Skip to main content
NCBI home page
Metabolic Engineering Communications logoLink to Metabolic Engineering Communications
. 2020 Nov 13;11:e00152. doi: 10.1016/j.mec.2020.e00152

Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae

Qingchun Ji a,1, Jie Mai a,1, Ying Ding a, Yongjun Wei b, Rodrigo Ledesma-Amaro c, Xiao-Jun Ji a,b,∗
PMCID: PMC7691175  PMID: 33294367

Abstract

The oleaginous non-conventional yeast Yarrowia lipolytica has enormous potential as a microbial platform for the synthesis of various bioproducts. However, while the model yeast Saccharomyces cerevisiae has very high homologous recombination (HR) efficiency, non-homologous end-joining is dominant in Y. lipolytica, and foreign genes are randomly inserted into the genome. Consequently, the low HR efficiency greatly restricts the genetic engineering of this yeast. In this study, RAD52, the key component of the HR machinery in S. cerevisiae, was grafted into Y. lipolytica to improve HR efficiency. The gene ade2, whose deletion can result in a brown colony phenotype, was used as the reporter gene for evaluating the HR efficiency. The HR efficiency of Y. lipolytica strains before and after integrating the ScRad52 gene was compared using insets with homology arms of different length. The results showed that the strategy could achieve gene targeting efficiencies of up to 95% with a homology arm length of 1000 bp, which was 6.5 times of the wildtype strain and 1.6 times of the traditionally used ku70 disruption strategy. This study will facilitate the further genetic engineering of Y. lipolytica to make it a more efficient cell factory for the production of value-added compounds.

Keywords: Homologous recombination, Non-homologous end-joining, Oleaginous yeast, Rad52, Yarrowia lipolytica

Highlights

  • •

    The gene ade2 was chose as the reporter gene for evaluating the HR efficiency.

  • •

    RAD52 governing the HR machinery in S. cerevisiae was grafted into Y. lipolytica.

  • •

    RAD52 could improve the HR efficiency of Y. lipolytica.

  • •

    It was better than the traditionally used ku70 disruption strategy.

1. Introduction

The oleaginous non-conventional yeast Yarrowia lipolytica, a generally recognized as safe (GRAS) microorganism, is an attractive production host with great promise for industrial applications (Liu et al., 2015; Ma et al., 2019, 2020; Muhammad et al., 2020). It has been used as a model organism to study the biosynthesis of peroxisomes, the accumulation of lipids, as well as the production and secretion of organic acids and heterologous proteins (Dulermo et al., 2017; Wang et al., 2020; Yu et al., 2012). Due to its ability to grow using hydrophobic carbon sources, as well as generate and accumulate large amounts of lipids, it is widely used as a production host for valuable bioproducts, such as fatty acids, omega-3 eicosapentaenoic acid, and other fatty acid derivatives (Ledesma-Amaro and Nicaud, 2016; Xu et al., 2016; Xue et al., 2013). In addition, it can secrete high levels of natural and heterologous proteins and has been used to produce various heterologous proteins, including proteases, lipases, and RNases (Dulermo et al., 2017; Madzak et al., 2014).

Targeted deletion or insertion of genes and functional nucleic acid sequences at the target sites of a microbial genome is not only an effective method to study the gene function and regulate gene expression, but also a basic tool for genome editing for strain improvement. With the deciphering of the whole-genome sequence of Y. lipolytica (Dujon et al., 2004), a series of platforms for molecular biology and genetics were established in this yeast, including transformation methods for gene deletion and integration (Larroude et al., 2018; Shi et al., 2018), which made it possible to redesign whole metabolic pathways (Abdel-Mawgoud et al., 2018). However, in Y. lipolytica, the vast majority of foreign genes are randomly inserted into the genome, and the probability of targeted insertion by homologous recombination (HR) is extremely low (Ding et al., 2019; Jang et al., 2018; Tsakraklides et al., 2015). When the 5′- and 3′-flanking regions of the target gene are 0.5–1.0 ​kb long, HR occurs only at a rate of 0–36% (Schwartz et al., 2017a). This is due to the fact that Y. lipolytica has a dominant non-homologous end-joining (NHEJ) recombination pathway that it uses to repair the DNA double strand breaks (DSB) rather than the HR pathway (Richard et al., 2015). These two repair mechanisms are independent and competitive (van Dyck et al., 1999). Most eukaryotic microorganisms use NHEJ as the main DSB repair pathway, and knocking out or destroying NHEJ-related genes is an effective strategy to increase the efficiency of gene targeting. However, many studies also showed that disrupting the NHEJ pathway can make the cells prone to mutation, which is not conducive to industrial application (Barnes and Rio, 1997; Kretzschmar et al., 2013). Moreover, the cell growth can be severely affected under non-optimal conditions, such as high temperature, ultraviolet irradiation and the presence of chemical DNA-damaging agents (Gandía et al., 2016; Zhang et al., 2015). In addition, in strains lacking components of the NHEJ pathway, the integration efficiency is highly dependent on the targeted gene locus.

