**2. Materials and Methods**

See Supplementary Material for tables of media, strains, plasmids, and oligonucleotides.

#### *2.1. Strains and Culture Conditions*

One ShotTM Top10 chemically competent *Escherichia coli*, One ShotTM *ccdB* SurvivalTM 2 T1R-competent *E*. *coli*, and One Shot™ OmniMAX™ 2 T1<sup>R</sup> *E*. *coli* were used as hosts for the construction of general plasmids and grown in LB medium with 50 mg·mL−<sup>1</sup> of the antibiotic carbenicillin at an incubation temperature of 37 ◦C. *Saccharomyces cerevisiae* strain CEN.PK was used to prepare competent yeast cells in YPAD medium and then utilized as the host for the expression plasmid assembly by homologous recombination in SM-URA medium at 28 ◦C. *Aspergillus oryzae* NSAR1 was used as the heterologous host for fungal transformation and metabolite production in DPY medium at 28 ◦C.

#### *2.2. Propionyl-CoA Toxicity Assessment to A. oryzae NSAR1*

Methionine, isoleucine, arginine, and sodium propionate were individually dissolved in deionized water and sterilized. The prepared solutions were then mixed into DPY agar media at increasing concentrations of 0, 10 mM, 25 mM, 50 mM, and 100 mM, respectively. The same amount of *A. oryzae* spore suspension was inoculated on the face of each DPY plate bearing the supplementary compound. The culture plates were grown in the incubator at 28 ◦C. After 2, 4, and 7 days, the growth state of each sample was recorded.

### *2.3. PCR-Based Gene Identification*

Fungal genomic DNA for PCR was extracted from mycelia of *A. oryzae* NSAR1 grown in DPY medium by using the GenElute plant genomic DNA kit (Sigma-Aldrich, Darmstadt, Germany). Using genomic DNA as the PCR template, a pair of specific 50 and 30 primers for gene identification were designed. PCR product purification was carried out by using the Wizard SV gel and PCR clean-up system (Promega, Madison, WI, USA). The PCR product was further identified by gene sequencing.

Total RNA was isolated from fresh 2-day-old cultured mycelia of *A. oryzae* NSAR1 grown in DPY medium by using the Quick-RNA™ Fungal/Bacterial Miniprep Kit according to the manufacturer0 s protocol (Zymo Research, Irvine, CA, USA). Single-stranded cDNA was prepared from total RNA by using the High-Capacity RNA-to-cDNA™ Kit (ThermoFisher, Waltham, MA, USA). Using the cDNA as a template, the PCR reaction with specific 50 and 30 primers for gene identification was carried out. For the gene identification under the condition of adding propionate, 50 mM of sodium propionate was supplemented into the *A. oryzae* culture one day before total RNA isolation.
