Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus

Abstract

BackgroundExotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear.ResultsWe compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants.ConclusionsWith the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0294-5) contains supplementary material, which is available to authorized users.

Effects of transcriptional mode on promoter substitution and tandem engineering for the production of epothilones in Myxococcus xanthus

“…The promoter with the biosynthetic gene cluster from S. cellulosum So0157-2 (Han et al 2013) was introduced by transposition insertion into the M. xanthus genome (Zhu et al 2015b). The production of epothilones controlled by this original promoter in M. xanthus is normally less than 1 mg/L, which was further improved by approximately ten times with the supplementation of methyl oleate in the fermentation medium (Yue et al 2017).…”

“…With its own original promoter P epo , the secondary metabolites epothilones are biosynthesized in the late growth stage and the stable stage, not only in the original producer S. cellulosum (Gong et al 2007) but also in the heterologous M. xanthus host (Zhu et al 2015b). The transcriptional mode of P epo is consistent with the epothilone production, i.e., P epo guides the epothilone gene expression majorly after the growth of cells, which is suggested to accumulate more epothilone products.…”

“…S6) (Yue et al 2017; Zhu et al 2015b). The transcriptional trends of different genes in different strains are shown in Fig.…”

Knockout Sudoku, a method for rapidly curating gene disruption collections

“…This transposon system has been reliably used to generate single-gene transposon insertion mutants in organisms such as Azospira suillum PS 22 , Dickeya dadantii 23 , Francisella tularensis 24 , Geobacter sulfurreducens 25 , Marinobacter subterrani 26 , Myxococcus xanthus [27][28][29] and S. oneidensis 3,21 . In principle any transposon system can be used with Knockout Sudoku given suitable modifications to the sequencing library generation primers (Experimental Design 10).…”

Knockout Sudoku, a method for rapidly curating gene disruption collections

“…The current version of Knockout Sudoku relies upon generation of a transposon insertion mutant library through mutagenesis with a modified pMiniHimar transposon delivered by conjugation with E. coli WM3064 3,21 . This transposon system has been reliably used to generate single-gene transposon insertion mutants in organisms such as Azospira suillum PS 22 , Dickeya dadantii 23 , Francisella tularensis 24 , Geobacter sulfurreducens 25 , Marinobacter subterrani 26 , Myxococcus xanthus [27][28][29] and S. oneidensis 3,21 . In principle any transposon system can be used with Knockout Sudoku given suitable modifications to the sequencing library generation primers (Experimental Design 10).…”