Consequently, strengthening the HR repair is also an effective approach for improving the gene targeting efficiency. The HR repair pathway is mediated by a class of conserved enzymes called DNA recombinase enzymes, most of which were initially identified in Saccharomyces cerevisiae (Campos-Góngora et al., 2013). Notably, the RAD51/RAD52 complex is a major participant in the targeted integration of foreign DNA in eukaryotes and plays a key role in the HR pathway (Di Primio et al., 2015) (Fig. 1). S. cerevisiae has very high HR efficiency (up to 100%), and ScRAD52 has been identified as an important component that plays a vital role in mitotic and meiotic recombination in this organism (McIlwraith and West, 2008; Mortensen et al., 2002; Resnick, 1969). The heterologous expression of S. cerevisiae HR related genes such as ScRad51 and ScRad52, in mammalian cells (e.g., monkey embryonic cells) and plant cells (e.g., Arabidopsis), or its combination with the CRISPR/Cas9 system, can greatly increase the efficiency of HR and reduce the off-target effects (Park et al., 1995; Shaked et al., 2005; Shao et al., 2017; Shcherbakova et al., 2000; Vispé et al., 1998; Yanez and Porter, 1999).

Fig. 1.

Fig. 1

Open in a new tab

Pathways of DNA double-strand break (DSB) repair by homologous recombination (HR) and non-homologous end joining (NHEJ) in Y. lipolytica. Y. lipolytica is more inclined to use the NHEJ pathway for double strand breaks (DSB) repair rather than the HR pathway. Destroying NHEJ-related proteins (KU70/KU80) can increase the efficiency of gene targeting. The RAD51/RAD52 complex plays a key role in the HR pathway.

Here, we focused on improving the HR efficiency of Y. lipolytica by grafting heterologous component that is crucial for the HR mechanisms of S. cerevisiae. Since ScRad52 is a key repair gene mainly responsible for DSB repair, we hypothesized that heterologous expression of the ScRAD52 protein in Y. lipolytica would improve its HR efficiency. To validate this concept, the ScRad52 gene was first heterologously expressed in Y. lipolytica and the HR efficiency was examined by knocking out the adenosine succinic acid synthase gene (ade2, YALI0B23188g) using cassettes with homology arms of different lengths. The corresponding endogenous gene of Y. lipolytica (YlRad52, YALI0F02431g) was further knocked out to explore its role in Y. lipolytica and whether the heterologous expression of ScRAD52 could replace the function of the native YlRAD52 protein. This work provides an effective strategy for precise genome editing, which may contribute to the molecular biology and genetics of Y. lipolytica and will greatly facilitate the rapid development of genome editing in other similar microorganisms.

2. Materials and methods

2.1. Strains and culture conditions

Y. lipolytica Po1f (ATCC MYA-2613), a leucine and uracil auxotrophic strain, was used as the base strain for all genome editing and expression experiments in this study. The ku70-disrupted Y. lipolytica strain Po1f-Δku70 was constructed using the URA3-blaster method and a ku70 deletion cassette as described by Jang et al. (2018). Escherichia coli DH5α was used for plasmid construction and propagation. It was grown in Luria-Bertani medium supplemented with 100 ​mg/L ampicillin at 37 ​°C. Yeast cultures were grown in YPD medium (20 ​g/L glucose, 10 ​g/L yeast extract, and 20 ​g/L tryptone) at 28 ​°C. The synthetic complete medium without uracil (SC-Ura) plates contained 20 ​g/L glucose, 6.7 ​g/L yeast nitrogen base (YNB) without amino acids (BBI, Sangon Biotech (Shanghai) Co. Ltd., China), 0.77 ​g/L CSM-Ura (Complete supplement mixture minus uracil, MP Biomedicals, USA), and 20 ​g/L agar. The synthetic complete medium without leucine (SC-Leu) plates contained 20 ​g/L glucose, 6.7 ​g/L YNB without amino acids (BBI, Sangon Biotech (Shanghai) Co. Ltd., China), 0.77 ​g/L CSM-Leu (Complete Supplement Mixture minus leucine, MP Biomedicals, USA), and 20 ​g/L agar. The URA3 maker was removed and selected against on SC ​+ ​FOA plates, which contained 6.7 ​g/L YNB, 0.77 ​g/L CSM (Complete Supplement Mixture, MP Biomedicals, USA), 0.8 ​g/L 5-fluoroorotic acid, and 20 ​g/L agar.

