Non-homologous End Joining-Mediated Insertional Mutagenesis Reveals a Novel Target for Enhancing Fatty Alcohols Production in Yarrowia lipolytica

Abstract

Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in Yarrowia lipolytica. One batch of strains transformed with fatty acyl-CoA reductase gene (FAR) showed significant differences (up to 70.53-fold) in fatty alcohol production. Whole-genome sequencing of the high-yield strain demonstrated that a new target YALI0_A00913g ("A1 gene") was disrupted by NHEJ-mediated integration of partial carrier DNA, and reverse engineering of the A1 gene disruption (YlΔA1-FAR) recovered the fatty alcohol overproduction phenotype. Transcriptome analysis of YlΔA1-FAR strain revealed A1 disruption led to strengthened protein synthesis process that was confirmed by sfGFP gene expression, which may account for enhanced cell viability and improved biosynthesis of fatty alcohols. This study identified a novel target that facilitated synthesis capacity and provided new insights into unlocking biosynthetic potential for future genetic engineering in Y. lipolytica.

Keywords: RNA-Seq; Yarrowia lipolytica; fatty alcohols; new target identification; non-homologous end joining-mediated integration.

Figure 1

Strains with different fatty alcohols yields were found in the same batch of transformed of FAR gene. (A) Schematic diagram of the fatty alcohol biosynthesis pathway engineering strategies in Yarrowia lipolytica. FAR, fatty acyl-CoA reductase gene from Marinobacter aquaeolei VT8; PEX10, the peroxisome synthesis gene. Fatty acyl-CoA reductase converts fatty acyl-CoA to fatty alcohols. The gene PEX10 was deleted to prevent the degradation of fatty alcohols. (B) Schematic diagram of the plasmid pINA1269-FAR used to transform Y. lipolytica. The FAR gene was driven by the Hp4d promoter and the plasmid was linearized by NotI and transformed into different basic strains and two transformants were randomly selected from each plate for fermentation (C). (C) Schematic diagram of establishing exogenous synthesis pathway of fatty alcohols. (D) Fatty alcohols production of strains from the same batch of transformants. All strains were cultured in rich YPD medium. Samples were taken at 72 h. Data are presented as mean ± SD of three biological replicates.

Figure 2

Whole-genome sequencing determined gene disruption in fatty alcohol high-yielding strains. YlA and YlP strains were applied to the whole-genome sequencing. The FAR gene marked in orange was integrated into the chromosome F as expected. The gene disruption caused by the integration of broken vector fragments into chromosomes A and E was marked with red vertical line, named A1 and E1 sites, respectively. The A1 site was located in the 118,335–118,543 region of chromosomes A, and the E1 site was in the 4,134,688–4,134,892 region of chromosomes E.

Figure 3

Reverse engineering of the candidate targets derived from WGS to identify beneficial gene for enhanced fatty alcohol production. (A) CRISPR/Cas-mediated knockout of candidate gene targets in wild-type Y. lipolytica strain ATCC 201249. The deletion of bases at A1 and E1 sites in ATCC 201249 genome were marked with red and strikethrough. The knockout location is contained within the location of the broken vector fragment insertion. (B) Schematic diagram of the plasmid pIntF-FAR used to transform Y. lipolytica. The FAR gene is driven by the TEFin promoter and flanked by 2 homology arms of IntF location. The plasmid was linearized by NotI and transformed into different basic strains and two transformants were randomly selected from each plate for fermentation. (C) The effect of A1 and E1 sites disruption on fatty alcohols production. All strains were cultured in rich YPD medium. Values are the mean of three biological replicates ± standard deviation (n = 3) after 72 h.

Figure 4

Transcriptome analysis of YlΔA1-FAR and WT-FAR. (A) Growth rate of YlΔA1-FAR strain with high fatty alcohols production compared with WT-FAR under rich medium conditions. All cultivations were performed in rich YPD medium for 108 h. Transcriptome analysis (RNA-Seq) was performed on the samples at 24 h (pointed by arrow). Results are presented as mean ± s. d. of three biological replicates. (B) Fold changes (log2) of genes differentially expressed (adjusted p < 0.05, |Fold change| > 1) in YlΔA1-FAR cells relative to WT-FAR cells. Genes at the positive side of the x axis are upregulated (highlighted in red), and genes at the negative side are downregulated (highlighted in blue). (C) KEGG pathways enrichment analysis. Gene sets were defined by KEGG pathways, which showed the percentage of genes that were either up- (red) or down-regulated (blue).

Figure 5

Changes in transcript levels of the protein synthesis process. The (A) RNA transport, (B) Ribosome and (C) Aminoacyl-tRNA synthesis pathway belong to the translation gene set, the (D) protein translocation, and (E) protein processing and (F) protein degradation pathway belong to the folding, sorting and degradation gene set. Among the six pathways, the up- and down-regulated pathways are marked with red and blue arrows, respectively. The boxes indicate fold-change in gene expression of YlΔA1-FAR compared to WT-FAR control. Red boxes indicate upregulated gene expression, and blue boxes indicate downregulation gene expression (p < 0.05).

Figure 6

Verification of improved protein expression by sfGFP gene expression. (A) Schematic diagram of the sfGFP insertion cassette used to transform Y. lipolytica. The sfGFP gene was driven by the TEFin promoter and stopped by the LIP1 terminator, flanking by two 500 bp homologous arms to IntF site. (B) The A1 gene disruption increased protein expression. Characterization of protein expression by green fluorescence intensity. All strains were cultured in SC-Leu medium. Values are the mean of three biological replicates ± standard deviation (n = 3) after 48 h. (C) Fluorescence images of the strains YGF01 and YGF02. The images of sfGFP fluorescence were observed by a fluorescence microscope (Olympus CX41, Japan).

Figure 7

Additional regularities for enhanced expression of exogenous protein. (A) Schematic diagram of transformed fragments with different promoters and homologous arms. (B) Schematic diagram of the distribution of IntX integration sites on the chromosome in Y. lipolytica. (C) Schematic diagram of the promoters with or without intron sequences. (D) The impact of different integration sites on protein expression from A1 disruption. Characterization of protein expression by green fluorescence intensity. The octothorpe (#) indicated the integration failure of sfGFP gene on chromosome B and C. (E) The impact of different promoters on protein expression from A1 disruption. Characterization of protein expression by green fluorescence intensity. All strains were cultured in SC-Leu medium. Values are the mean of three biological replicates standard deviation (n = 3) after 48 h.