Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea

Abstract

Background Two reference strains have been sequenced from the mushroom Coprinopsis cinerea, monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 (ade8-1) and a para-aminobenzoic acid (PABA)-auxotrophy in AmutBmut (pab1-1) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph, respectively. Results Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8+ vector pCcAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pCcAde8 in single-vector and co-transformations with ade8+-selection were similarly high, unlike for trp1+ plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pCcAde8 helped in co-transformations of trp1 strains with a trp1+-selection vector to overcome suicidal effects by transferred trp1+. Co-transformation rates of pCcAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26–42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase. Conclusions ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. pCcAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8+ successive rounds of transformation possible.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene

“…Plasmids pYSK7, pBD5, and pCRII have been characterized in previous reports (Dörnte and Kües 2016 ; Dörnte et al 2020 ; Rühl et al 2018 ). Considering the close evolutionary affinity between C. cinerea okayama7#130 and C. cinerea FA2222, the genomic information of C. cinerea FA2222 is referenced to that of C. cinerea okayama7#130 (genome accession number: GCA_000182895.1).…”

“…The C. cinerea protoplast preparation and transformation were performed according to the method previously reported by Dörnte et al ( 2020 ). Briefly, the protoplasts were prepared based on the enzymatic digestion of C. cinerea oidia until the enzymatic extent reached 40–60%.…”

Seryl-tRNA Synthetase Shows a Noncanonical Activity of Upregulating Laccase Transcription in Trametes hirsuta AH28-2 Exposed to Copper Ion

“…The ThserRS overexpression vector pYSK7- Ov and the silencing vector pYSK7- An were constructed based on the recombinant plasmid pYSK7, as previously described ( 32 , 63 ). In particular, a 1,353-bp full-length cDNA and a 362-bp partial cDNA (from +398 to +759 bp) of ThserRS were amplified using the primer pairs of Ov- ThserRS -F and Ov- ThserRS -R and An- ThserRS -F and An- ThserRS -R, respectively (Table S1).…”

Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea

“…Unlike white and brown rot fungi which decompose wood, litter decomposers obtain carbon from nonwoody plant biomass. In C. cinerea , the availability of a self-fertile homokaryotic strain (AmutBmut pab1-1 ), along with widely used selection markers, including auxotrophic marker genes, facilitate genetic studies ( 19 , 23 , 24 ).…”