Δcda
- Streptomyces coelicolor M1152
Δact Δred Δcpk Δcda rpoB[C1298T] rpsL[A262G C271T]
These Streptomyces superhosts were constructed from Streptomyces coelicolor M145. Four exogenous secondary metoblite gene clusters were deleted (actinorhodin, prodiginine, CPK and CDA) to remove competition of carbon and nitrogen resources. For M1152, point mutations were introduced into rpoB and rpsL to help increase production of biosynthetic gene clusters.
Production of heterologously expressed gene clusters: Once a gene cluster has been transferred into a heterologous host, production of potential metabolites can be tested. It can be useful to test a range of growth conditions and different media (both solid and liquid) containing different types of carbon and nitrogen sources.
[1] Zaburannyi, N. et al. (2014) ‘Insights into naturally minimised Streptomyces albus J1074 genome’ BMC genomics 15:97
[2] Gomez-Escribano, J. P. and Bibb, M. J. (2011) ‘Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters’ Microbial Biotechnology, 4(2):207-15. doi: 10.1111/j.1751-7915.2010.00219.x.
