iGEM 2022 — Team nnu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering-Success{% endblock %}
{% block lead %}Sharing and collaboration are core values of iGEM. We encourage you to reach out and work with other teams on difficult problems that you can more easily solve together.{% endblock %}
{% block page\_content %}

# Engineering Success

Team:CUG-China/Notebook - 2022.igem.org

[Overview](#Overview)
[Cycle 1](#Cycle 1. Constructing the EPA producer)
[Cycle 2](#Cycle 2. Inhibition of β-oxidation pathway)
[Cycle 3](#Cycle 3. Screening for different sources of elongases/desaturates)
[Cycle 4](#Cycle 4. Increasing the copy numbers of target genes)
[Cycle 5](#Cycle 5. Replacement of promoter)

# **Overview**

---

We introduced an entire EPA biosynthesis pathway into *Yarrowia lipolytica* *Po1f*, which is capable of guiding the conversion from the C18:2 to EPA step by step. In addition, many strategies were applied to enhance the EPA content, including inhibiting β-oxidation pathway, screening for different sources of elongases/desaturates, increasing the copy numbers of target genes, and replacement of promoter. All BioBricks were registered and tested, and most of them work well to perform their functions as expected.

# **Cycle 1. Constructing the EPA producer**

---

**Design**

Because *Y. lipolytica* *Po1f* is lack of EPA biosynthesis-related enzymes, we attempted to create an EPA biosynthesis pathway in *Y. lipolytica* *Po1f* by integrating heterogenous genes from *Euglena gracilis* and *Pythium aphanidermatum*. Here, three genes encoding ∆-9 elongase (*EgElo9*), ∆-8 desaturase (*EgDes8*) and ∆-5 desaturase (*EgDes5*), respectively, from *E. gracilis*, and a ∆-17 desaturase gene (*PaDes17*) from *P. aphanidermatum* were selected as candidate genes. In order to express the heterogenous genes in *Y. lipolytica Po1f*, the coding sequences (CDS) of selected genes were optimized based on the codon usage bias of *Y. lipolytica Po1f* (http://www.kazusa.or.jp/codon/). The native strong constitutive promoter PTEF was used to express these genes.

**Build**

In order for *Y. lipolytica Po1f* to produce EPA, ∆-9 elongase, ∆-8 desaturase and ∆-5 desaturase needed to be expressed in *Y. lipolytica*. Firstly, the CDS of *EgElo9* [BBa\_K4343095](http://parts.igem.org/Part%3ABBa_K4343095), *EgDes8* [BBa\_K4343096](http://parts.igem.org/Part%3ABBa_K4343096), *EgDes5* [BBa\_K4343097](http://parts.igem.org/Part%3ABBa_K4343097) and *PaDes17* [BBa\_K4343098](http://parts.igem.org/Part%3ABBa_K4343098) were optimized, and strong constitutive promoter (PTEF) fragments were subcloned into the linear pUC-HUH together by the enzyme digestion and ligation, yielding the desired plasmids pUC-HUH-EgElo9 [BBa\_K4343066](http://parts.igem.org/Part%3ABBa_K4343066), pUC-HUH-EgDes8 [BBa\_K4343067](http://parts.igem.org/Part%3ABBa_K4343067), pUC-HUH-EgDes5 [BBa\_K4343068](http://parts.igem.org/Part%3ABBa_K4343068), pUC-HUH-PaDes17 [BBa\_K4343069](http://parts.igem.org/Part%3ABBa_K4343069). Take the process of constructing pUC-HUH-EgElo9 for instance, i) the DNA fragment of *EgElo9* was synthesized with the restriction enzyme cutting site *BsaI*, and PCR-amplified was used to plus the restriction enzyme cutting site *BsaI* on the vector pUC-HUH; ii) then, T4 ligase was used to connect the DNA fragment of *EgElo9* and the vector pUC-HUH. iii) next, the reaction mixture was transformed to *E. coli* DH5α; iv) corrected colonies were verified by PCR and sequenced; v) the correctly sequenced plasmid was digested by the nuclease *SwaI* to obtain a linearized fragment, which was transferred into the *Y. lipolytica Po1f*. A complete overview of the detailed processes visit our Part.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-1.png)

Fig. 1 (A) Construction of target plasmid. (B) Constructing expression vectors of ∆-9 elongase, ∆-8 desaturase, ∆-5 desaturase and ∆-17 desaturase. (C) The PCR validation diagram of pUC-HUH-EgElo9.

**Test**

The percentages of different fatty acids in TFAs in the recombinant strains were detected by gas chromatography (GC) analysis. Results showed that the *Po1f-1* strain that has been integrated with ∆-9 elongase and ∆-8 desaturase genes produced eicosadienoic acid (EDA; C20: 2n-6) and DGLA at 20.8% and 8.5% of the TFAs, respectively, indicating the two enzymes were successfully expressed in the *Y. lipolytica Po1f*. Moreover, transforming all the four heterogenous genes into *Y. lipolytica Po1f* led to EPA production at 2.4% of the TFAs.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-2.png)

Fig. 2 (A) Fatty acid profile of the *Y . lipolytica* wild-type strain *Po1f* determined by Gas Chromatography analysis. (B) Different fatty acid percentage of TFAs.

**Learn**

In this cycle, we learned that codon optimization is helpful strategy to improve the success rate of heterogenous gene expression. We were able to assemble the two DNA fragments (CDS and promoter) with the backbone to obtain a full construct. Then the plasmid was linearized and transformed into the receptor state of *Y. lipolytica Po1f*. Besides, the linearized fragments were randomly integrated into the genome by non-homologous End Joining (NHEJ). The recombinant strains were endowed ability to successfully produce EPA, suggesting our design idea is flexible.

# **Cycle 2. Inhibition of β-oxidation pathway**

---

**Design**

Fatty acid β-oxidation is the process of breaking down a long-chain acyl-CoA molecule to acetyl-CoA molecules. Therefore, blockade of β-oxidation pathway contribute to accumulation of PUFAs. In order to disrupt this pathway, the homologous recombination strategy will be utilized to delete the key gene *PEX10* of β-oxidation pathway in *Po1f-2* stain. However, the number of selection markers that can be used for gene editing is limited. In this study, we employed a marker recycling system that allows the recycle of the selection marker *URA3* in successive transformations in *Y. lipolytica Po1f*. Our *URA3* sequence was flanked by the same 1000-bp *hisG* sequence. *URA3* can be removed from the genome by homologous recombination when the cell is grown in the presence of 5-fluoroorotic acid (5-FOA), a chemical that is converted into a toxin by *URA3* activity.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-3.png)

Fig. 3 The recycle of the selection marker *ura3* in successive transformations in *Y. lipolytica Po1f*.

