CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

Error-prone DnaE2 Balances the Genome Mutation Rates in Myxococcus xanthus DK1622.

dnaE is an alpha subunit of the tripartite protein complex of DNA polymerase III that is responsible for the replication of bacterial genome. The dnaE gene is often duplicated in many bacteria, and the duplicated dnaE gene was reported dispensable for cell survivals and error-prone in DNA replication in a mystery. In this study, we found that all sequenced myxobacterial genomes possessed two dnaE genes. The duplicate dnaE genes were both highly conserved but evolved divergently, suggesting their importance in myxobacteria. Using Myxococcus xanthus DK1622 as a model, we confirmed that dnaE1 (MXAN_5844) was essential for cell survival, while dnaE2 (MXAN_3982) was dispensable and encoded an error-prone enzyme for replication. The deletion of dnaE2 had small effects on cellular growth and social motility, but significantly decreased the development and sporulation abilities, which could be recovered by the complementation of dnaE2. The expression of dnaE1 was always greatly higher than that of dnaE2 in either the growth or developmental stage. However, overexpression of dnaE2 could not make dnaE1 deletable, probably due to their protein structural and functional divergences. The dnaE2 overexpression not only improved the growth, development and sporulation abilities, but also raised the genome mutation rate of M. xanthus. We argued that the low-expressed error-prone DnaE2 played as a balancer for the genome mutation rates, ensuring low mutation rates for cell adaptation in new environments but avoiding damages from high mutation rates to cells.

[Spatial variation of soil properties and quality evaluation for arable Ustic Cambosols in central Henan Province].

A GIS-based 500 m x 500 m soil sampling point arrangement was set on 248 points at Wenshu Town of Yuzhou County in central Henan Province, where the typical Ustic Cambosols locates. By using soil digital data, the spatial database was established, from which, all the needed latitude and longitude data of the sampling points were produced for the field GPS guide. Soil samples (0-20 cm) were collected from 202 points, of which, bulk density measurement were conducted for randomly selected 34 points, and the ten soil property items used as the factors for soil quality assessment, including organic matter, available K, available P, pH, total N, total P, soil texture, cation exchange capacity (CEC), slowly available K, and bulk density, were analyzed for the other points. The soil property items were checked by statistic tools, and then, classified with standard criteria at home and abroad. The factor weight was given by analytic hierarchy process (AHP) method, and the spatial variation of the major 10 soil properties as well as the soil quality classes and their occupied areas were worked out by Kriging interpolation maps. The results showed that the arable Ustic Cambosols in study area was of good quality soil, over 95% of which ranked in good and medium classes and only less than 5% were in poor class.