iGEM 2022 — Team njxdf-chn

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## Inspirations

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* [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)

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# Engineering Success

Overview

    To achieve the production of 2-PE, we firstly characterized and identified the optimal phenylacetaldehyde reductase (PAR) by screening the entire genome of Y. lipolytica. Next, we refactored the Ehrlich Pathway to improve 2-PE production. To further improve 2-PE yield, we attempted to delete the competing pathways.

Cycle 1: Screening and identified the optimal phenylacetaldehyde reductase

#### Design

    To obtain an optimized phenylacetaldehyde reductase, we firstly screened genome of Y. lipolytica by using amino sequences of the rose phenylacetaldehyde reductase PARL, and got eight putative phenylacetaldehyde reductases, encoding by YALI0D08844g (PAR1, BBa\_K4297009), YALI0F09097g (PAR2, BBa\_K4297010), YALI0F24937g (PAR3, BBa\_K4297011), YALI0D07062g (PAR4, BBa\_K4297012), YALI0D12386g (PAR5, BBa\_K4297013), YALI0C20251g (PAR6, BBa\_K4297014), YALI0D11616g (PAR7, BBa\_K4297015), and YALI0D08778g (PAR8, BBa\_K4297016). Moreover, we also synthesized the rose phenylacetaldehyde reductase gene PARL (BBa\_K4297017) with the codon optimization. It has been reported that alcohol dehydrogenases could perform the similar function as the phenylacetaldehyde reductase. Therefore, we also investigate the performances of alcohol dehydrogenases in Y. lipolytica. By screening Genbank (https://www.ncbi.nlm.nih.gov/genbank/), we got eight alcohol dehydrogenases, namely YALI0D25630g (ADH1, BBa\_K4297001), YALI0E17787g (ADH2, BBa\_K4297002), YALI0A16379g (ADH3, BBa\_K4297003), YALI0A15147g (ADH4, BBa\_K4297004), YALI0E07766g (ADH5, BBa\_K4297005), YALI0E15818g (ADH6, BBa\_K4297006), YALI0D02167g (ADH7, BBa\_K4297007), and YALI1C17782g (ADH8, BBa\_K4297008).

![](https://static.igem.wiki/teams/4297/wiki/es/es1.jpg)

Fig. 1 The results of screening the phenylacetaldehyde reductase in Y. lipolytica

#### Build

    To identify the optimal phenylacetaldehyde reductase/alcohol dehydrogenase, we need to express these genes individually in Y. lipolytica and perform the whole-cell biocatalytic conversion of phenylacetaldehyde. For this, seventeen plasmids need to be constructed, including pYXLP’-PAR1 (BBa\_K4297042), pYXLP’-PAR2 (BBa\_K4297043), pYXLP’-PAR3 (BBa\_K4297044), pYXLP’-PAR4 (BBa\_K4297045), pYXLP’-PAR5 (BBa\_K4297046), pYXLP’-PAR6 (BBa\_K4297047), pYXLP’-PAR7 (BBa\_K4297048), pYXLP’-PAR8 (BBa\_K4297049), pYXLP’-PARL (BBa\_K4297050), pYXLP’-ADH1 (BBa\_K4297034), pYXLP’-ADH2 (BBa\_K4297035), pYXLP’-ADH3 (BBa\_K4297036), pYXLP’-ADH4 (BBa\_K4297037), pYXLP’-ADH5 (BBa\_K4297038), pYXLP’-ADH6 (BBa\_K4297039), pYXLP’-ADH7 (BBa\_K4297040), pYXLP’-ADH8 (BBa\_K4297041), and pYXLP’-ADH9 (BBa\_K4297063). Here, we take the process of constructing pYXLP’-PAR1 (BBa\_K4297042) as an example: i) firstly, the DNA fragment of PAR1 (BBa\_K4297009) was PCR-amplified by primers PAR1\_F and PAR1\_R; ii) next, plasmid pYLXP’ (BBa\_K4297032) was digested by the nuclease BsaI to get the linearized pYLXP’; iii) then, pYXLP’-PAR1 was obtained by Golden Gate Assembly of linearized pYLXP’ and the PCR-amplified PAR1 fragment; iv) the reaction mixture of Golden Gate Assembly was transformed to E. coli DH5α; v) corrected colonies were verified by PCR and sequenced by Sangon Biotech (Shanghai, China).