2.2. General molecular biology techniques

The plasmids and primers used in this study are listed in Table 1 and Supplementary Table 1. Genomic DNA from the yeast strains was purified using an E.Z.N.A.® Yeast DNA Kit (Omega Bio-Tek Inc., USA). The transformation of Y. lipolytica was performed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Orange, CA). Gene deletion was performed by HR based on direct repeats, using URA3 as a counter-selection marker on SC ​+ ​FOA plates. The recombinant strains were spread on SC ​+ ​FOA plates and cultured for 2–3 days. Strains expressing URA3 did not grow in the presence of 5-FOA.

Table 1.

Strains and plasmids used in this study.

Strains or plasmids Genotypes or properties Sources
Strains
E. coli DH5α supE44 ΔlacU169 (ϕ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Takara
Y. lipolytica Po1f (ATCC MYA-2613) MATa, leu2-270, ura3-302, xpr2-322, axp1-2 ATCC
Y. lipolytica Po1f-Δku70 Po1f, Δku70 This work
Y. lipolytica Po1f-ScRad52 Po1f, ScRad52::A08 This work
Y. lipolytica Po1f-Δku70-ScRad52 Po1f, Δku70, ScRad52::A08 This work
Y. lipolytica Po1f-ΔYlRad52 Po1f, ΔYlRad52 This work
Y. lipolytica Po1f-Δku70-ΔYlRad52 Po1f, Δku70,ΔYlRad52 This work
Y. lipolytica Po1f-ScRad52 -ΔYlRad52 Po1f, ScRad52::A08, ΔYlRad52 This work
Y. lipolytica Po1f-Δku70-ScRad52-ΔYlRad52 Po1f, Δku70, ScRad52::A08, ΔYlRad52 This work
Plasmids
pUC57 ColE1 Ori, LacZ, Amp GenScript
pUC-Leu-A08 A08 upstream and downstream homology arms and Leu auxotrophic selection marker in pUC57 This work
pUC-Leu-A08-ScRad52 PYlRad52-ScRad52 -TYlRad52 This work
pUC-HUH HisG-URA3-HisG (HUH) in pUC57 This work
pUC-HUH-ΔYlRad52 YlRad52-up-HUH-YlRad52-down This work
pUC-HUH-Δade2-100 ade2-100-up-HUH-ade2-100-down This work
pUC-HUH-Δade2-250 ade2-250-up-HUH-ade2-250-down This work
pUC-HUH-Δade2-500 ade2-500-up-HUH-ade2-500-down This work
pUC-HUH-Δade2-1000 ade2-1000-up-HUH-ade2-1000-down This work
Open in a new tab

2.3. Construction of expression cassettes expressing ScRad52

The S. cerevisiae Rad52 sequence (GenBank accession No. CAA86623.1) was codon-optimized for expression in Y. lipolytica and ordered as fully synthetic DNA (GeneScript, Nanjing, China, Supplementary Table 2). The vector pUC-Leu-A08-ScRad52 expressing the gene of interest under the control of the endogenous Rad52 gene promoter PYlRad52 and terminator TYlRad52 as generated using the ClonExpress™ MultiS one step cloning kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. The promoter PYlRad52 and terminator TYlRad52 were respectively amplified by PCR using the primer pairs PYlRad52-F/R and TYlRad52-F/R with genomic DNA extracted from Y. lipolytica. The codon-optimized ScRad52 gene was then amplified using the primers ScRad52-F/R. The PCR-amplified sequences were digested with PacI and SnaBI, and then one-step cloned into the digested pUC-Leu-A08 backbone to generate the recombinant vector pUC-Leu-A08-ScRad52. We used the A08 gene site in the Y. lipolytica genome whose disruption is known to not affect cell growth (Schwartz et al., 2017b). All the newly constructed plasmids were confirmed by DNA sequencing.

2.4. Construction of disruption cassettes

Disruption cassettes targeting YlRad52 (YALI0F02431g) and ade2 (YALI0B23188g) were constructed according to the URA3-blaster protocol (Jang et al., 2018). For the construction of the YlRad52 deletion cassette, 2 ​kb 5′- and 3′- homology arms of the YlRad52 gene, named YlRad52-up and YlRad52-down, were respectively amplified from Y. lipolytica genomic DNA using the primer pairs YlRad52-up-F/R and YlRad52-down-F/R. The resulting PCR fragments were respectively digested with EcoRI and PacI, and one-step cloned into the pUC-HUH vector containing the HisG-URA3-HisG (HUH) blaster cassette, to generate the vector pUC-HUH-ΔYlRad52.

For the construction of the ade2 deletion cassettes with 5′- and 3′-homology arms of 1000, 500, 250 and 100 bp, the homology fragments were respectively amplified from the yeast genomic DNA using the primer combinations 1000-up-F, 500-up-F, 250-up-F, 100-up-F/Ade2-up-R and Ade2-down-F/1000-down-R, 500-down-R, 250-down-R, 100-down-R. The obtained PCR fragments were respectively digested with EcoR I and Pac I, and one-step cloned into the pUC-HUH vector containing the HisG-URA3-HisG blaster cassette, to generate the vectors of pUC-HUH-Δade2-1000, pUC-HUH-Δade2-500, pUC-HUH-Δade2-250, and pUC-HUH-Δade2-100.

