Heterologous Expression-Mediated Activation of Silent Gene Clusters

Heterologous expression is a strategy to induce gene expression to produce a new natural product under specific conditions by linking a silenced gene to an efficient promoter and then introducing it into a heterologous host with simple genetic manipulation. Since promoter replacement strategies cannot be used in strains where genetic manipulation systems are difficult to establish, the heterologous expression has become another widely used strategy for targeted activation of silenced gene clusters. Lifeasible can use Saccharomyces cerevisiae and Aspergillus spp. as heterologous hosts to achieve expression of fungal silencing gene clusters and is working to develop more diverse heterologous hosts to accommodate fungal silencing gene expression of specific origin.

Figure 1. Overview of the heterologous expression of natural product BGCs. (Tao W, et al., 2019)

Heterologous expression of gene clusters means cloning the entire silent biosynthetic gene cluster into a plasmid, expressing it using a heterologous host, and then finalizing the isolation, purification, and structural identification of the metabolites by analyzing the fermentation broth of the expression host containing the cloned plasmid as well as the control host.

• Direct Cloning. The most direct approach to heterologous expression is to directly clone the target silent biosynthetic gene cluster into a heterologous host. Some strategies based on DNA recombination for direct cloning of large segments of biosynthetic gene clusters have been widely used. We can provide yeast transformation coupled recombination systems, Red/ET-mediated homologous recombination, Cas9-associated chromosome fragment capture, and point-specific recombination to help our customers achieve heterologous expression of silenced gene clusters.
• Reconstructing biosynthetic gene clusters. When silenced biosynthetic gene clusters are still not activated after direct cloning and splice transfer into a heterologous host, heterologous expression is often achieved by altering the original regulatory elements to reconstitute the biosynthetic gene cluster. The most common reconfiguration strategy is to replace the natural promoter with a constitutive promoter.

Currently, the main heterologous hosts that we can provide for the expression of fungal silencing gene clusters are Saccharomyces cerevisiae and Aspergillus spp. fungi.

• Saccharomyces cerevisiae is a widely studied unicellular fungus with essential applications in the heterologous activation of fungal silencing gene clusters. We can use synthetic biology techniques to modulate the metabolic flow of Saccharomyces cerevisiae and construct different chassis cells to provide efficient heterologous hosts for activating gene clusters for different types of natural product biosynthesis.
• We have obtained several strains of Aspergillus spp. with nutritional defects by UV mutagenesis and gene knockout techniques. Aspergillus spp. fungi can accurately identify and remove intron sequences from fungal genes. We provide services to activate many fungal silent gene clusters using Aspergillus spp. fungi as heterologous hosts.

Lifeasible is dedicated to activating silent gene clusters by solving problems in heterologous expression processes such as the transfer of genetic pathways, functional gene expression, protein activity, substrate supply of enzymes, and effects on host cell metabolism. As your trusted partner, we can meet all your fungal phylogenetic analysis needs and provide you with efficient and high-quality services. If you want to know the details, please contact us.