CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea.

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15310%2F21%3A73604520" target="_blank" >RIV/61989592:15310/21:73604520 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0168165620302649" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0168165620302649</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jbiotec.2020.09.028" target="_blank" >10.1016/j.jbiotec.2020.09.028</a>

Jazyk výsledku

angličtina

Název v původním jazyce

CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea.

Popis výsledku v původním jazyce

Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5&apos;-phosphate decarboxylase involved in pyrimidine biosynthesis and the a-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.

Název v anglickém jazyce

CRISPR/Cas9 genome editing in ergot fungus Claviceps purpurea.

Popis výsledku anglicky

Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5&apos;-phosphate decarboxylase involved in pyrimidine biosynthesis and the a-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10611 - Plant sciences, botany

Projekt

<a href="/cs/project/EF16_019%2F0000827" target="_blank" >EF16_019/0000827: Rostliny jako prostředek udržitelného globálního rozvoje</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>S - Specificky vyzkum na vysokych skolach

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

JOURNAL OF BIOTECHNOLOGY

ISSN

0168-1656

e-ISSN

—

Svazek periodika

325

Číslo periodika v rámci svazku

JAN

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

14

Strana od-do

341-354

Kód UT WoS článku

000603417700039

EID výsledku v databázi Scopus

2-s2.0-85094573746