2.5. Determination of the homologous recombination efficiency

The ade2 gene was selected as the reporter, because its deletion causes the accumulation of phosphoribosyl amino imidazole, resulting in the disrupted mutants becoming brown (Supplementary Fig. 1). The HR efficiency was estimated by counting the number of ade2- colonies arising from cells transformed with the ade2 disruption cassettes with different homology arm lengths (100, 250, 500 and 1000 bp) (Ugolini and Bruschi, 1996). The HR efficiency at the ade2 locus was determined via the ratio of brown colonies. Transformation with the disruption cassettes was repeated three times.

3. Results

3.1. Construction of recombination strains expressing ScRad52

The expression cassette encoding the ScRad52 gene driven by the endogenous promoter PYlRad52 and URA3-blaster expression cassette were integrated into the A08 locus in the genomes of the Y. lipolytica strains Po1f and Po1f-Δku70. The resulting strains Y. lipolytica Po1f-ScRad52 and Po1f-Δku70-ScRad52 were successfully selected on SC-Ura plates, and further confirmed by colony PCR as well as genomic DNA isolation followed by PCR. Microscopic examination and colony morphology observation throughout the growth cycle indicated that the engineered Y. lipolytica was not morphologically different from the parental strain.

3.2. ScRad52 expression enhanced the homologous recombination efficiency

To explore the effect of heterologous expression of the ScRad52 gene on the HR efficiency of Y. lipolytica, we selected genes whose deletion results in significant phenotypic changes (e.g. color, fluorescence, etc.) as target sites. In yeast, the adenylosuccinate synthetase gene ade2 is responsible for the conversion of phosphoribosyl aminoimidazole (AIR) into 5-amino 4-carboxyimidazoleribose (CAIR). The deletion of ade2 causes the accumulation of AIR, which turns the corresponding yeast colonies brown (Fig. 2). This phenotype was used for easy visual selection in both S. cerevisiae and Y. lipolytica (Ugolini and Bruschi, 1996; Verbeke et al., 2013; Wagner et al., 2018). Therefore, the ade2 was chosen as the target site for gene knockout in this study.

Fig. 2.

Fig. 2

Open in a new tab

Screening method based on deleting the adenylosuccinate synthetase gene (ade2) involved in adenine biosynthesis in Y. lipolytica. The deletion of the ade2 gene results in a brown colony phenotype. PRPP: phosphoribosyl pyrophosphate; PRA: 5-phosphoribosylamine, GAR: glycinamide ribotide, FGAR: formyl glycinamide ribotide, FGAM: formyl glycinamide ribotide, AIR: phosphoribosyl aminoimidazole, CAIR: 5-amino 4-carboxyimidazole ribotide. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

By using the URA3 blaster method with homology arms of different targeting the ade2 gene as a single variable, knockout plasmids containing different homology arm lengths were constructed via a single-fragment one-step assembly method. After the recombinant plasmid was linearized and introduced into Y. lipolytica, the positive transformants were screened according to their visible phenotype and confirmed using colony PCR. The HR efficiency of Y. lipolytica strains, including Po1f-ScRad52 and Po1f-Δku70-ScRad52, as well as the parental strains Po1f and Po1f-Δku70, was obtained by calculating the ratio of the colonies with ade2 knockout to the total number of strains on the plate, that is, the ratio of brown mutants.

As shown in Table 2, when the ade2 knockout was performed with homology arms of 1000 bp, HR efficiency of the engineered strains Y. lipolytica Po1f-ScRad52 and Po1f-Δku70-ScRad52 with heterologous HR components from S. cerevisiae reached 90% and 95%, respectively. By contrast, the HR efficiency of the control strains Y. lipolytica Po1f and Po1f-Δku70 was only 14.7% and 60%, respectively. The best strain with the highest HR efficiency for ade2 deletion was Y. lipolytica Po1f-Δku70-ScRad52. Its HR efficiency reached up to 95%, which was 6.5 times of the wild-type strain Y. lipolytica Po1f (Table 2). These results indicated that ScRAD52, the main component of the HR machinery in S. cerevisiae, was functional and improved the HR efficiency in Y. lipolytica, simplifying genome editing.

Table 2.

Homologous recombination efficiency of different Y. lipolytica strains using different homology arm lengths.

