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. 2018 Jan 29;17(1):15.
doi: 10.1186/s12934-018-0867-1.

CRISPR/dCas9-mediated Transcriptional Improvement of the Biosynthetic Gene Cluster for the Epothilone Production in Myxococcus Xanthus

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CRISPR/dCas9-mediated Transcriptional Improvement of the Biosynthetic Gene Cluster for the Epothilone Production in Myxococcus Xanthus

Ran Peng et al. Microb Cell Fact. .
Free PMC article

Abstract

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.

Keywords: Activator proteins; Biosynthetic gene cluster; CRISPR/dCas9 activation; Epothilones; Myxococcus xanthus; Promoter; Transcriptional improvement; sgRNAs.

Figures

Fig. 1
Fig. 1
Expression of mxdcas9 in mutant YL1610 (DK1622::pSWdmxCas9gfp) and the ZE9 control, measured by RT-qPCR amplification of the dcas9 gene (a) or fluorescence under a fluorescence microscope with the 500 ms exposure time (b). In this experiment, the M. xanthus strain ZE9 was used as a control
Fig. 2
Fig. 2
Design of the sgRNA and the mxdCas9-activator fusion for RNA-guided transcriptional activation. a A physical map of the promoter region for the epothilone biosynthetic gene cluster, showing the places of five designed spacers. The epothilone genes are shown in green arrow, and the two TSSs are shown with red lines. The different spacer sequences are shown in bars with different colors. b The molecular structures of epothilones A and B. c Design of sgRNA scalfold, containing a 20-nt specific targeting sequence, the tracrRNA scaffold and a U6 terminator. d The ω subunit protein was designed to fuse to the C-terminal of the mxdCas9 with a linker. This fusion gene is under the control of T7A1 promoter. The two mutation sites (D10A and H840A) are shown
Fig. 3
Fig. 3
Activation effects of the epothilone gene cluster by the ω subunit of RNA polymerase. a The yields of epothilones A and B and their summation in mutants with the p41sg plasmids containing different spacers (YL1612, 95-nt TSS1/coding; YL1613, 93-nt TSS1/noncoding; YL1614, 120-nt TSS2/noncoding; YL1615, 103-nt TSS1/coding; YL1616, 106-nt TSS2/coding) and the ancestral strain M. xanthus ZE9. b RT-qPCR analysis of expression levels of the seven genes for the biosynthesis of epothilones in different mutants at 48 h of incubation. The expressions of the seven genes of the gene cluster for the biosynthesis of epothilones in M. xanthus ZE9 were each set as 1, and the expressions of the seven genes of mutant strains were shown as the relative expressions to that of their corresponding genes in ZE9. The error bars represent the standard deviation of three independent experiments. For statistical analysis between the ancestral strain and mutant strains, the signals of ** and * mean p < 0.01 and p < 0.05, respectively. c RT-qPCR analysis of the expression levels of the seven genes for the biosynthesis of epothilones of M. xanthus ZE9 at 48 h of incubation. The expression of epoA gene was set as 1, and the expressions of the other six genes in ZE9 were the relative expressions to that of epoA
Fig. 4
Fig. 4
Effects of different activation proteins (ω, α, σ54, CarQ) on the growth (a), epothilone production (b) and the gene expression (c) of different M. xanthus mutants (a). The growth of different M. xanthus mutants on CYE plate. Mutants: YL1611 (ZE9::pSWdmxCas9-ω), YL1616 (ZE9::pSWmxdCas9-ω-p41sg5), YL1617 (ZE9::pSWmxdCas9-α), YL1618 (ZE9::pSWmxdCas9-σ54), YL1619 (ZE9::pSWmxdCas9-CarQ), YL1620 (ZE9::pSWmxdCas9-α-p41sg5), YL1621 (ZE9::pSWmxdCas9-σ54-p41sg5), YL1622 (ZE9::pSWmxdCas9-CarQ- p41sg5). b The yield and the summation of epothilones A and B in mutants (YL1616, YL1620, YL1621, YL1622) and M. xanthus ZE9. c RT-qPCR analysis of the seven genes for the biosynthesis of epothilones in M. xanthus strains at 48 h of incubation. The expressions of the seven genes in ZE9 were each set as 1, and the expressions of seven genes in mutant strains were the relative expressions to them. The error bars represent the standard deviation of three independent experiments. For statistical analysis between the ancestral type strain and mutant strains, the signals of ** and * mean p < 0.01 and p < 0.05, respectively
Fig. 5
Fig. 5
Activation of the epothilone gene cluster under the control of copper-inducible promoter. a The yield of epothilones A and B in mutants YL1616, YL1624 (25 μM CuSO4), YL1624 (120 μM), YL1624 (200 μM) and M. xanthus ZE9. b RT-qPCR analysis of the expression levels of seven genes of mutants YL1616, YL1624 (25 μM), YL1624 (120 μM), YL1624 (200 μM). The expressions of the seven genes in M. xanthus ZE9 at 48 h of incubation were set as 1, and the expressions of the seven genes of mutant strains were the relative expressions on them. The error bars represent the standard deviation of three independent experiments. For statistical analysis between wild type strain and mutant strains, ** and * mean p < 0.01 and p < 0.05, respectively

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References

    1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315:1709–1712. doi: 10.1126/science.1138140. - DOI - PubMed
    1. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–170. doi: 10.1126/science.1179555. - DOI - PubMed
    1. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41:7429–7437. doi: 10.1093/nar/gkt520. - DOI - PMC - PubMed
    1. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, et al. Genome-Scale CRISPR-Mediated control of gene repression and activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014.09.029. - DOI - PMC - PubMed
    1. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, Iyer EP, Lin S, Kiani S, Guzman CD, Wiegand DJ, et al. Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015;12:326–328. doi: 10.1038/nmeth.3312. - DOI - PMC - PubMed

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