![](https://static.igem.wiki/teams/4297/wiki/es/es2.jpg)

Fig. 2 Constructing expression vectors of phenylacetaldehyde reductases/alcohol dehydrogenases.a) The process of Golden Gate Assembly; b) The colony of plasmid pYLXP’-PAR1 transformed into E. coli DH5α; c) Corrected colonies were verified by PCR; d) The map of plasmid pYLXP’-PAR1; e) The sequenced result of plasmids from Sangon Biotech.

Table 1. Primers used for constructing expression vectors

![](https://static.igem.wiki/teams/4297/wiki/es/es-table-1.jpg)

#### Test

    Next, we transformed plasmids into Y. lipolytica and perform the whole-cell conversion of phenylacetaldehyde. For transforming plasmids into Y. lipolytica, we used the lithium acetate method, which has been described as previously reported. In brief, one milliliter cells was harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 uL transformation solution, containing 90 uL 50% PEG4000, 5 uL lithium acetate (2M), 5 uL boiled single stand DNA (salmon sperm, denatured) and 5 uL DNA products (including 200-500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39 oC for 1 h, then spread on selected plates. It should be noted that the transformation mixtures needed to be vortexed for 15 seconds every 15 minutes during the process of 39 oC incubation. The selected marker is leucine in our project. As a result, we successfully transformed plasmids into Y. lipolytica and obtained engineering strains po1g pYLXP’-PAR1, po1g pYLXP’-PAR2, po1g pYLXP’-PAR3, po1g pYLXP’-PAR4, po1g pYLXP’-PAR5, po1g pYLXP’-PAR6, po1g pYLXP’-PAR7, po1g pYLXP’-PAR8, po1g pYLXP’-PARL, po1g pYLXP’-ADH1, po1g pYLXP’-ADH2, po1g pYLXP’-ADH3, po1g pYLXP’-ADH4, po1g pYLXP’-ADH5, po1g pYLXP’-ADH6, po1g pYLXP’-ADH7, and po1g pYLXP’-ADH8.

    For prepare the whole-cell biocatalyst, cells were harvested during the exponential growth phase (48 h) from the shake flask cultivation. Then, cells were washed twice with 100 mM phosphate buffer (pH 7.0), and resuspended to an OD600 of 4 in the same buffer. Next, whole-cell biocatalytic conversion of phenylacetaldehyde was performed in 20-ml glass tube containing 1 mL of cell suspension (OD600=4) and 1 mL phenylacetaldehyde-water solution (2 g/L phenylacetaldehyde) at 30 oC and 250 r.p.m. for 4 h. One hundred microliter of cell suspension was sampled every 1 h for 2-PE and penylacetate measurements. The whole-cell biocatalytic conversion of 1 g/L phenylacetaldehyde by aforementioned strains gave 2-PE titers of 762.71, 703.62, 759.37, 786.58, 435.61, 437.33, 399.11, 501.10, 379.28, 509.34, 723.74, 617.95, 413.53, 389.78, 430.07, 394.16, 507.33, and 394.50 mg/L, thus indicating that PAR4 is the optimal for 2-PE production in Y. lipolytica.

    We successfully transformed plasmids into Y. lipolytica and obtained engineering strains po1g pYLXP’-PAR1, po1g pYLXP’-PAR2, po1g pYLXP’-PAR3, po1g pYLXP’-PAR4, po1g pYLXP’-PAR5, po1g pYLXP’-PAR6, po1g pYLXP’-PAR7, po1g pYLXP’-PAR8, po1g pYLXP’-PARL, po1g pYLXP’-ADH1, po1g pYLXP’-ADH2, po1g pYLXP’-ADH3, po1g pYLXP’-ADH4, po1g pYLXP’-ADH5, po1g pYLXP’-ADH6, po1g pYLXP’-ADH7, and po1g pYLXP’-ADH8. The whole-cell biocatalytic conversion of 1 g/L phenylacetaldehyde by these strains gave 2-PE titers of 762.71, 703.62, 759.37, 786.58, 435.61, 437.33, 399.11, 501.10, 379.28, 509.34, 723.74, 617.95, 413.53, 389.78, 430.07, 394.16, 507.33, and 394.50 mg/L, thus indicating that PAR4 is the optimal for 2-PE production in Y. lipolytica.