Y. lipolytica strains HR efficiency with different homology arm length (%)
100 bp 250 bp 500 bp 1000 bp
Po1f-Δku70-ScRad52-ΔYlRad52 0 ​± ​0.0 0 ​± ​0.0 4.1 ​± ​0.2 6.7 ​± ​0.3
Po1f 0 ​± ​0.0 2.0 ​± ​0.1 8.2 ​± ​0.4 14.7 ​± ​0.8
Po1f-ScRad52-ΔYlRad52 1.8 ​± ​0.1 11.9 ​± ​0.6 32.8 ​± ​1.6 53.3 ​± ​2.5
Po1f-Δku70 3.3 ​± ​0.2 13.3 ​± ​0.7 43.7 ​± ​2.2 60.0 ​± ​3.0
Po1f-ScRad52 6.1 ​± ​0.3 22.6 ​± ​1.1 52.6 ​± ​2.6 90.0 ​± ​4.5
Po1f-Δku70-ScRad52 11.9 ​± ​0.6 32.8 ​± ​1.6 65.3 ​± ​3.3 95.0 ​± ​4.8
Open in a new tab

3.3. Effect of homology arm length on homologous recombination efficiency

Disruption cassettes with homology arms of 1000, 500, 250, and 100 bp targeting the ade2 were constructed. As shown in Table 2, when the disruption cassette with 1000 bp homology regions was respectively used for ade2 deletion, the percentage of transformants generated by HR in the strains Po1f-ScRad52 and Po1f-Δku70-ScRad52 reached 90% and 95%, respectively. However, when the length of homology arms was less than 500 bp, the HR efficiency dropped sharply. At 100 bp, the HR efficiency of Y. lipolytica Po1f-ScRad52 and Po1f-Δku70-ScRad52 decreased to 6.1% and 11.9% respectively, which was only 6.8% and 12.5% of that with homology arms of 1000 bp (Fig. 3). The extension of homology arm length on each side of the disruption cassette increased the HR efficiency, which was consistent with previous reports (Choo et al., 2014; Oguro et al., 2017; Verbeke et al., 2013).

Fig. 3.

Fig. 3

Open in a new tab

Homologous recombination efficiency of wild-type Y. lipolytica Po1f and different engineered strains. Disruption cassettes with different homology arm lengths targeting the adenylosuccinate synthetase encoding gene (ade2) were constructed respectively. The length of the homology arms from left to right is 100, 250, 500, and 1000 bp. The S. cerevisiae derived ScRad52 was integrated into the genome of the Y. lipolytica strains Po1f and Po1f-Δku70. Additionally, YlRad52 was knocked out to investigate the function of endogenous Rad52 in Y. lipolytica. Homology arms of different lengths were used to evaluate the homologous recombination efficiency of the strains. The homologous recombination efficiency was determined by the ratio of brown colonies with successful ade2 deletion. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Effect of endogenous Rad52 disruption on homologous recombination efficiency

In order to further investigate the effect of the endogenous Rad52 (YlRad52) on the HR efficiency, and whether the heterologous ScRad52 can completely replace YlRad52 in Y. lipolytica, the YlRad52 gene was knocked out using the URA3 blaster method in the aforementioned Y. lipolytica strains. The growth rate of the engineered strains with the YlRad52 gene knockout was slightly lower than that of the YlRad52 wild-type strains. In addition, for the two strains Po1f-ScRad52-ΔYlRad52 and Po1f-Δku70-ScRad52-ΔYlRad52, there was almost no difference of colony morphology compared to the parental strains. However, the strains Po1f-ΔYlRad52 and Po1f-Δku70-ΔYlRad52 formed smaller and sharper colonies on YPD plates compared with the other two strains Po1f-ScRad52-ΔYlRad52 and Po1f-Δku70-ScRad52-ΔYlRad52 which express the ScRad52 gene. This may be explained by the fact that Rad52 is important for nuclear integrity in Y. lipolytica. This result is similar to a report by Campos-Gongora et al. (2013). The expression of the ScRad52 gene alleviates the damage to nuclear integrity caused by the deletion of the endogenous Y. lipolytica Rad52 to some extent. Consequently, the colony morphology of Po1f-ScRad52-ΔYlRad52 and Po1f-Δku70-ScRad52-ΔYlRad52 was not significantly different from the original strains.

The HR efficiency of the strains Po1f-ScRad52-ΔYlRad52 and Po1f-Δku70-ScRad52-ΔYlRad52 was significantly lower thanthe strains with their own YlRad52 genes (Table 2 and Fig. 3). When the homology arm length was 1000 bp, the HR efficiency of strain Po1f-ScRad52-ΔYlRad52 reached 53.3%, which was only 40.8% that of Po1f-ScRad52. Moreover, the HR efficiency of Po1f-Δku70-ScRad52-ΔYlRad52 was 6.7%, which was even lower than that of the wild-type strain Y. lipolytica Po1f. Compared with Po1f-Δku70-ScRad52, the HR efficiency of Po1f-Δku70-ScRad52-ΔYlRad52 was reduced by 92.9%, and there was practically no recombination with homology arms of 250 and 100 bp. These results show that the YlRad52 gene plays a crucial role in the native HR mechanism of Y. lipolytica, which is consistent with the previous report that the Rad52 gene in is crucial for DNA repair in this yeast (Campos-Gongora et al., 2013).

