Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System.
The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the scarless and antibiotic marker-free deletion of the gene encoding the tyrosinase MelC, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated ΔmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants.
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Timo
T
Wolf
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Tetiana
T
Gren
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Eric
E
Thieme
Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Daniel
D
Wibberg
Product Supply, Bayer Pharma AG, Friedrich Ebert Str. 217-475, 42117 Wuppertal, Germany.
Till
T
Zemke
Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Alfred
A
Pühler
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Electronic address: Joern.Kalinowski@Cebitec.Uni-Bielefeld.DE.
Jörn
J
Kalinowski
D039903:Actinobacteria / Q000235:genetics* / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D008543:Melanins / Q000235:genetics; D010957:Plasmids / Q000235:genetics
D008543:Melanins; C041877:eumelanin
Actinomycetes; Actinoplanes; CRISPR/Cas9; Genome editing; Genome engineering; Tyrosinase