![](https://static.igem.wiki/teams/4297/wiki/es/es3.jpg)

Fig. 3 Identifying the optimal phenylacetaldehyde reductase/alcohol dehydrogenase. a) The morphology analysis of engineering strains; b) Performing the whole-cell biocatalytic conversion; c) The 2-PE titer of the whole-cell biocatalytic conversion

#### Learn

    Specifically, the prominent bioconversion performances and relatively few byproducts generation are the typical characteristics of high rigidity metabolism pathway (Larroude et al., 2018; Markham & Alper, 2018; Wang et al., 2020). Therefore, the critical problems in this work are to improve Ehrlich pathway efficiency and reduce byproducts generation.

Cycle 2: Overexpressing the Ehrlich pathway

#### Design

    L-phenylalanine will be converted to 2-PE through four enzyme-dependent processes by the Ehrlich pathway: (i) L-phenylalanine is transaminated to phenylpyruvate by transaminases with 2-oxoglutarate as the amine-receptor; (ii) phenylpyruvate is further converted to phenylacetaldehyde by phenylpyruvate decarboxylases; (iii) and finally, phenylacetaldehyde is reduced to 2-PE by alcohol dehydrogenases or phenylacetaldehyde reductase with consuming one molecule NADH. In Y. lipolytica, the transaminases and the phenylpyruvate decarboxylase are yliARO8 (encoded by gene YALI0E20977g) or ylARO9 (encoded by gene YALI0C05258g) and ylARO10 (encoded by gene YALI0D06930g), respectively. To get an efficient Ehrlich pathway, we adopted the stepwise pathway engineering.

#### Build

    To get an efficient Ehrlich pathway, we successfully constructed six plasmids, including pYLXP’-ylARO10 (BBa\_K4297053), pYLXP’-ylARO8 (BBa\_K4297051), pYLXP’-ylARO9 (BBa\_K4297052), pYLXP’-ylARO10-ylARO8 (BBa\_K4297054), pYLXP’-ylARO10-ylARO9 (BBa\_K4297055), and pYLXP’-ylARO10-ylARO8-PAR4 (BBa\_K4297056). Further, we transformed these plasmids into po1g and obtained po1g1, po1g2, po1g3, po1g4, po1g5, and po1g6.

![](https://static.igem.wiki/teams/4297/wiki/es/es4.jpg)

Fig. 4 The method of constructing the plasmid to overexpress the Ehrlich pathway. a) Constructing multi-genes expression vectors construction; b) The map of plasmids pYLXP’-ylARO10-ylARO8 and pYLXP’-ylARO10-ylARO9

#### Test

    Firstly, the recognized genes ylARO8 (BBa\_K4297018), ylARO9 (BBa\_K4297019) and ylARO10 in Ehrlich pathway were overexpressed in Y. lipolytica po1g under the control of strong constitutive promoter pTEP with intron via plasmid pYLXP’, respectively. Shaking flask results showed that overexpression of genes ylARO8 and ylARO9 (strain po1g1 and po1g2) has no effect on the 2-PE production. However, overexpression of gene ylARO10 (strain po1g3) has a remarkable increase of 2-PE titer, which reached 922.86 mg/L and is the 1.33-fold of the control. Further, we constructed plasmids pYLXP’-ylARO10-ylARO8 (BBa\_K4297054) and pYLXP’-ylARO10-ylARO9 (BBa\_K4297055), and transformed into po1g, obtaining strains po1g4 and po1g5. However, shake flask results showed no significant differences in 2-PE production between these two strains. In addition, we constructed the plasmid pYLXP’-ylARO10-ylARO8-PAR4 (BBa\_K4297056) and transformed this plasmid into po1g, obtaining strain po1g6. As a result, overexpression of Ehrlich pathway has a remarkable increase of 2-PE titer, which reached 1086.86 mg/L and is the 1.17-fold of the control.

![](https://static.igem.wiki/teams/4297/wiki/es/es5.jpg)

Fig. 5 Overexpressing the Ehrlich pathway. a) The 2-PE titer, yield, and cell growth of strains po1g1, po1g2, and po1g3; b) The colony of po1g6; c) The 2-PE titer, yield, and cell growth of strains po1g4, po1g5, and po1g6