4. Discussion

With the development of artificial nucleases in recent years, the possibilities of performing accurate genome editing based on HR have been greatly expanded. Although the efficiency is still relatively low, double-strand breaks can be introduced at practically any target sits, but there are great differences in knockout efficiency for different organisms (Ran et al., 2013; Rong et al., 2014). RAD52 protein is considered to be the crucial recombinase of the HR pathway. Previous studies have reported that integrating the S. cerevisiae RAD52 can significantly enhance the efficiency of HR in mammalian cells (Shao et al., 2017). In the present study, the gene encoding RAD52 from S. cerevisiae was codon-optimized and expressed in Y. lipolytica. The results showed that the heterologous expression of ScRad52 significantly improved the HR efficiency of Y. lipolytica, and the endogenous Rad52 gene also plays a crucial role in the Y. lipolytica HR mechanism. When the homology arm length was 1000 bp, the HR efficiency of the strains, Po1f, Po1f-Δku70, Po1f-ScRad52, and Po1f-Δku70-ScRad52 was 14.7%, 60%, 90%, and 95%, respectively. The highest HR efficiency was observed in the strain Po1f-Δku70-ScRad52, and it was 6.5 times of the wildtype strain Po1f as well as 1.6 times of the traditionally used ku70 disruption strategy. However, when the he endogenous Rad52 gene (YlRad52) of Y. lipolytica was knocked out, the HR efficiency was reduced. Although the mechanism of HR mediated by YlRad52 is currently unclear, the results show that it plays a very important role in nuclear integrity and DSB repair. Interestingly, the HR efficiency of the Po1f-Δku70-ScRad52-ΔYlRad52 strain was lower than that of Po1f-ScRad52-ΔYlRad52, which may be due to the deletion of the KU70 protein related to the NHEJ mechanism in this strain, further compromising nuclear integrity and DNA repair in this strain compared to Po1f-ScRad52-ΔYlRad52. In addition, in the engineered strain with YlRad52 knockout, the increase of homology arm length improved the HR efficiency. Therefore, HR mediated by YlRAD52 plays a very important role in nuclear integrity and DNA repair.

In conclusion, the HR efficiency of Y. lipolytica was improved by integrating the key gene ScRad52 governing HR in S. cerevisiae. The HR efficiency of the best engineered strain Po1f-Δku70-ScRad52 reached 95% with 1000 bp homology arms. Additionally, the results proved that the endogenous YlRAD52 plays an important role in HR. Generally, most industrially important non-model eukaryotic microorganisms have a complicated genetic background and are inherently inefficient in terms of HR, making it difficult to achieve large-scale, high-throughput gene editing in these cells. This study provides a reference for improving the HR efficiency of Y. lipolytica and other similar eukaryotic microorganisms, which can further accelerate the genome editing and metabolic engineering of important industrial hosts.

Author statement

We have reviewed the final version of the manuscript and approve it for publication. To the best of our knowledge and belief, this manuscript has not been published in whole or in part nor is it being considered for publication elsewhere.

Declaration of competing interest

The authors declare no financial or commercial conflict of interest.

Acknowledgement

This work was financially supported by the National Science Fund for Excellent Young Scholars of China (21922806), the National Key Research and Development Program of China (2018YFA0903700), the National Natural Science Foundation of China (21776131, 22061130203), the Six Talent Peaks Project in Jiangsu Province of China (2018-SWYY-047), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTD1814), Key Research and Development Program of Jiangsu Province (2020), and the BBSRC grant (BB/R01602X/1), the Newton Advanced Fellowship (NAF∖R1∖201187), and the British Council/Newton Fund Institutional Links (527429894).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mec.2020.e00152.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (88.7KB, docx)