**Build**

Firstly, we removed the selection marker *ura3* of *Po1f-2* by reverse selection on the YPD media with 5-FOA. The survival clone was uridine/uracil auxotrophic mutant and served as the recipient strain for *PEX10* deletion. In order to knock out the *PEX10* gene, the upstream and downstream sequences (both 100bp) flanking *PEX10* were PCR-amplified, then they were subcloned into the linear pUC-HUH together by the *BsaI* enzyme digestion and T4 ligase ligation, yielding the knockout plasmids pUC-HUH-ΔPEX10. The PCR was used to verify the deletion of *PEX10* (Validation primer F: TCCGACGAcactcgtcttct; Validation primer R: TATCATTAACCAGCTTTGAT). The obtained vector was sequenced by Sangon Biotech (Shanghai, China). The correctly sequenced plasmid was digested by the nuclease *SwaI* to obtain a linearized fragment, which was transformed into *Po1f-2*, generating the strain *Po1f-3*.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-4.png)

Fig. 4 (A)The deletion process of *PEX10* gene. (B) PCR validation diagram for verifying whether the *PEX10* was knocked out on the genome.

**Test**

The percentages of different fatty acids in TFAs in the related strains were analyzed by GC. In comparison to *Po1f-2*, the *PEX10* deletion stain produce more EPA at 4.5% of the TFAs, demonstrating inhibition of β-oxidation pathway indeed contributes to EPA accumulation in *Y. lipolytica Po1f*.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-5.png)

Fig.5 Different fatty acid percentage of TFAs in *Po1f-3*.

**Learn**

In this cycle, we learned how to recycle the *ura3* selection marker by the 5-FOA-meidated reverse selection and how to delete the target gene by homologous recombination strategy. In addition, we realized that inhibition of the catabolic pathway of the target product was a very effective strategy in microbial metabolic engineering.

# **Cycle 3. Screening for different sources of elongases/desaturates**

---

**Design**

Studies have demonstrated that elongases and desaturases form different species have different catalytic activities and substrate specificities for fatty acid substrates, so the selection of elongases/desaturases with high substrate specificity is a significant strategy to improve the synthesis of EPA.

**Build**

To screen the optimal elongases/ desaturates, the optimized CDS and strong constitutive promoter fragments were subcloned into the linear pUC-HUH together. Take ∆-12 desaturase for example, pUC-HUH-Intc-FmDes12 [BBa\_K4343077](http://parts.igem.org/Part%3ABBa_K4343077), pUC-HUH-Intc-CcDes12 [BBa\_K4343078](http://parts.igem.org/Part%3ABBa_K4343078) and pUC-HUH-Intc-PaDes12 [BBa\_K4343079](http://parts.igem.org/Part%3ABBa_K4343079) were constructed. i) Firstly, the upstream and downstream squences (both 100bp) of flanking *IntC* were PCR-amplified. The three fragments, the upstream, the downstream and *FmDes12* were subcloned into the linear pUC-HUH by T4 ligase, obtaining the targeted integration plasmid pUC-HUH-Intc-FmDes12 [BBa\_K4343077](http://parts.igem.org/Part%3ABBa_K4343077). ii) Next, the obtained vector was sequenced by Sangon Biotech (Shanghai, China), iii) the correctly sequenced plasmid was digested by the nuclease *SwaI* to obtain a linearized fragment, which was transferred into the *Y. lipolytica Po1f* to obtain *Po1f-4*. Besides, ∆-9 desaturase was integrated at the *Scp2* site of the genome of *Po1f-4*, yielding the desired plasmid pUC-HUH-Scp2-BgElo9 [BBa\_K4343080](http://parts.igem.org/Part%3ABBa_K4343080), pUC-HUH-Scp2-EhElo9 [BBa\_K4343081](http://parts.igem.org/Part%3ABBa_K4343081), pUC-HUH-Scp2-IgASE1 [BBa\_K4343082](http://parts.igem.org/Part%3ABBa_K4343082), pUC-HUH-Scp2-IgASE2 [BBa\_K4343083](http://parts.igem.org/Part%3ABBa_K4343083), pUC-HUH-Scp2-PpElo9 [BBa\_K4343084](http://parts.igem.org/Part%3ABBa_K4343084) and pUC-HUH-Scp2-PsElo9 [BBa\_K4343085](http://parts.igem.org/Part%3ABBa_K4343085). And C16/18 elongase was also integrated at the *Scp2* site of the genome of *Y. lipolytica Po1f*, yielding the desired plasmid pUC-HUH-Scp2-MaElo2 [BBa\_K4343086](http://parts.igem.org/Part%3ABBa_K4343086), pUC-HUH-Scp2-MaElo16 [BBa\_K4343087](http://parts.igem.org/Part%3ABBa_K4343087), pUC-HUH-Scp2-PaElo16 [BBa\_K4343088](http://parts.igem.org/Part%3ABBa_K4343088) and pUC-HUH-Scp2-rElo2 [BBa\_K4343089](http://parts.igem.org/Part%3ABBa_K4343089), respectively. The above plasmids were linearized by enzyme cleavage and transformed into the corresponding competent cell of *Y. lipolytica*.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-6.png)

Fig.6 (A) Construction of plasmids of elongases/ desaturates from different sources. (B) PCR validation diagram for *FmDes12* integrated in the genome. (C) Squencing of *FmDes12*, *EhElo9* and *MaElo16*.

**Test**

GC analysis indicated that the enzyme activity of *FmDes12* was the highest among all the tested ∆-12 desaturases due that the *FmDes12*-expressing strain displayed the conversion rate (81.1%) of C18:2 to C18:1. Besides, *IgASE2* and *EhElo9* performed better to promote the synthesis of C20:2 from C18:2. It is worth mentioning that all the four tested C16/18 elongases, and all have catalytic activity for C18:1, however, only *MaElo2* and *rElo2* can efficiently convert C16:0 to C18:0.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-7.png)

Fig.7 Different fatty acid percentage of TFAs produced by different sources of ∆-12 desaturase, ∆-9 elongase and C16/18 elongase.