#### Learn

    The results of shaking flask strain po1g1, po1g2 and po1g3 strongly suggested that the reaction catalyzed by phenylpyruvate decarboxylase ylARO10 is a limiting-step in Ehrlich pathway. Moreover, YlARO8 and ylARO9 are both transaminases, which catalyze the transamination of L-phenylalanine with different amine-receptors, including 2-oxoglutarate and pyruvate. Therefore, it is necessary to identify the optimized transaminases. We constructed plasmids pYLXP’-ylARO10-ylARO8 (BBa\_K4297054) and pYLXP’-ylARO10-ylARO9 (BBa\_K4297055), and transformed into po1g, obtaining strains po1g4 and po1g5. However, shake flask results showed no significant differences in 2-PE production between these two strains. On the basis of these results, we speculated that Y. lipolytica has the obstacle in catalyzing phenylacetaldehyde to 2-PE. Therefore, we overexpressed the obtained PAR4 by constructing pYLXP’-ylARO10-ylARO8-PAR4 (BBa\_K4297056). In addition, as suggested by the mathematical model, we focus on experimentally enhancing pathway rigidity. Next, to further increase the 2-PE production, we turn to block competitive pathways.

Cycle 3: Enhancing the production of 2-PE by blocking competitive pathways

#### Design

    To further improving the production of 2-PE, we turned to remove the competitive metabolic pathways. For the convenience of the genetic manipulations, we used stain po1f (the derivative of po1g, auxotroph of leucine and uracil) for following manipulations. Specifically, the main byproduct was phenylacetate, which was synthesized by aldehyde dehydrogenases ALD2 and ALD3 with phenylacetaldehyde as the precursor and generation of one mole NADH. Thus, we orderly knocked out genes ALD2 (YALI0D07942g) and ALD3 (YALI0F04444g) in po1fk.

#### Build

    A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported (Lv et al., 2020; Lv et al., 2019). Here, we take the process of constructing the gene knockout vectors pYXLP’-loxp-uras-ALD2 as an example. For performing gene knockout, the upstream and downstream sequences (both 1000 bp) flanking ALD2 were PCR-amplified. These two fragments, the loxP-Ura/Hyr-loxP cassette (digested from plasmid pYLXP’-loxP-Ura/Hyr by BasI), and the residual plasmid backbone of pYLXP’-loxP-Ura/Hyr were joined by Golden Gate assembly, obtaining the knockout plasmids pYXLP’-loxp-uras-ALD2. The obtained vector was sequenced by Sangon Biotech (Shanghai, China).

![](https://static.igem.wiki/teams/4297/wiki/es/es6.jpg)

Fig. 6 Constructing the gene knockout vectors.a) The map of plasmids pYXLP’-loxp-uras-ALD2 (BBa\_K4297057) and pYXLP’-loxp-uras-ALD3 (BBa\_K4297058); b) The results of sequencing plasmids pYXLP’-loxp-uras-ALD2 and pYXLP’-loxp-uras-ALD3

Table2 Primers used for constructing the gene knockout vectors

![](https://static.igem.wiki/teams/4297/wiki/es/es-table-2.jpg)

#### Test

    The gene knockout cassettes were PCR-amplified from plasmids pYXLP’-loxp-uras-ALD2 (BBa\_K4297057) and pYXLP’-loxp-uras-ALD3 (BBa\_K4297058), and further transformed into Y. lipolytica. The positive transformants were determined by colony PCR. Next, plasmid pYLXP’-Cre (BBa\_K4297059) was introduced into the positive transformants and promoted the recombination of loxP sites, which recycle the selected marker. Finally, the intracellular plasmid pYLXP’-Cre was evicted by incubation at 30 oC for 48h. Here, Ura means the uracil marker. We successfully deleted genes ALD2 and ALD3, and obtained strain po1fk3. Next, we performed the shaking flask. For performing shake flask cultivations, seed culture was carried out in the shaking tube with 2 mL seed culture medium at 30 oC and 250 r.p.m. for 48 h. Then, 0.8 mL of seed culture was inoculated into the 250 mL flask containing 35 mL of fermentation medium and grown under the conditions of 30 oC and 250 r.p.m. for 144 h. One milliliter of cell suspension was sampled every 24 h for OD600, glucose, 2-PE, L-phenylalanine, and penylacetate measurements. Moreover, seed culture medium used in this study was the yeast complete synthetic media regular media CSM containing: glucose 20.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 5.0 g/L, and CSM-Leu or CSM-Ura 0.74 g/L. The nitrogen-limited media CSM contained: glucose 40.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 1.1 g/L, CSM-Leu 0.74 g/L, and appropriate L-phenylalanine. As a result, shaking flask of po1fk3 showed an increased 2-PE titer, which was 1846.49 mg/L, 1.70-fold of the control.

