Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

The overall goal of this procedure is to develop effective genetic engineering methods for an unconventional yeast species, including construction of transforming DNA, DNA transformation, transformant selection, and targeted gene deletion by homologous recombination. We've established a highly vision gene deletion technique in the yarrowia lipolytica PO1G strain with potential applications in bio fuel and bio chemical production. The main advantage of this technique over existing methods like PCR-mediated gene disruption is that large amounts of gene deletion crescents and the wide range of selectable markers are available.

In this study, we report the genetics transformation protocol for the yarrowia lipolytica PO1G strain that is efficient, easy, reputed, and reproducible. The yarrowia lipolytica PO1G KU70 deletion strain constructed in this study show a very efficient homologous recombination frequency which enables faster and easier screenings of targeted gene deletions. The first step in this procedure is to amplify, by PCR, the LEU2 expression cassette from a Y.lipolytica expression vector and introduce LoxP sites into the 5-prime and 3-prime ends of the LEU2 cassette.

A BamHI restriction site is also introduced in primer one for subsequent steps of the cloning process. After purifying the LoxP promoter LEU2 terminator LoxP cassette, three prime-A overhangs are added to the purified PCR product. The A-tailed PCR product is then ligated into a TA cloning vector to yield the plasma T-LEU2.

The second PCR step is to amplify the one kilobase 5-prime upstream sequence of the KU70 gene using primers three and four from the Y.lipolytica PO1G genomic DNA. After that, the double digest both the purified PCR product and T-LEU2 plasmid with SacII and BamHI enzymes and ligate the purified and digested PCR product to the SacII BamHI sites of T-LEU2 to yield the plasmid T-LEU2-5E. The third PCR step is to amplify the one kilobase 3-prime downstream sequence of the KU70 gene using primers five and six from the Y.lipolytica PO1G genomic DNA.

Then double digest both the purified PCR product and the T-LEU2-53 plasmid with NotI and NdeI enzymes and ligate the purified and digested PCR product to the NotI NdeI sites of T-LEU2-5E to yield the T-KO plasmid. Lastly, digest the T-KO plasmid with SacII and NdeI enzymes to produce the disruption cassette. Begin this procedure by preparing competent Y.lipolytica PO1G cells.

Inoculate a colony of Y.lipolytica PO1G strain from a fresh yeast extract peptone dextrose or YPD plate in 10 milliliters of YPD medium in a 100 milliliter flask. Incubate in a 30 degree Celsius shaking incubator at 225 RPM for 20 hours until saturation. On the following day, pellet the cells by cetrifuging for five minutes at 5000 times G at room temperature.

Wash the cells with 20 milliliters of TE buffer and pellet the cells again. After removing the supernatant, resuspend the cells in one milliliter of 0.1 molar lithium acetate and incubate for 10 minutes at room temperature. Divide the competent cells in to 100 microliter aliquots in sterile 1.5 milliliter tubes.

Proceed immediately to the transformation procedure by gently mixing 10 microliters of denatured salmon sperm DNA and one to five micrograms of the purified disruption cassette together with 100 microliters of competent cells. Incubate at 30 degrees Celsius for 15 minutes. Add 700 microliters of 40%polyethylene glycol for 1000.

Mix well, and incubate in a 30 degree Celsius shaking incubator at 225 RPM for 60 minutes. Next, heat chuck the transformation mixture by placing the tube in a 39 degree Celsius water bath for 60 minutes. After 60 minutes, add one milliliter of YPD medium to the tube and allow the cells to recover for two hours at 30 degrees Celsius at 225 RPM.

Centrifuge at 9000 times G for one minute at room temperature. Remove the supernatant and resuspend the pellet in one milliliter of TE buffer. Centrifuge the cells again and discard the supernatant.

Resuspend the pellet in 100 microliters of TE buffer and plate on to leucine-deficient plate. Incubate the plate at 30 degrees Celsius for two to three days. Prior to performing CRE recombinase mediated marker rescue, the CRE expression plasmid is constructed as described in the text protocol.

Some of the features of this episomally replicating plasmid include a CRE recombinase gene, a hygromycin B-resistance marker, and an ampicillin-resistance gene. To begin this procedure transform the CRE expression plasmid into competent cells of the KU70 knockout strain following the transformation protocol demonstrated earlier. Plate the transformed cells on to YPD plus hygromycin B plates and incubate at 30 degrees Celsius for two to three days.

After performing colony PCR, as described in the text protocol to identify positive colonies, pick a positive clone to inoculate two milliliters of YPDH medium and incubate in a 30 degree Celsius shaking incubator at 225 RPM overnight to saturation. On the following day, measure the OD 600 of the culture with a spectrophotometer. When the cells are at an OD 600 of about 15, harvest by centrifugation at 5000 times G for five minutes at room temperature.

Remove the supernatant, add two milliliters of sterile water and spin again. Remove the supernatant and resuspend the cells in YPD medium Reinoculate the cells in to two milliliters of YPD medium with an initial OD 600 of 0.1. And allow the cells to grow in a 30 degree Celsius incubator at 225 RPM overnight.

On the following day, streak the overnight cell culture on to YPD plates to isolate single colonies. Incubate at 30 Celsius until colonies appear. Replica plate colonies on to YPD, YPDH, and Leucine-deficient plates.

Incubate the plates at 30 degrees Celsius for two days. A schematic of the KU70 knockout plasmid is shown. Digest of the plasmid with SacII and NdeI enzymes produces the disruption Cassette.

Gel electrophoresis confirms the presence of a 4.3 kilobase band, which is the expected size of the disruption cassette. The KU70 gene deletion in Y.lipolytica PO1G is confirmed by PCR. Lanes one to 10 contain PCR products amplified from the genomic DNA of transformants and lane 11 contains PCR products from the wild type strain.

Lane seven illustrates a positive knockout while lane one shows a false positive. The selection marker gene in the KU70 knockout strain was eventually removed by the expression of CRE recombinase. Positive marker-free transformants were verified by their growth on only a YPD plate.

To verify the increase of the gene deletion rate, after deletion of the KU70 gene, 11 target genes were individually deleted using the same procedure in the KU70 knockout strain. Each gene disruption was confirmed by PCR and a representative result is shown here with lanes one, two and four identified as positive knockouts. The yarrowia lipolytica PO1G KU70 deletion platinum strain provides a more efficient system and a better choice for applications in pathway engineering and strain optimization when constructing other chromosomal deletion mutants.

After watching this video, you should have a comprehensive understanding of the targeted single-gene deletion strategy through homologous recombination in the yarrowia lipolytica PO1G cells.