Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074

Bohdan Bilyk, Liliya Horbal, Andriy Luzhetskyy

Efforts to construct the Streptomyces host strain with enhanced yields of heterologous product have focussed mostly on engineering of primary metabolism and/or the deletion of endogenous biosynthetic gene clusters. However, other factors, such as chromosome compactization, have been shown to have a significant influence on gene expression levels in bacteria and fungi. The expression of genes and biosynthetic gene clusters may vary significantly depending on their location within the chromosome. Little is known about the position effect in actinomycetes, which are important producers of various industrially relevant bioactive molecules. To demonstrate an impact of the chromosomal position effect on the heterologous expression of genes and gene clusters in Streptomyces albus J1074, a transposon mutant library with randomly distributed transposon that includes a β-glucuronidase reporter gene was generated. Reporter gene expression levels have been shown to depend on the position on the chromosome. Using a combination of the transposon system and a φC31-based vector, the aranciamycin biosynthetic cluster was introduced randomly into the S. albus genome. The production levels of aranciamycin varied up to eightfold depending on the location of the gene cluster within the chromosome of S. albus J1074. One of the isolated mutant strains with an artificially introduced attachment site produced approximately 50% more aranciamycin than strains with endogenous attBs. In this study, we demonstrate that expression of the reporter gene and aranciamycin biosynthetic cluster in Streptomyces albus J1074 varies up to eightfold depending on its position on the chromosome. The integration of the heterologous cluster into different locations on the chromosome may significantly influence the titre of the produced substance. This knowledge can be used for the more efficient engineering of Actinobacteria via the relocation of the biosynthetic gene clusters and insertion of additional copies of heterologous constructs in a suitable chromosomal position.