Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis

Abstract

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

“…To characterize and compare elements for Cas9 and sgRNA expression in C. tropicalis , a set of eight promoters ( P GAP1 , P FBA1 , P ADH2 , P PGK1 , P ENO1 , P HXT7 , P GPM1 , and P TEF1 ) were identified and cloned from C. tropicalis ATCC 20336 genomic DNA, and then corresponding green fluorescent protein reporter gene ( yeGFP3 ) expression cassettes were constructed (Figure a). These cassettes were inserted into the uracil‐auxotrophic host C. tropicalis XZX and integrated at the carnitine acetyltransferase gene ( CAT ) locus (L. Zhang et al, ). To quantitatively determine the strengths of these promoters, transformants were cultured with glucose or methyl laurate as the sole carbon source and fluorescence levels were measured.…”

“…To create a CRISPR–Cas9 system for C. tropicalis , a CRISPR–Cas9 expression cassette was designed and constructed (Figure a). Based on previous studies (Vyas et al, ; L. Zhang et al, ), the Cas9 gene from Streptococcus pyogenes was codon‐optimized and chemically synthesized. The synthetic Cas9 gene ( CtCas9 ) was tagged on its 3′ terminus with a triple repeat of the SV40 nuclear localization signal, and placed under control of the GAP1 promoter and ENO1 terminator.…”

“…First, the FBA1 promoter ( P FBA1 ) and terminator ( T FBA1 ) were amplified by PCR from the genomic DNA of C. tropicalis ATCC 20336 using primers cP FBA1 F and P FBA1 R, and T FBA1 F and cT FBA1 R, respectively. The open reading frame (ORF) of the yeGFP3 gene was amplified by PCR from plasmid Ts‐ CAT2 ‐ gda324 ‐ URA3 ‐ P GAP1 ‐ yeGFP3 ‐ T GAP1 (L. Zhang et al, ) using primers FBAGF and FBAGR. Secondly, the three fragments were used as templates for fusion PCR using interior primers P FBA1 F and T FBA1 R. The PCR product was purified using the AxyPrep DNA Gel Extraction Kit (Axygen, Suzhou, China), digested with Pst I and Sal I, and then inserted into similarly digested plasmid Ts‐ CAT2 ‐ gda324 ‐ URA3 (L. Zhang et al, ) to replace the gda fragment.…”

“…The resulting plasmid was named Ts‐ CAT2 ‐ P FBA1 ‐ yeGFP3 ‐ T FBA1 ‐ URA3 . The integrated GFP expression cassette ( CAT2 ‐ P FBA1 ‐ yeGFP3 ‐ T FBA1 ‐ URA3‐CAT2 ) was isolated as described previously (L. Zhang et al, ). A similar strategy was used for the construction of other GFP expression cassettes.…”

“…The DSBs induced by Cas9 dramatically improve the efficiency of in vivo assembly of multiple DNA fragments and subsequent integration into a target locus (Jakociunas et al, ). The one‐step assembly and integration method is more economical and efficient (Kuijpers et al, ) than classical methods, in which gene expression cassettes are assembled in vitro using restriction digestion and ligation methods (Engler, Kandzia, & Marillonnet, ; L. Zhang et al, ) or overlap assembly strategies (Gibson et al, ; Li & Elledge, ), and then inserted into the host and integrated at a chromosomal locus by homologous recombination. However, the rapid and high‐efficiency in vivo assembly method has not been available for nonconventional yeast, especially for C. tropicalis .…”