The codon‐optimized genes, encoding the pyruvate dehydrogenase complex and lipoate‐protein ligase from Enterococcus faecalis, were ordered from GenScript (sequences can be found in Supporting information, Table S5). The genes encoding ATP‐dependent citrate lyase and the mitochondrial citrate transport protein, were amplified from the genomic DNA of Yarrowia lipolytica DSM‐8218, obtained from the DSMZ collection (www.dsmz.de ). All of the primers, biobricks and plasmids constructed and used in this study can be found in Supporting information, Tables S1–S3.
The EasyClone‐MarkerFree vectors were created by amplifying the EasyClone 2.0 vectors6 with primers that were designed to attach to either side of the selection markers, creating a fragment that no longer contained the marker. These fragments were then ligated to form the marker‐less vectors. Seven of the resulting vectors (named ”Intermediate vectors“ in Table S3) contained PAM sites in the integration regions, which were removed by site‐directed mutagenesis using the QuikChange II XL Site‐Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturers' protocol.
gRNA cassettes targeting particular integration loci (chromosomal coordinates can be found in Supporting information, Table S4) were ordered as double‐stranded gene blocks from IDT DNA. These cassettes were amplified using primers 10525(TJOS‐62 [P1F]) and 10529(TJOS‐65 [P1R]) and USER‐cloned into pCfB2926 (pTAJAK‐71)15 to give single gRNA helper vectors. For construction of triple gRNA helper vectors, three gRNA cassettes were amplified using three primer pairs (10525(TJOS‐62 [P1F]) and 10530(TJOS‐66 [P2R]) for the first, 10526(TJOS‐63 [P2F]) and 10531(TJOS‐67 [P3R]) for the second, and 10527(TJOS‐64 [P3F]) and 10529(TJOS‐65 [P1R]) for the third gRNA cassette) and cloned into pCfB2926 (p‐TAJAK‐71) 15 . Single gRNA helper vectors for Ethanol Red were constructed by PCR amplification of the template plasmid pCfB3041 using primers indicated in Supporting information, Table S1 as described in 17 . All of the cloning steps for creating gRNA helper vectors and EasyClone‐MarkerFree vectors were performed in E. coli. Correct cloning was confirmed by Sanger sequencing.
For expression of the Cas9 gene we used an episomal vector pCfB2312 with CEN‐ARS replicon and KanMX resistance marker17 .
The EasyClone‐MarkerFree vectors for expression of fluorescent protein or 3HP pathway genes were cloned as described in5 , 26 . The vectors were linearized with NotI, the integration fragment (part of the expression vector without E. coli ori and AmpR) was gel‐purified and transformed, along with a gRNA helper vector, into yeast carrying the Cas9 plasmid (pCfB2312) via the lithium acetate method 27 . After the heat shock the cells were recovered for two hours in YPD medium and then plated on YPD agar containing 200 mg/L G418 and 100 mg/L nourseothricin. For yeast transformations with a single vector we routinely use 500 ng of the linear integration fragment along with 500 ng of the relevant gRNA helper plasmid. For yeast transformations with three vectors we use 1 µg of linear integration fragments and 1 µg of triple gRNA helper plasmid. Correct integration of the vectors into the genome was verified by colony PCR using primers listed in Supporting information, Table S1.
The EasyClone‐MarkerFree vectors were created by amplifying the EasyClone 2.0 vectors
gRNA cassettes targeting particular integration loci (chromosomal coordinates can be found in Supporting information, Table S4) were ordered as double‐stranded gene blocks from IDT DNA. These cassettes were amplified using primers 10525(TJOS‐62 [P1F]) and 10529(TJOS‐65 [P1R]) and USER‐cloned into pCfB2926 (pTAJAK‐71)
For expression of the Cas9 gene we used an episomal vector pCfB2312 with CEN‐ARS replicon and KanMX resistance marker
The EasyClone‐MarkerFree vectors for expression of fluorescent protein or 3HP pathway genes were cloned as described in
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