In fusion method

bPAC mutation is a gift from Shiqiang Gao, who works at Department of Biology, Institute for Molecular Plant Physiology and Biophysics, Biocenter, ulius-Maximilians-University of Würzburg, Julius-von-Sachs-Platz 2, 97,082 Würzburg, Germany. Plasmids were made using the Infusion method (Takara Bio United States, Inc.). PCR fragments were amplified using PrimerStar (normal PCR or site-directed mutagenesis) or Taq (random mutagenesis) DNA polymerases. When needed, overlap PCR was exploited to generate the intact DNA fragment for Infusion. All PCR primers were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). DNA sequence for Green cGull and mitochondrial targeting peptide was synthesized by Genscript. To express fluorescent proteins or sensors in bacterial or mammalian cells, cDNAs of FPs or sensors were subcloned into pNCS or pCAG vector (Chu et al., 2016 (link)). The pNCS and pCAG-mEGFP were kept in our lab. For mitochondrial localization of G-Flamp2, DNA coding sequences for mitochondrial targeting peptide of COX8A (G4S)10 and G-Flamp2 was overlapped and cloned into pCAG vector. All constructs were confirmed by DNA sequencing (Sangon Biotechnology Co., Ltd., Shanghai, China).

For overexpression in EPC2 cells, the wild-type and identified mutants of DSP or PPL were synthesized into a pLVX-IRES-puro lentiviral vector by the In-Fusion method (Takara). The EPC2 cells were grown and transduced in KSFM. The viruses, obtained from a 60 mm dish of HEK293T cells, were precipitated using Lenti-X concentrator solution and resuspended in 300 μl of KSFM. KSFM (150 μl) was mixed with polybrene to a final concentration of 5 μg/ml and used to transduce EPC2 cells grown to 60–70% confluency on a 6-well plate by centrifugation at 2000 g at room temperature for 1 h. All cells underwent puromycin selection (1 μg/ml) for 1–2 weeks and were later kept in puromycin at 0.5 μg/ml. The puromycin was removed 24 h prior to the beginning of the experiment. Expression levels were validated by quantitative PCR and western blot analyses.

Yeast strains listed in S2 Table were grown in yeast extract-peptone-dextrose (YPD) or appropriate synthetic complete (SC) dropout medium at 30°C. Gene deletions were constructed by PCR-mediated one-step replacement with kanMX4, natMX6 or hygMX6 cassettes. C-terminal epitope-tagging of endogenous genes was achieved by integrating 3xHA-TRP, 3xV5-kanMX6, or 13xMyc-kanMX6 in place of the stop codon. To generate yeast strains expressing GST-Fob1, a GST-FOB1 ORF segment was PCR amplified from pJSS92-8 (pGEX6P1 expressing GST-Fob1) and subcloned into HindIII/XbaI sites of pYES2.URA using the In-Fusion method (Takara). The rdnΔΔ yeast strain NOY891carrying pNOY353 was maintained on SC-trp 2% galactose [45 (link)]. Primers used in strain construction are listed in S3 Table.

The pTRKH3-slpGFP vector (Addgene, Watertown, Massachusetts, United States) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert the T7 transcriptional terminator at BamHI/EcoRV sites, and insert the linker sequence containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using In-Fusion method (Clontech, Mountain View, CA, United States). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the L. paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/ml) selection. Both parental plasmid (pLP) and NAPE-PLD expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa, and Sharpe (MRS) broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain) both supplemented with erythromycin 5 μg/ml (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37°C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/ml was obtained through a colony number correction for the dilution factor.

The pTRKH3-slpGFP vector (Addgene, Watertown, MA, USA) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert T7 transcriptional terminators at BamHI/EcoRV sites, and insert linker sequences containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using the In-Fusion method (Clontech, Mountain View, CA, USA). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the Lactobacillus paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/mL) selection. Both parental plasmid (pLP) and NAPE-PLD-expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa and Sharpe (MRS)-broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain), both supplemented with erythromycin 5 μg/mL (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37 °C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/mL were obtained through a colonies number correction for the dilution factor.

S. cerevisiae strains (W303 matA bar1 Δ, origin—established cell line from Dr. Brian Chait’s laboratory at The Rockefeller University) were created by transformation with a variation of the plasmid pPrA-dCas9 + gRNA-ARS305 with the GAL1 promoter and either dCas9 or enhanced dCas9 (1.1) (edCas9 (1.1)). These plasmids were assembled from PCR fragments using the In-Fusion method (Clontech) followed by mutagenesis to change the 18 bp guide RNA target sequence (Fu et al., 2014 (link)). The ARS305-1 target sequence is GTTGGTAGCACTTTGATG, and the ARS305-2 target sequence is CCAGTTTCATGTACTGTC (the target site used for the sequencing study). Enhanced dCas9 (1.1) was generated by site-directed mutagenesis in the same manner as the guide RNAs.
The ChAP control strain was generated by homologous recombination to generate a genomic deletion of the ARS305 locus. The ARS305 consensus sequence was replaced with a Kluyveromyces lactis URA3 gene + promoter. Selection for knockout cells was done on media lacking uracil. Cells were screened by PCR to verify URA3 replacement at the correct genomic location. This strain was transformed with the same dCas9 + gRNA plasmid as the matched experimental strain.
For detailed contextual information regarding the ARS305 locus, gRNA target sequences, and PCR primers see Fig. S1.