**Learn**

In this cycle, we learned that employing elongases/desaturases with high substrate specificity is an effective strategy to improve EPA synthesis. This cycle provided an effective strategy and a good basis for us to further increase the yield of EPA.

# **Cycle 4. Increasing the copy numbers of target genes**

---

**Design**

Based on the push-pull strategy in microbial metabolic engineering, the accumulated intermediates (C16:0, C18:1, C18:2) of EPA biosynthesis pathway need to be reduced by genetic modify for elevating EPA yield. We planned to transform the ∆-12 desaturase, ∆-9 elongase and C16/18 elongase from other organisms into the genome of *Y. lipolytica Po1f* by random integration strategy to reduce the content of intermediates.The resulting strains with the highest EPA yield were analyzed by qPCR to confirm the copy number of target genes.

**Build**

To obtain engineered strains for efficient EPA production, seven plasmids were successfully constructed, including pUC-HUH-MaElo2 [BBa\_K4343072](http://parts.igem.org/Part%3ABBa_K4343072), pUC-HUH-FmDes12 [BBa\_K4343073](http://parts.igem.org/Part%3ABBa_K4343073), pUC-HUH-EhElo9 [BBa\_K4343074](http://parts.igem.org/Part%3ABBa_K4343074), pUC-HUH-IgASE2 [BBa\_K4343075](http://parts.igem.org/Part%3ABBa_K4343075), pUC-HUH-EgDes8 [BBa\_K4343067](http://parts.igem.org/Part%3ABBa_K4343067), pUC-HUH-EgDes5 [BBa\_K4343068](http://parts.igem.org/Part%3ABBa_K4343068) and pUC-HUH-PaDes17 [BBa\_K4343069](http://parts.igem.org/Part%3ABBa_K4343069). Then these plasmids were linearized by the nuclease *SwaI* and transformed into the competent cell of *Y. lipolytica* to obtain *Po1f-17*, *Po1f-18*, *Po1f-19* and *Po1f-20*. All the final plasmids were verified by PCR and commercial sequencing. A complete overview of the detailed processes visit our results.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-8.png)

Fig.8 Corrected colonies were verified by PCR and sequencing.

**Test**

After GC analysis, the *Po1f-18* strain integrated with 3 copies of *MaElo2* and 2 copies of *FmDes12* can produce at 9.6% of TFAs. Besides, the incorporation of 3 copies of the *EhElo9*, 3 copies of the *IgASE2* and 2 copies of the *EgDes8* elevated the EPA content to 16.3%. Finally, employing 2 copies of *EgDes5* and 2 copies of *PaDes17* further increased the percentage of EPA to 22.4%. All the above results indicated that increasing the copy number of target genes contributed to a significant increase in EPA ratio.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results8.png)

Fig.9 (A) Different fatty acid percentage of TFAs produced by random integration of the best strains of elongases/desaturases. (B) Optimal copy numbers detected by QPCR. (C) Fatty acid profiles of the *Po1f-3* and *Po1f-20* determined by Gas Chromatography analysis.

**Learn**

In term of supplementing activity of related enzymes, improving the encoding gene expression level is a routine way. In this cycle, we learned that increasing the copy numbers of gene (*MaElo2*,*FmDes12*,*IgASE2*,*EgDes8*, *EgDes5* and *PaDes17*) in the host genome by multiple-round transformation can effectively increase gene expression level and product contents. In comparison to the alternative strategy, promoter replacement, the strategy was more convenient and efficient due to omission of promoter search and new plasmid construction.

# **Cycle 5. Replacement of promoter**

---

**Design**

After communicating with our cooperative partner, a group of university of science and technology of China, we plan to adopt their suggestion to replace the originally used promoter PTEF with the other strong promoter PGAP for further improving the EPA proportion in TFA. In addition, several other strong constitutive promoters (PEXP, PYAT, PFBA in and PTEF in) were selected for probing the variation of different promoters on EPA content.

**Build**

To test whether different promoter strengths can effectively control EPA production, PTEF was cleaved by the nuclease *BsaI*. And different strong constitutive promoters (PGAP, PEXP, PYAT, PFBA in and PTEF in) were amplified by PCR. Then they were subcloned into the linear pUC-HUH-Scp2-EhElo9 by T4 ligase and the above plasmids were linearized by the nuclease *SwaI*. The obtained plasmid was sequenced by Sangon Biotech (Shanghai, China). Next, the correctly sequenced plasmid was digested by the nuclease *SwaI* to obtain a linearized fragment, which was transformed into *Po1f-3*.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/engineeringsuccess-10.png)

Fig.10 (A) Constructing expression vectors of ∆-9 elongase under the control of different promoters. (B) Corrected colonies were verified by PCR and sequencing.

**Test**

As shown in Figure, these engineered strains integrated with new promoters had no showed elevated EPA yield compared with their parent strains, which demonstrates that the selected candidate promoters did not play positive role in our case.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results9.png)

Fig.11 Different fatty acid percentage of TFAs produced by engineered strains transferred of EhElo9 controlled by different promoters.

**Learn**

Although promoters, PGAP, PEXP, PYAT, PFBA in and PTEF in, were not as superior in *Y. lipolytica* as the original promoters (PTEF), it does not mean that the promoter replacement strategy does not work in our project. As a next plan, RNA-seq method can be considered to screen the specific strong promoters in the EPA fermentation conditions for continually improving the expression of target genes.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Sharing and collaboration are core values of iGEM. We encourage you to reach out and work with other teams on difficult problems that you can more easily solve together.{% endblock %}
{% block page\_content %}

# Experiments

Our experiment was mainly consisted of five parts: constructing plasmids with different target genes, transforming yeast strains, knocking out the gene *PEX10*, regulating the culture and fermentation conditions, conducting the quantitative real-time PCR, extracting and detecting the products.

