All genes were PCR amplified with PrimerSTAR Max DNA Polymerase (TaKaRa Bio). Each gene was assembled with the respective plasmid using In-Fusion Cloning Kit (TaKaRa Bio). Chromosomal in-frame deletions of pobA were individually carried out as reported earlier [18 (link)]. MNX1 from Candida parapsilosis CBS604 and AS from Rauvolfia serpentine were codon optimized and synthesized by Genewiz (Suzhou, China). To substitute gene phzA and phzB with MNX1 and AS, a modified gene deletion version was used to amplify a 400–500 bp DNA fragment of phzA upstream, the open reading frame (ORF) of MNX1, AS and 400–500 bp DNA fragment of phzB downstream, above genes, were cloned into pk18mobsacB using In-Fusion Cloning Kit (TaKaRa Bio).
Other gene deletion or substitution derivatives were constructed in the corresponding strains using a similar method. Plasmid pBBR-MNX1–AS was constructed as follows. First, the gene set of MNX1 and AS was PCR-amplified using pk18-MNX1–AS as the template. The amplified gene set was cloned into pBBR1MCS digested with XhoI and XbaI under the control of Plca promoter. Other candidate genes were similarly cloned into pBBR1MCS, individually. The corresponding accession numbers of nucleotide sequence data were shown in Additional file1 , Table S2.
Other gene deletion or substitution derivatives were constructed in the corresponding strains using a similar method. Plasmid pBBR-MNX1–AS was constructed as follows. First, the gene set of MNX1 and AS was PCR-amplified using pk18-MNX1–AS as the template. The amplified gene set was cloned into pBBR1MCS digested with XhoI and XbaI under the control of Plca promoter. Other candidate genes were similarly cloned into pBBR1MCS, individually. The corresponding accession numbers of nucleotide sequence data were shown in Additional file
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