References

  1. Abdel-Mawgoud A.M., Markham K.A., Palmer C.M., Liu N., Stephanopoulos G., Alper H.S. Metabolic engineering in the host Yarrowia lipolytica. Metab. Eng. 2018;50:192–208. doi: 10.1016/j.ymben.2018.07.016. [DOI] [PubMed] [Google Scholar]
  2. Barnes G., Rio D. DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 1997;94:867–872. doi: 10.1073/pnas.94.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Campos-Góngora E., Andaluz E., Bellido A., Ruiz-Herrera J., Larriba G. The RAD52 ortholog of Yarrowia lipolytica is essential for nuclear integrity and DNA repair. FEMS Yeast Res. 2013;13:441–452. doi: 10.1111/1567-1364.12047. [DOI] [PubMed] [Google Scholar]
  4. Choo J.H., Han C., Kim J.Y., Kang H.A. Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus. Biotechnol. Lett. 2014;36:2059–2067. doi: 10.1007/s10529-014-1576-4. [DOI] [PubMed] [Google Scholar]
  5. Di Primio C., Galli A., Cervelli T., Zoppe M., Rainaldi G. Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52. Nucleic Acids Res. 2005;33:4639–4648. doi: 10.1093/nar/gki778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ding Y., Wang K.F., Wang W.J., Ma Y.R., Shi T.Q., Huang H., Ji X.J. Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering. Appl. Microbiol. Biotechnol. 2019;103:4313–4324. doi: 10.1007/s00253-019-09802-2. [DOI] [PubMed] [Google Scholar]
  7. Dujon B., Sherman D., Fischer G., Durrens P., Casaregola S., Lafontaine I. Genome evolution in yeasts. Nature. 2004;430:35–44. doi: 10.1038/nature02579. [DOI] [PubMed] [Google Scholar]
  8. Dulermo R., Brunel F., Dulermo T., Ledesma-Amaro R., Vion J., Trassaert M., Thomas S., Nicaud J.M., Leplat C. Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica. Microb. Cell Factories. 2017;16:31. doi: 10.1186/s12934-017-0647-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gandía M., Xu S., Font C., Marcos J.F. Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum. Fungal Biol. 2016;120:317–323. doi: 10.1016/j.funbio.2015.11.001. [DOI] [PubMed] [Google Scholar]
  10. Jang I.S., Yu B.J., Jang J.Y., Jegal J., Lee J.Y. Improving the efficiency of homologous recombination by chemical and biological approaches in Yarrowia lipolytica. PloS One. 2018;13 doi: 10.1371/journal.pone.0194954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kretzschmar A., Otto C., Holz M., Werner S., Hübner L., Barth G. Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining. Curr. Genet. 2013;59:63–72. doi: 10.1007/s00294-013-0389-7. [DOI] [PubMed] [Google Scholar]
  12. Larroude M., Rossignol T., Nicaud J.M., Ledesma-Amaro R. Synthetic biology tools for engineering Yarrowia lipolytica. Biotechnol. Adv. 2018;36:2150–2164. doi: 10.1016/j.biotechadv.2018.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ledesma-Amaro R., Nicaud J.M. Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Prog. Lipid Res. 2016;61:40–50. doi: 10.1016/j.plipres.2015.12.001. [DOI] [PubMed] [Google Scholar]
  14. Liu H.H., Ji X.J., Huang H. Biotechnological applications of Yarrowia lipolytica: past, present and future. Biotechnol. Adv. 2015;33:1522–1546. doi: 10.1016/j.biotechadv.2015.07.010. [DOI] [PubMed] [Google Scholar]
  15. Ma J., Gu Y., Marsafari M., Xu P. Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform. J. Ind. Microbiol. Biotechnol. 2020 doi: 10.1007/s10295-020-02290-8. [DOI] [PubMed] [Google Scholar]
  16. Ma Y.R., Wang K.F., Wang W.J., Ding Y., Shi T.Q., Huang H., Ji X.J. Advances in the metabolic engineering of Yarrowia lipolytica for the production of terpenoids. Bioresour. Technol. 2019;281:449–456. doi: 10.1016/j.biortech.2019.02.116. [DOI] [PubMed] [Google Scholar]
  17. Madzak C., Gaillardin C., Beckerich J.M. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J. Biotechnol. 2004;109:63–81. doi: 10.1016/j.jbiotec.2003.10.027. [DOI] [PubMed] [Google Scholar]
  18. McIlwraith M.J., West S.C. DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair. Mol. Cell. 2008;29:510–516. doi: 10.1016/j.molcel.2007.11.037. [DOI] [PubMed] [Google Scholar]
  19. Mortensen U.H., Erdeniz N., Feng Q., Rothstein R. A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52. Genetics. 2002;161:549–562. doi: 10.1093/genetics/161.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Muhammad A., Feng X., Rasool A., Sun W., Li C. Production of plant natural products through engineered Yarrowia lipolytica. Biotechnol. Adv. 2020;43:107555. doi: 10.1016/j.biotechadv.2020.107555. [DOI] [PubMed] [Google Scholar]
  21. Oguro Y., Yamazaki H., Ara S., Shida Y. Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains. Curr. Genet. 2017;63:751–763. doi: 10.1007/s00294-017-0679-6. [DOI] [PubMed] [Google Scholar]
  22. Park M.S. Expression of human RAD52 confers resistance to ionizing radiation in mammalian cells. J. Biol. Chem. 1995;270:15467–15470. doi: 10.1074/jbc.270.26.15467. [DOI] [PubMed] [Google Scholar]