# **A Constructing Plasmids with Different Target Genes**

---

# **Materials and Instruments**

The Microspectrophotometer

The PCR Amplifier

Dry thermostat

Thermostat water bath

The centrifuge

Centrifuge tubes

Preservation tubes

*E. coli* competent cells (DH5-α)

*Bsa I* restriction enzyme

LB ampicillin solid medium (100 μg/mL ampicillin)

40% glycerol

DNA Extraction Mini Kit

T4 ligase (T4DLig)

T4 ligase buffer (10× T4 DHA Ligase Buffer)

# **1 Obtaining the target gene and skeleton**

Our experiment required appropriate enzyme genes and plasmid skeletons. After consulting relevant literature, we designed plasmids on SnapGene software, chose appropriate DNA segments and plasmid skeletons, determined the restriction sites added on the two sides of different target genes, and designed relevant PCR primers.

(1)The total reaction volume of PCR is 25 μl, including 12.5 μL primer STAR max enzyme, 1 μL\*2 primers, 0.7 μL template, and 9.8μL double distilled water (ddH2O).

(2)Purification of PCR products and DNA fragments: PCR products and DNA fragments were purified with DNA Extraction Mini Kit (Nanjing Vazyme Biotech Co., Ltd).

①Add the triploid volume of Buffer GL to PCR stock solution, mix with pipette.

②Transfer the solution to the adsorption column, centrifugate the tube at 12000 ×g for 1min, then discard the liquid, and put the adsorption column back into the empty collection tube.

③Add 700 μL Buffer W2 to the adsorption column, centrifugate the tube at 12000 ×g for 1min, and discard the liquid.

④Repeat step ③ once.

⑤Put the adsorption column back into the empty collecting tube and centrifugate it at 12000 ×g for 1min.

⑥Take out the adsorption column, put it in a clean 1.5 ml centrifuge tube, open the cover and heat it at 65℃ for 1 min.

⑦Add 30 μl of ddH2O and centrifuge at 12000 ×g for 1min to get the solution containing the purification of PCR products.

(3)The concentration of target gene or skeleton DNA was measured by microspectrophotometer.

# **2 Ligating DNA fragments through T4 ligase**

Different target genes were ligated with corresponding promoter/termination/plasmid skeleton at 16℃ using T4 ligase. The recombinant plasmids were transformed in *E. coli* competent DH5-α, and cultured at 37℃ overnight. The positive transformants were transferred to the LB ampicillin solid medium.

(1)The total volume of the T4-ligation system was 20 μL, including (number of base pairs\*0.02/ concentration) ng of plasmid, (number of base pairs \*0.04/ concentration) ng of target gene, 2 μL of T4 ligase, ddH2O, and the mixture was bathed at 16℃ for 4-6 h.

(2)The recombinant plasmid was transformed into the competent state of *E. coli*.

①10 μl mixture of ligation mixture was injected into DH-5α competent which was then placed on ice for 30 min.

②Heat shock under 42 ℃ for 1.5 min.

③Place the competent on ice for 3 minutes.

(3)The *E. coli* were then spread on LB ampicillin solid medium.

# **3 Verifying the recombinant plasmid**

Ampicillin was used to screen the *E. coli* carried the recombinant plasmids. Monoclonal *E. coli* grown on LB ampicillin solid medium were selected and then diagnostic PCR was performed to verify the recombinant plasmids. Lastly, the recombinant plasmids were sent to a professional institution for gene sequencing.

(1)Diagnostic PCR

①Total volume of each colony PCR was 10 μl, including 5 μl Taq enzyme, 0.5 μl F and R primers, and 4 μl ddH2O.

②Pick monoclone with sterilized toothpicks into the PCR tube as the templates in the clean bench.

③Mixtures were placed in the PCR Amplifier (Taq enzyme program) and amplified for 30 cycles.

(2)Select an appropriate Marker to indicate the positive band.

(3)Pick and culture the positive colony in LB ampicillin liquid medium for 12h. Extract the plasmids and send to professional institutions for sequencing.

(4)Strain preservation: Mix the *E. coli* culture liquid having correct sequencing results and 40% glycerol in a 1:1 ratio, and stored in a refrigerator at -80℃.

# **B Constructing Engineered *Y. lipolytica***

---

# **Materials and Instruments**

The Microspectrophotometer

The visible spectrophotometer

Restriction enzymes

DNA Extraction Mini Kit

Centrifuge tube

Dry thermostat

YNB solid medium for fermentation (20 g/L glucose, 5g/L ammonia sulfate, 1.7 g/L YNB without amino acids and ammonium sulfate, 0.4 g/L uracil, or 0.4 g/L L-leucine and 25 g/L agar)

YPD liquid medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose)

Frozen-EZ Yeast Transformation II Kit

# **1 Preparing yeast competent state**

Yeast Competent cells were prepared according to the method of Frozen-EZ Yeast Transformation II Kit.

(1)Seeds: Inoculate monoclonal yeast to a conical flask with 10 mL YPD liquid medium and grow under 30℃ at 220 rpm until its OD600 reaching between 0.8-1.

(2)Take 1 mL of the medium into a centrifuge tube in the clean bench, and centrifugate at 4000 ×g for 3min.

(3)Remove the supernatant in the clean bench, add 1mL EZ1 solution, resuspend the strain, and centrifugate at 4000 ×g for 3 min.

(4)Remove the supernatant in the clean bench, add 100 μL EZ2 solution, and resuspend the strain.

(5)Divide the solution into two parts, 50 μl each, and store them in the refrigerator at -80 ℃.

# **2 The transformation of *Y. lipolytica***

We digested the constructed plasmid to obtain a linear DNA fragment containing target gene, *URA/Leu* gene and *HisG tag*, which was then transferred to the yeast competent cells. The engineered *Y. lipolytica* was obtained by screening.

Since the Yeasts we used were nutrition-deficient (uracil or leucine deficient) strains, once the linear DNA fragment carrying *URA/Leu* gene was successfully transformed and integrated into *Y. lipoltiyca*, the transformants could grow on the YNB solid medium. In addition, we placed the HisG tag at both ends of *URA* gene to recycle the *URA* gene by adding 5-Fluoroorotic Acid (5-FOA) to the medium to get *URA* deficient strain allowing for the next round transformation.

(1)Digest the plasmids with the total volume of 50 μl, including 4 μg of plasmids, 2 μL×2 of enzyme, 5 μL of 10X buffer, ddH2O, incubated under 37℃ for 4-5 h.

(2)Agarose gel electrophoresis and purification.

①Cut the gel containing required fragments and then put it into the 2 ml centrifuge tube.