![](https://static.igem.wiki/teams/4297/wiki/es/es7.jpg)

Fig. 7 Blocking the pathway of phenylacetate synthesis. a) Deleting genes ALD2 and ALD3 in po1fk; b) The 2-PE titer, yield, and cell growth of strain po1fk3

#### Learn

    Further investigations on relieving the 2-PE toxicity and promoting cell growth could be performed for improving 2-PE production. Major considerations include increasing the 2-PE tolerance of Y. lipolytica, optimizing the medium composition, and ameliorating fermentation processes. In general, mutagenesis method with appropriate screening technique is an efficient and convenient way to improve tolerance of engineering strains. In addition, using small amounts of organic nitrogen sources, such as peptone and yeast extract, is conducive to cell growth without inhibiting the Ehrlich pathway.

#### References

    Larroude, M., Rossignol, T., Nicaud, J.M., Ledesma-Amaro, R. 2018. Synthetic biology tools for engineering Yarrowia lipolytica. Biotechnol Adv, 36(8), 2150-2164.

    Lv, X., Wu, Y., Tian, R., Gu, Y., Liu, Y., Li, J., Du, G., Ledesma-Amaro, R., Liu, L. 2020. Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control. Metab Eng, 59, 106-118.

    Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.

    Markham, K.A., Alper, H.S. 2018. Synthetic Biology Expands the Industrial Potential of Yarrowia lipolytica. Trends Biotechnol, 36(10), 1085-1095.

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# Experiments

Overview

    In this project, we attempted to engineer the nonconventional oleaginous yeast Yarrowia lipolytica (Y. lipolytica) as a competitive platform host to produce 2-PE. As a result, the heterologous production of 2-PE from Y. lipolytica is considered as an economically viable alternative to plant extraction.

#### 1. Conditions of shaking flask

    All engineering strains were performed the optimized screening before shake flask cultivations. For performing shake flask cultivations, seed culture was carried out in the shaking tube with 2 mL seed culture medium at 30 oC and 250 r.p.m. for 48 h. Then, 0.8 mL of seed culture was inoculated into the 250 mL flask containing 35 mL of fermentation medium and grown under the conditions of 30 oC and 250 r.p.m. for 144 h. One milliliter of cell suspension was sampled every 24h for OD600, glucose, 2-PE, L-phenylalanine, and penylacetate measurements.

    Seed culture medium used in this study was the yeast complete synthetic media regular media CSM containing: glucose 20.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 5.0 g/L, and CSM-Leu or CSM-Ura 0.74 g/L. Two types of fermentation medium were used in our work includes nitrogen-limited media CSM and YNB. The nitrogen-limited media CSM contained: glucose 40.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, ammonium sulfate 1.1 g/L, CSM-Leu 0.74 g/L, and appropriate L-phenylalanine. The nitrogen-limited media YNB contained: glucose 40.0 g/L, yeast nitrogen base (without ammonium sulfate) 1.7 g/L, leucine or uracil 0.2 g/L, and appropriate L-phenylalanine.

#### 2. The whole-cell biocatalytic conversion of phenylacetaldehyde

    For prepare the whole-cell biocatalyst, cells were harvested during the exponential growth phase (48 h) from the shake flask cultivation. Then, cells were washed twice with 100 mM phosphate buffer (pH 7.0), and resuspended to an OD600 of 4 in the same buffer. Next, whole-cell biocatalytic conversion of phenylacetaldehyde was performed in 20-ml glass tube containing 1 mL of cell suspension (OD600=4) and 1 mL phenylacetaldehyde-water solution (2 g/L phenylacetaldehyde) at 30 oC and 250 r.p.m. for 4 h. One hundred microliter of cell suspension was sampled every 1 h for 2-PE and penylacetate measurements.

#### 3. Yeast transformation and optimized screening of high-producing strains

    The standard protocols of Y. lipolytica transformation by the lithium acetate method were described as previously reported (Liu et al., 2019; Lv et al., 2019). In brief, one milliliter cells was harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 uL transformation solution, containing 90 uL 50% PEG4000, 5 uL lithium acetate (2M), 5 uL boiled single stand DNA (salmon sperm, denatured) and 5 uL DNA products (including 200-500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39 oC for 1 h, then spread on selected plates. It should be noted that the transformation mixtures needed to be vortexed for 15 seconds every 15 minutes during the process of 39 oC incubation. The selected markers, including leucine and uracil, were used in this study. All engineering strains after genetic manipulations were performed optimized screening by the shaking tube cultivations, and the optimal strain was used to perform shaking flask (these data have been shown in Supplementary Materials).

