Nourseothricin

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 vectors 6 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 marker 17.
The EasyClone‐MarkerFree vectors for expression of fluorescent protein or 3HP pathway genes were cloned as described in 5, 26. The vectors were linearized with NotI, the integration fragment (part of the expression vector without E. coli ori and Amp^R) 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.

Saccharomyces cerevisiae strains were transformed according to Gietz and Woods (2002 ). Mutants were selected on solid YP medium (demineralized water, 10 g·L⁻¹ Bacto yeast extract, 20 g·L⁻¹ Bacto peptone, 2% (w/v) agar), supplemented with 200 mg·L⁻¹ G418, 200 mg·L⁻¹ hygromycin B or 100 mg·L⁻¹ nourseothricin (for dominant markers) or on SM supplemented with appropriate auxotrophic requirements (Verduyn et al.1992 (link)). In all cases, gene deletions and integrations were confirmed by colony PCR on randomly picked colonies, using the diagnostic primers listed in Table S1 (Supplementary data). Integration of cas9 into the genome was achieved via assembly and integration of two cassettes containing cas9 and the natNT2 marker into the CAN1 locus. The cas9 cassette was obtained by PCR from p414-TEF1p-cas9-CYC1t (DiCarlo et al.2013b (link)), using primers 2873 & 4653. The natNT2 cassette was PCR amplified from pUG-natNT2 with primers 3093 & 5542. 2.5 μg cas9 and 800 ng natNT2 cassette were pooled and used for each transformation. Correct integration was verified by colony PCR (Supplementary data) using the primers given in Table S1 (Supplementary data), the resulting strains have been deposited at EUROSCARF. IMX719 was constructed by co-transformation of pUDR022 (see below) with genes required for functional Enterococcus faecalis PDH expression (Kozak et al.2014b (link)). The gene cassettes were obtained by PCR using plasmids pUD301–pUD306 as template (Table 2) with the primers indicated in Table S1 (Supplementary data) and the ACS1 dsDNA repair fragment, obtained by annealing two complementary single-stranded oligos (6422 & 6423). After confirmation of the relevant genotype (Fig. 4B), the pUDR022 plasmid was removed as explained in Supplementary data.