②Add 500 μL Buffer GL and then incubate in the thermostat water bath at 65°C for 4-6 min. Mix the solution every 2-3 min until the gel is completely melted. The following steps are the same in [A-1- (2)]

③Add 700 uL Buffer W2 to the adsorption column, centrifugate the tube at 12,000 ×g for 1min, discard the waste liquid.

④Repeat step ③ once。

⑤Put the adsorption column back into the empty collecting tube and centrifugate the tube at 12,000 ×g for 1 min.

⑥Take out the adsorption column, put it in a clean 1.5 ml centrifuge tube, open the cover and heat it at 65℃ for 1 min.

⑦Add 30 μl of ddH2O and centrifugate the tube at 12000 ×g for 1min to get the solution containing the purified PCR products.

(3)Measure the concentration of target gene or skeleton DNA by microspectrophotometer, and the required concentration should be over 50 ng/μL.

(4)Transform the linearized DNA segment into *Y. lipolytica*. During the transformation, add 10 μl lineage DNA segment and 500 μL of EZ3 solution into the prepared yeast competent cells, incubate at 28℃ for 1h.

(5)Spread 250 μL of the mix on YNB (including Leu) fermentation solid medium, grow at 30℃ for 48-96 h.

# **C knocking out the gene of *PEX10***

---

# **Materials and Instruments**

The Microspectrophotometer

The PCR Amplifier

Dry thermostat

The centrifuge

Centrifuge tubes

Preservation tubes

*E. coli* competent cells (DH5-α)

LB ampicillin solid medium(100 μg/mL ampicillin)

Restriction enzymes

DNA Extraction Mini Kit

YNB solid screening medium

Frozen-EZ Yeast Transformation II Kit

40% glycerol

yeast lysate

100% absolute ethanol

75% absolute ethanol

# **1 Constructing *PEX10* knockout plasmids**

Set the gene *PEX10* homology arm and construct the "gene knockout plasmid", using the same steps as [A].

# **2 Transformation of gene *PEX10* knockout plasmids**

We transformed the gene *PEX10* knockout plasmid into the competent cells of yeast and knocked out the gene *PEX10* by homologous recombination.

(1)Prepare the yeast competent cells, the same as [B-1].

(2)Transform gene *PEX10* knockout plasmids, the same as [B-2].

# **3 Verifying whether the gene of *PEX10* was knocked out**

(1)Preparation before the experiment

①We randomly selected 12-16 yeast transformers and cultivated them in 5 mL YPD liquid medium at 30℃, 220rpm for 12h (OD600 must under 0.6).

②Preparation of yeast lysate (200mM lithium acetate + 1% SDS solution)

(2)Genome extraction of *Y. lipolytica*

①Place 500-1000 μL of yeast solution in 1.5mL centrifuge tube, centrifugate the tube at 15000 ×g for 2 min, discard supernatant.

②Add 100 uL of yeast lysate, resuspend the strain, and incubate at 75℃ for 10min, mix the solution at the same time.

③300 uL of 100% absolute ethanol was added to the above solution to precipitate DNA, totally mixed the solution, centrifugate the tube at 15000 ×g for 5min, and then discarded the supernatant.

④400u L 75% ethanol was added, resuspend the strain, centrifugate the tube at 15000 ×g for 5min, and then discarded the supernatant.

⑤The centrifuge tube was placed in a dry thermostat at 65℃ for 10 min to evaporate the residual ethanol.

⑥add 50 μL ddH2O to resuspend the precipitation, centrifugate the tube at 15000×g for 1min. The supernatant was genome DNA of *Y. lipolytica*.

(3)The PCR system

①Total PCR system was 10 μl, including 5 μl of Taq enzyme, 0.5 μl of F and R primers each, 1uL of template and 3uL of ddH2O.

②The PCR tubules were placed in the PCR Amplifier (Taq enzyme program) and amplified for 30 cycles.

(4)After PCR, we chose appropriate markers, performed agarose gel electrophoresis to whether *PEX10* was successfully knocked out.

# **D Optimizing the Culture and Fermentation Conditions**

---

# **Materials and Instruments**

Conical flask

The visible spectrophotometer

Preservation tubes

40% glycerol

YPD liquid medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose)

YNB solid screening medium (20 g/L glucose, 5g/L ammonia sulfate, 1.7 g/L YNB without amino acids and ammonium sulfate, 0.4 g/L uracil, or 0.4 g/L L-leucine and 25 g/L agar)

YNB fermentation medium (2.5 g/L yeast extract, 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate), 60 g/L glucose)

# **Preparation before the experiment**

We inoculated monoclonal yeast on YNB solid screening medium (20-25 monoclones selected at one transformation) into YPD liquid medium, then incubated them at 30℃, 220 rpm for 24h to obtain seed solution.

# **Experimental procedure**

(1)We obtained seeds as the steps mentioned before and 40% glycerol at a ratio of 1:2 to protect the yeast. We named the yeast in the format of strain-gene-date.

(2)Transfer 500 uL seed medium into 50 mL of YPD fermentation medium, incubated them at 30℃ 220 rpm for 96 h in a conical flask.

# **E Conducting the Quantitative Real-time PCR(qPCR)**

---

# **Materials and Instruments**

The PCR Amplifier

# **Steps**

(1)After measuring the extracted yeast genomes described above in [C-3-(2)] by NanoDrop, we calculated and diluted its concentration to 1-3ng /μL,using ddH2O.

(2)We designed the primers for test and reference, using SIGMA online design of qPCR website. In this experiment, we used G6PDH as the reference primer. (Website: http://www.oligoarchitect.com/OligoArchitect/LoginServlet)

Preparation of the qPCR reaction system:10 μL of 2xSYBR qPCR Mix, 0.4 μL of primer F(10 μM), 0.4 μL of primer R(10 μM), 0.4 μL of genome (1-3 ng/μL), and dd H2O up to 25 μL.

(4)PCR program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 20s, 56 ℃ annealing for 20s, and 72℃ for 20s at extension stage. A total of 40 cycles; 56 ℃ temperature gradient rise (1℃ / min) measurement of the melting curve.

(5)The acquired data were calculated according to Eq: C copy number = 2 - (CTTarget-CTReference)

# **F Analyzing and Detecting the Products**

---

# **Materials and Instruments**

1M sodium hydroxide-methanol solution

Concentrated sulfuric acid

n-hexane

Gas chromatograph

Centrifuge tube

The centrifuge

96-well plate shaker

Vials

The visible spectrophotometer

# **Steps**

After fermentation, we extracted the total fatty acids of the engineered *Y. lipolytica* and detected the content and proportion of various fatty acids with gas chromatograph.