  23. Ran F.A., Hsu P.D., Lin C.Y., Gootenberg J.S. Double nicking by RNA-guided CRISPR/Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–1389. doi: 10.1016/j.cell.2013.08.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Resnick M.A. Genetic control of radiation sensitivity in Saccharomyces cerevisiae. Genetics. 1969;62:519. doi: 10.1093/genetics/62.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Richard G.F., Kerrest A., Lafontaine I., Dujon B. Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination. Mol. Biol. Evol. 2005;22:1011–1023. doi: 10.1093/molbev/msi083. [DOI] [PubMed] [Google Scholar]
  26. Rong Z., Zhu S., Xu Y., Fu X. Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template. Protein & Cell. 2014;5:258–260. doi: 10.1007/s13238-014-0032-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwartz C., Frogue K., Ramesh A., Misa J., Wheeldon I. CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica. Biotechnol. Bioeng. 2017;114:2896–2906. doi: 10.1002/bit.26404. [DOI] [PubMed] [Google Scholar]
  28. Schwartz C., Shabbir-Hussain M., Frogue K., Blenner M., Wheeldon I. Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth. Biol. 2017;6:402–409. doi: 10.1021/acssynbio.6b00285. [DOI] [PubMed] [Google Scholar]
  29. Shaked H., Melamed-Bessudo C., Levy A.A. High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene. Proc. Natl. Acad. Sci. U.S.A. 2005;102:12265–12269. doi: 10.1073/pnas.0502601102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shao S., Ren C., Liu Z., Bai Y. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int. J. Biochem. Cell Biol. 2017;92:43–52. doi: 10.1016/j.biocel.2017.09.012. [DOI] [PubMed] [Google Scholar]
  31. Shcherbakova O.G., Lanzov V.A., Ogawa H., Filatov M.V. Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells. Mutat. Res. 2000;459:65–71. doi: 10.1016/s0921-8777(99)00059-2. [DOI] [PubMed] [Google Scholar]
  32. Shi T.Q., Huang H., Kerkhoven E.J., Ji X.J. Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system. Appl. Microbiol. Biotechnol. 2018;102:9541–9548. doi: 10.1007/s00253-018-9366-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsakraklides V., Brevnova E., Stephanopoulos G., Shaw A.J. Improved gene targeting through cell cycle synchronization. PloS One. 2015;10 doi: 10.1371/journal.pone.0133434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ugolini S., Bruschi C.V. The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr. Genet. 1996;30:485–492. doi: 10.1007/s002940050160. [DOI] [PubMed] [Google Scholar]
  35. van Dyck E., Stasiak A.Z., Stasiak A., West S.C. Binding of double-strand breaks in DNA by human Rad52 protein. Nature. 1999;398:728–731. doi: 10.1038/19560. [DOI] [PubMed] [Google Scholar]
  36. Verbeke J., Beopoulos A., Nicaud J.M. Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol. Lett. 2013;35:571–576. doi: 10.1007/s10529-012-1107-0. [DOI] [PubMed] [Google Scholar]
  37. Vispé S., Cazaux C., Lesca C., Defais M. Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation. Nucleic Acids Res. 1998;26:2859–2864. doi: 10.1093/nar/26.12.2859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wagner J.M., Williams E.V., Alper H.S. Developing a piggyBac transposon system and compatible selection markers for insertional mutagenesis and genome engineering in Yarrowia lipolytica. Biotechnol. J. 2018;13 doi: 10.1002/biot.201800022. [DOI] [PubMed] [Google Scholar]
  39. Wang J., Ledesma-Amaro R., Wei Y., Ji B., Ji X.J. Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica – a Review. Bioresour. Technol. 2020;313:123707. doi: 10.1016/j.biortech.2020.123707. [DOI] [PubMed] [Google Scholar]
  40. Xu P., Qiao K., Ahn W.S., Stephanopoulos G. Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc. Natl. Acad. Sci. U.S.A. 2016;113:10848–10853. doi: 10.1073/pnas.1607295113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xue Z., Sharpe P.L., Hong S.P., Yadav N.S., Xie D., Short D.R. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat. Biotechnol. 2013;31:734–740. doi: 10.1038/nbt.2622. [DOI] [PubMed] [Google Scholar]
  42. Yanez R., Porter A. Gene targeting is enhanced in human cells overexpressing hRAD51. Gene Ther. 1999;6:1282. doi: 10.1038/sj.gt.3300945. [DOI] [PubMed] [Google Scholar]
  43. Yu Z., Du G., Zhou J., Chen J. Enhanced α-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by an improved integrated fed-batch strategy. Bioresour. Technol. 2012;114:597–602. doi: 10.1016/j.biortech.2012.03.021. [DOI] [PubMed] [Google Scholar]
  44. Zhang J., Mao Z., Xue W., Li Y. Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger. Curr. Microbiol. 2011;62:1342–1346. doi: 10.1007/s00284-010-9853-5. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.docx (88.7KB, docx)

Articles from Metabolic Engineering Communications are provided here courtesy of Elsevier

RESOURCES