Figure 1.

Development of a recyclable pyrG marker in M. circinelloides. (A) Construction of a pyrG blaster marker with repeats of 237 bp on 5′ and 3′ end. The short repeat (237 bp) in the 3′ end was tandemly incorporated to enhance the excision of the marker gene. The resulting pyrG blaster marker can be amplified by PCR with M13 forward and reverse primers. (B) Homologous replacement of the carRP gene with the pyrG blaster marker. The loss of the carRP gene results in a failure to produce carotenoids in M. circinelloides. Therefore, the carRPΔ mutants display white colonies as indicated by the red arrows on the plate, whereas wild-type cells form yellow colonies. Although the target gene is disrupted, only a portion of the nuclei contains the carRPΔ allele because the spores are multinucleated with aseptate hyphae. Transformants that stably form white colonies were selected to obtain the carRPΔ mutant with a homozygous karyotype. Gene size is not to scale.