(1)Measure the OD600 of each conical flask by visible spectrophotometer, based on which we calculated the volume of each flask fermentation broth to be inoculated (To final OD600=4). For example, the OD600 of the first conical flask is 8.5, the volume would be 4/8.5=470 uL.

(2)According to the above method, we took the corresponding volume of the fermentation broth from each bottle into different centrifuge tubes, centrifugated at 12,000 ×g for 2 min, and discard the supernatant.

(3)Add 500 μL 1M sodium hydroxide-methanol solution in each tube, mix, and incubate in a 96-well plate shaker for 4-5h.

(4)Add 40 μL concentrated sulfuric acid to each tube, then add 500 μL hexane. Then again incubate in a 96-well plate shaker for 10 min to get fully reacted.Centrifugate at 12000 ×g for 2 min. Then absorbe the upper organic layer and injected it into a vial.

(5)Detected the content of various fatty acids with the Gas chromatograph. In the end, we collected the data and analyzed the results.

{% endblock %}

# **1. Construction of an engineered *Yarrowia lipolytica* to produce Eicosapentaenoic acid (EPA)**

---

The fatty acid profile of the *Yarrowia lipolytica* wild-type strain *Po1f* indicated that it can produce palmitic acid (C16:0, 14.9%),
palmitoleic acid (C16:1, 6.9%), stearic acid (C18:0, 5.0%), oleic acid (C18:1, 53.8%) and linoleic acid (LA, C18:2,19.3%)，which
account for more than 99 percentage in total fatty acid. However, omega-3 polyunsaturated fatty acids cannot be produced by *Po1f*
(Fig. 1). In theory, the introduction of either the Δ-6 pathway or the Δ-9 pathway into the wild-type *Y. lipolytica* *Po1f* could
produce Eicosapentaenoic acid (EPA) in the presence of desaturases and elongases (Fig. 2). However, the elongation step from
γ-linolenic acid (GLA; C18:3n-6) to dihomo-γ-linolenic acid (DGLA; C20:3n-6) was rate limiting, contributing to a large accumulation
of GLA (Xue, Z. et al., 2013). Thus, Δ-9 pathway was selected for the *Y. lipolytica* wild- type strain *Po1f* (*Ura-*, *Leu-*) to produce EPA.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-1.jpg)

Fig.1 (A) Fatty acid profile of the *Y . lipolytica* wild-type strain *Po1f* determined by Gas Chromatography
analysis. (B) Different fatty acid percentage of TFAs.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-2.jpg)

Fig. 2 Schematic diagram of the aerobic pathways for EPA biosynthesis. The native pathway in *Y. lipolytica* is
indicated with the green arrow, and the engineered ∆-9 pathway for EPA biosynthesis is indicated with the yellow arrow.

In Δ-9 pathway, four enzymes are required for the biotransformation of LA to EPA. Therefore, one copy of codon-optimized ∆-9 elongase from
*Euglena gracilis* (*EgElo9*) and one copy of ∆-8 desaturase from *Euglena gracilis*(*EgDes8*) under the control of strong promoter PTEF were
integrated into the genome of *Y. lipolytica* wild-type strain *Po1f* by non-homologous End Joining (NHEJ), yielding engineered strain *Po1f-1*.
And the percentage of eicosadienoic acid (EDA; C20: 2n-6) and DGLA were 20.8% and 8.5%, respectively (Fig. 3A-B). Furthermore, one copy of
∆-5 desaturase from *Euglena gracilis* (*EgDes5*) and one copy of ∆-17 desaturase from *Pythium aphanidermatum*(*PaDes17*) continued to be
integrated into the genome of the engineered strain *Po1f-1*, yielding engineered strain *Po1f-2*. Fig. 3A-B showed that the engineered strain
*Po1f-2* was able to produce 16.3% of arachidonic acid (ARA; C20:4 n-6) and 2.4% of EPA in the TFAs, which enabled the heterologous synthesis
of EPA in the *Po1f*.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-3.jpg)

Fig.3 (A) Different fatty acid percentage of TFAs in *Po1f-1* and *Po1f-2*. (B) Fatty acid profiles of the *Po1f-1*
and *Po1f-2* determined by Gas Chromatography analysis.

Furthermore, free fatty acids can be converted to short-chain fatty acyl-coenzyme A via the β-oxidation pathway in the peroxisome, and
deletion of the peroxisome generator peroxin-10 (*PEX10*) would disrupt the β-oxidation pathway, thereby preventing the degradation of
free fatty acids. Therefore, homologous recombination was used to knock out *PEX10* of the genome of the engineered strain *Po1f-2* to
construct the engineered strain *Po1f-3*. The GC analysis illustrated that EPA content in the TFAs was further enhanced to 4.5%, which is
about 1.88 times more than that of the engineered strain *Po1f-2* (Fig. 4).

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-4.jpg)

Fig.4 Different fatty acid percentage of TFAs in *Po1f-3*.

# **2. Screening of different sources of elongases/desaturases**

---

Fig. 1 revealed that C18:1 and C18:2 of the engineered strain *Po1f* accounted for a high percentage of TFAs, among which C18:1 accounting for
approximately more than 50% of the TFAs. Besides, Δ-12 desaturase is able to convert C18:1 to C18:2 by adding a double bond to the carbon
chain through dehydrogenation, which suggests that the activity of endogenous Δ-12 desaturase of *Y. lipolytica* *Po1f* was low (Fig. 1).
Moreover, studies have demonstrated that elongases and desaturases form different sources have different catalytic activities and substrate
specificities for fatty acid substrates, so the selection of elongases/desaturases with high substrate specificity is a significant
strategy to improve the synthesis of EPA. For example, heterologous expression of a highly substrate-specific Δ6 desaturase in *Mortierella
alpina* contributed to a remarkable 26-fold increase in EPA content (Shi, H. et al., 2016). *Po1f* was used as the starting strain and the
∆-12 desaturase from *Fusarium moniliforme* (*FmDes12*), *Coprinus cinereus* (*CcDes12*), and *Parietichytrium sp.* (*PaDes12*) were integrated at the
Intc site of its genome (Damude, H. G. et al., 2006; Ishibashi, Y. et al., 2021a; Zhang, S. et al., 2007), the engineered strains were
named *Po1f-4*, *Po1f-5* and *Po1f-6* respectively. Besides, GC analysis was used to illustrate the conversion of endogenous fatty acids
catalyzed by different ∆-12 desaturase. Fig. 6 indicated that the enzyme activity of *FmDes12* was the best, which results in C18:1
decreasing from 53.8% to 9.5%, while C18:2 reached 62.7% of TFAs. And the ratio of C18:2/C18:1 went from 0.378 to 6.62, with a conversion
rate of 81.1% for C18:1. While the conversion of *CcDes12* from C18:1 to C18:2 was 62.7% and *PaDes12* was the least effective catalyst for
only 37% conversion, so *Po1f-4* was used to perform subsequent validation of elongases/desaturases effects (Fig. 5).

