Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster

Abstract

The TRP1

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system

“…Inactivation of TRP1 (trp − ) resulted in Y. lipolytica tryptophan auxotrophy, and the trp − mutant was unable to grow on a synthetic complete (SC) medium lacking tryptophan [6]. The disruption efficiency achieved with pCAS1yl-trp was calculated based on the number of cells on the agar plate with or without tryptophan.…”

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

“…The mutant strains with HpTRP1 deletion (Δ Hptrp1 ) showed Trp − auxotrophy as expected. However, surprisingly, the growth of the Δ Hptrp1 mutants was severely retarded on both the complex YPD and the synthetic complete (SC) media (Figure 2A, b), which was not observed for other yeasts, such as S. cerevisiae and Yarrowia lipolytica (Cheon et al , 2003). The growth defect of Δ Hptrp1 mutants on complex media could be fully recovered by reintroduction of the wild‐type HpTRP1 gene, as shown in the DL1‐LdT strain transformed with vector pDTMOX containing HpTRP1 as a selectable marker, or by additional supplementation of tryptophan in the media.…”

“…In yeasts, the most routinely used marker gene for counter‐selection is URA3 (Boeke et al , 1987). Recently, the S. cerevisiae and Y. lipolytica TRP1 genes encoding phosphoribosylanthranilate isomerase were also shown to be effective counter‐selection markers (Cheon et al , 2003; Toyn et al , 2000). We tested whether disruption of HpTRP1 conferred resistance to 5‐fluoroanthranilic acid (5‐FAA), which has been used for counter‐selection of mutation in tryptophan biosynthesis genes (Toyn et al , 2000).…”

“…To construct a HpTRP1–lacZ blaster containing the lacZ repeat region upstream and downstream of HpTRP1 , the 166 bp Pvu II/ Ava I C‐terminal fragment of LacZ , excised from pBluescript KS + , was subcloned into the Sma I and Sal I sites of pBS‐HpTRP1 to generate pBS‐HpTRP1‐LZ. To construct a pBHpTRP1‐tc vector containing a HpTRP1 ‐tc blaster with a 585 bp direct repeat of the tetracycline gene (tc), the YlTRP1 region ( Xho I– Afl II fragment) of vector pBTERP2 (Cheon et al , 2003) was replaced with the HpTRP1 gene fragment amplified with primer set TRP1_1F_SalI/TRP1_4B_AflII (5′‐gactcacttaa g aagaagctgattgctggc ‐3′).…”

Deus ex Candida genetics: overcoming the hurdles for the development of a molecular toolbox in the CTG clade

“…This compound, toxic for prototrophic cells, permits in most cases an easy counterselection of ura3 or ura5 genotypes from a randomly mutagenized population. In the same way, recent advances have demonstrated the potential of 5-fluoroanthranilate for counter-selection of trp1, trp3, trp4 or trp5 genotypes in yeast (Toyn et al, 2000;Cheon et al, 2003Cheon et al, , 2009Lebel et al, 2006;Jones et al, 2008).…”