2.2 Construction of engineered strain
All S. cerevisiae strains used in this study were derived from CEN.PK113-11C (MATa SUC2 MAL2-8c his3Δ1 ura3-52 ). The primers used in this study were listed in Table S3. Firstly, strain YHM01 was obtained via integrating Cas9 gene expression box at neutral site XI of FFA producing strain YJZ06 [19]. ScYan01 (ura3 Δ) and ScYan08 (his3 Δ) were obtained by in situ restoration of gene HIS3 and URA3 in strain YHM01, respectively. Furthermore, other auxotrophic strains were constructed based on ScYan01, adopting the CRISPR/Cas9 system with different sgRNA and donor DNA (approximately 500 bp upstream and downstream homologous arms) [16], obtaining engineered strains with double deficient genes of URA3 and other screening marker. Finally, the single auxotrophic strains were obtained by in siturepair of URA3 gene. Gene HIS3 was in situsupplemented in strain ScYan08, generating strain ScYan09 as the positive control, without any deletions of auxotrophic genes. The obtained engineering strains were tested on SD + / - amino acid or nucleotide (20 mg/L) culturing at 30℃ for 2 - 3 days, as shown in Figure S1.