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-5.jpg)

Fig.5 Different fatty acid percentage of TFAs produced by different sources of ∆-12 desaturase.

In addition to the high proportion of C18:1, C18:2 also accounts for high percentage of the TFAs, especially up to 62.7% in *Po1f-4*. To
reduce the percentage of C18:2, six different sources of ∆-9 elongase gene from *Blattella germanica* (*BgElo9*), *Emiliania huxleyi* (*EhElo9*),
*Isochrysis galbana* (*IgASE1*), *Isochrysis galbana* (*IgASE2*), *Pavlova pinguis* (*PpElo9*), and *Pavlova salina* (*PsElo9*) were selected (Juárez,
M. P., 2004; Li, M. et al., 2012; Petrie, J. R. et al., 2010; Qi, B. et al., 2002), which were reported as elongases with a preference
for C18:2 in the previous study. Similarly, *Po1f-4* was used as the starting strain and the ∆-9 desaturase above were integrated at the
Scp2 site of its genome, the engineered strains were named *Po1f-7*, *Po1f-8*, *Po1f-9*, *Po1f-10*, *Po1f-11*, and *Po1f-12* respectively. Fig. 6
indicated that the enzyme activities of *IgASE2* and *EhElo9* were the best, with 40.0% and 35.2% of C20:2 generated, respectively. Besides,
C18:2 produced by them decreased from 62.7% to 22.2% and 32.8%, and the conversion of C18:2 to C20:2 reached 63.8% and 56.1%,
respectively. *IgASE1* and *BgElo9* showed the worst enzyme activities towards C18:2. The conversion of C18:2 to C20:2 under the catalytic
action of *IgASE1* was around 26.3%, with only 16.5% of C20:2 produced. However, *Po1f-7* rarely produced C20:2, so *BgElo9* was thought to
have no preference towards C18:2 (Fig. 6). Taken together, *IgASE2* and *EhElo9* had a strong preference for C18:2 and could significantly
promote the synthesis of C20:2.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-6.jpg)

Fig.6 Different fatty acid percentage of TFAs produced by different sources of ∆-9 elongase.

In addition to exploring the substrate preferences of the two main fatty acid in *Po1f-3*, it was found that the content of C16:0 accounted
for about 15% of the TFAs of strain *Po1f-3*. Thus, C16/18 elongase from *Mortierella alpina* (*MaElo2*), *Misgurnus anguillicaudatus* (*MaElo16*),
*Parietichytrium sp.* (*PaElo16*), and *rat* (*rElo2*) were integrated at the Scp2 site of its genome (Chen, J. et al., 2018; Ishibashi,
Y. et al., 2021b; Wang, F. et al., 2019; Yazawa, H. et al., 2011), and the engineered strains were named *Po1f-13*, *Po1f-14*, *Po1f-15*, and
*Po1f-16* respectively. It was indicated that *MaElo2* and *rElo2* were the most effective catalysts for C16:0, with the C16:0 content reduced
to 10.5% and 12.4% of TFAs, respectively. It is a remarkable fact that the four C16/18 elongases also have catalytic activity for C18:1
as well because the percentage of C18:2 they produced were almost doubled, which suggested that the C16/18 elongases may have both
elongase and desaturase activities (Fig. 7).

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-7.jpg)

Fig.7 Different fatty acid percentage of TFAs produced by different sources of C16/18 elongase.

# **3. Optimization of EPA yield using push-pull strategy**

---

# **3.1 Increasing gene copy number**

In order to reduce the amounts of intermediates and increase the amount of EPA, elongases/desaturases above were randomly integrated in
the genome. Besides, the optimal copy numbers of the elongases/desaturases were detected by QPCR. Firstly, *MaElo2* was randomly
integrated into the *Po1f-3* genome because the content of C16:0 accounted for about 15% of the TFAs of strain *Po1f-3*. And it was found
that *MaElo2* with three copies (*Po1f-17*) showed the best result and EPA percentage of total fatty acids it produced was 7.3% (Fig. 8A-B).
Similarly, *Po1f-18* was achieved by integrating two copies of *FmDes12* and the proportion of EPA it obtained was 9.6% (Fig. 8A-B).

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results8.png)

Fig.8 (A) Different fatty acid percentage of TFAs produced by random integration of the best strains of
elongases/desaturases. (B) Optimal copy numbers detected by QPCR. (C) Fatty acid profiles of the *Po1f-3* and *Po1f-20* determined by
Gas Chromatography analysis.

Furthermore, *EhElo9*, *IgASE2* and *EgDes8* were randomly integrated in the genome of *Po1f-18* to obtain *Po1f-19*. It can be found that EPA
*Po1f-19* produced made up 16.3% of TFAs, and the integration of 3 copies of the *EhElo9*, 3 copies of the *IgASE2* and 2 copies of the *EgDes8*
gene in this strain was demonstrated by QPCR (Fig. 8A-B). *Po1f-20* was further randomly integrated with 2 copies of *EgDes5* and 2 copies
of *PaDes17* in the genome of the engineered strain *Po1f-19*, and EPA proportion reached 22.4% of the TFAs (Fig. 8A-B).

# **3.2 Optimizing promoters**

In addition to increasing copy numbers, regulating the strength of promoters is also a potential strategy for adjusting EPA content.
Therefore, *Po1f-3* was used as the starting strain, *EhElo9* was ultilized to verify whether the strength of promoter had an effect on the
EPA ratio. And different strong constitutive promoters including PGAP, PEXP, PYAT, PFBA in and PTEF in was used to instead of PTEF,
which was integrated at the Scp2 site of *Po1f-3* genome respectively. However, it was found that there was no significant change in fatty
acid content under different promoters (Fig. 9), which demonstrates that the promoter optimization did not have a significant effect on
EPA yield.

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results9.png)

Fig.9 Different fatty acid percentage of TFAs produced by engineered strains transferred of *EhElo9* controlled
by different promoters.

# **4. Modification of *Yarrowia lipolytica* for co-production of EPA and DHA**

---

In order to obtain an engineered strain for co-production of DHA and EPA production, *Po1f-20* was used as the starting strain. Besides,
∆-5 elongase and Δ-4 desaturase were identified to convert EPA to DHA, so ∆-5 elongase from *Thraustochytrium sp.* and Δ-4 desaturase from
*Schizochytrium sp.* were randomly integrated in the genome of *Po1f-20* to produce *Po1f-21*. The GC analysis indicated that the percentage
of EPA and DHA the engineered strain produced were 2.5% and 18.9% of TFAs respectively, indicating successful heterologous synthesis of
DHA and EPA in the *Yarrowia lipolytica* (Fig. 10).

![Pulpit rock](https://static.igem.wiki/teams/4343/wiki/results-10.jpg)

Fig.10 (A) Fatty acid profiles of the *Po1f-20* and *Po1f-21* determined by Gas Chromatography analysis.
(B) Different fatty acid percentage of TFAs in *Po1f-20* and *Po1f-21*.

# **References**

---

Chen, J., Cui, Y., Yan, J., Jiang, J., Cao, X., and Gao, J., 2018. Molecular characterization of elongase of very long-chain fatty acids
6 (elovl6) genes in *Misgurnus anguillicaudatus* and their potential roles in adaptation to cold temperature. Gene. 666, 134-144.

Damude, H. G., Zhang, H., Farrall, L., Ripp, K. G., Tomb, J. F., Hollerbach, D., and Yadav, N. S., 2006. Identification of bifunctional
Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants. Proceedings of the National Academy
of Sciences. 103, 9446-9451.

Ishibashi, Y., Goda, H., Hamaguchi, R., Sakaguchi, K., Sekiguchi, T., Ishiwata, Y., Okita, Y., Mochinaga, S., Ikeuchi, S., Mizobuchi,
T., Takao, Y., Mori, K., Tashiro, K., Okino, N., Honda, D., Hayashi, M., and Ito, M., 2021a. PUFA synthase-independent DHA synthesis
pathway in *Parietichytrium sp.* and its modification to produce EPA and n-3DPA. Commun Biol. 4, 1378.

Ishibashi, Y., Goda, H., Hamaguchi, R., Sakaguchi, K., Sekiguchi, T., Ishiwata, Y., Okita, Y., Mochinaga, S., Ikeuchi, S., Mizobuchi,
T., Takao, Y., Mori, K., Tashiro, K., Okino, N., Honda, D., Hayashi, M., and Ito, M., 2021b. PUFA synthase-independent DHA synthesis
pathway in *Parietichytrium sp.* and its modification to produce EPA and n-3DPA. Communications Biology. 4, 1378.

Juárez, M. P., 2004. Fatty acyl-CoA elongation in *Blatella germanica* integumental microsomes. Archives of Insect Biochemistry and
Physiology. 56, 170-178.

Li, M., Ou, X., Yang, X., Guo, D., Qian, X., Xing, L., and Li, M., 2012. Cloning and identification of a novel C18-Δ9 polyunsaturated
fatty acid specific elongase gene from DHA-producing *Isochrysis galbana* H29. Biotechnology and Bioprocess Engineering. 17, 22-32.

Petrie, J. R., Mackenzie, A. M., Shrestha, P., Liu, Q., Frampton, D. F., Robert, S. S., and Singh, S. P., 2010. Islation of three novel
long-chain polyunsaturated fatty acid Δ9-Elongases and the transgrntic assembly of the the entire palova salina docosahexaenoic acid
pathway in *Nicotiana benthamiana1*. Journal of Phycology. 46, 917-925.

Qi, B., Beaudoin, F., Fraser, T., Stobart, A. K., Napier, J. A., and Lazarus, C. M., 2002. Identification of a cDNA encoding a novel C18-Δ9
polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, *Isochrysis galbana 1*.
FEBS Letters. 510, 159-165.

Shi, H., Chen, H., Gu, Z., Zhang, H., Chen, W., and Chen, Y. Q., 2016. Application of a delta-6 desaturase with alpha-linolenic acid
preference on eicosapentaenoic acid production in *Mortierella alpina*. Microb Cell Fact. 15, 117.

Wang, F., Bi, Y., Diao, J., Lv, M., Cui, J., Chen, L., and Zhang, W., 2019. Metabolic engineering to enhance biosynthesis of both
docosahexaenoic acid and odd-chain fatty acids in *Schizochytrium sp.* S31. Biotechnol Biofuels. 12, 141.

Xue, Z., Sharpe, P. L., Hong, S. P., Yadav, N. S., Xie, D., Short, D. R., Damude, H. G., Rupert, R. A., Seip, J. E., Wang, J., Pollak,
D. W., Bostick, M. W., Bosak, M. D., Macool, D. J., Hollerbach, D. H., Zhang, H., Arcilla, D. M., Bledsoe, S. A., Croker, K., McCord,
E. F., Tyreus, B. D., Jackson, E. N., and Zhu, Q., 2013. Production of omega-3 eicosapentaenoic acid by metabolic engineering of
*Yarrowia lipolytica*. Nat Biotechnol. 31, 734-40.

Yazawa, H., Kamisaka, Y., Kimura, K., Yamaoka, M., and Uemura, H., 2011. Efficient accumulation of oleic acid in *Saccharomyces
cerevisiae* caused by expression of rat elongase 2 gene (*rELO2*) and its contribution to tolerance to alcohols. Appl Microbiol Biot. 91,
1593-1600.

Zhang, S., Sakuradani, E., Ito, K., and Shimizu, S., 2007. Identification of a novel bifunctional Δ12/Δ15 fatty acid desaturase from
a basidiomycete, *Coprinus cinereus* TD#822-2. FEBS Letters. 581, 315-319.