#### 4. Expression vectors construction and pathway assembly

    The YaliBrick plasmid pYLXP’ was used as the expression vector in this study(Wong et al., 2017). Plasmid constructions were performed by using preciously described methods(Lv et al., 2019). In brief, recombinant plasmids of pYLXP’-XX (a single gene expression) were obtained by Golden Gate method using linearized pYLXP’ and the appropriate PCR-amplified DNA fragment. Multi-genes assembly was achieved by restriction enzyme digestion subcloning based on the application of isocaudamers AvrII and NheI(Wong et al., 2017). All genes were respectively expressed by the TEF promoter with intron sequence and XPR terminator. The modified DNA fragments and plasmids were sequenced by Quintarabio. The endonucleases used in this research were purchased from Thermo Fisher Scientific or NEB.

#### 5. Gene knockout

    A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported(Fickers et al., 2003). For performing gene knockout, the upstream and downstream sequences (both 1000 bp) flanking the deletion targets were PCR-amplified. These two fragments, the loxP-URA-loxP cassette (digested from plasmid pYLXP’-loxp-URA by by BasI), and the residual plasmid backbone of pYLXP’-loxp-URA were ligase, obtaining the gene knockout plasmids pYLXP’-loxP-URA-XX. The obtained plasmids were sequenced by Sangon Biotech Co., Ltd (Shanghai, China). Next, the gene knockout cassettes were PCR-amplified from construction plasmids pYLXP’-loxp-URA-XX, and further transformed into Y. lipolytica. The positive transformants were determined by colony PCR. Next, plasmid pYLXP’-Cre was introduced into the positive transformants and promoted the recombination of loxP sites, which recycle the selected marker. Finally, the intracellular plasmid pYXLXP’-Cre was evicted by incubation at 30 oC for 48h.

#### 6. Quantification of cell density, 2-PE, penylacetate, L-phenylalanine

    Cell densities were monitored by measuring the optical density at 600 nm (OD600). The concentrations of 2-PE, penylacetate, and L-phenylalanine were measured by high-performance liquid chromatography (HPLC) through Agilent HPLC 1220 equipped with a ZORBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μm, Agilent) and a VWD detector. The analysis was performed at 215 nm under 40 oC column temperature with a mobile phase comprising 50% (v/v) methanol in water at a flow rate of 0.5 mL/min.

#### References

    Fickers, P., Le Dall, M.T., Gaillardin, C., Thonart, P., Nicaud, J.M. 2003. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods, 55(3), 727-37.

    Liu, H., Marsafari, M., Wang, F., Deng, L., Xu, P. 2019. Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica. Metab Eng, 56, 60-68.

    Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.

    Wong, L., Engel, J., Jin, E., Holdridge, B., Xu, P. 2017. YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metab Eng Commun, 5, 68-77.

    Xu, P., Li, L.Y., Zhang, F.M., Stephanopoulos, G., Koffas, M. 2014. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. PNAs, 111(31), 11299-11304.

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# Results

1. Screening and identified the optimal phenylacetaldehyde reductase

#### What work we have done:

    Screening the phenylacetaldehyde reductases/alcohol dehydrogenases in Y. lipolytica and constructing their expression vectors (please see the part of engineering success).

    Identifying the optimal phenylacetaldehyde reductase PAR4 by performing the whole-cell biocatalyst (please see the part of engineering success).

#### Results:

    To obtain an optimized alcohol dehydrogenase or phenylacetaldehyde reductase, we firstly screened genome of Y. lipolytica by using amino sequences of the rose phenylacetaldehyde reductase PARL, and got eight putative phenylacetaldehyde reductase, which encoding genes include YALI0D08844g (PAR1, BBa\_K4297009), YALI0F09097g (PAR2, BBa\_K4297010), YALI0F24937g (PAR3, BBa\_K4297011), YALI0D07062g (PAR4, BBa\_K4297012), YALI0D12386g (PAR5, BBa\_K4297013), YALI0C20251g (PAR6, BBa\_K4297014), YALI0D11616g (PAR7, BBa\_K4297015), and YALI0D08778g (PAR8, BBa\_K4297016). We also synthesized the rose phenylacetaldehyde reductase gene PARL with the codon optimization. These candidates were overexpressed by plasmid pYLXP’, respectively, and transformed into Y. lipolytica po1g, obtaining strains PAR1, PAR2, PAR3, PAR4, PAR5, PAR6, PAR7, PAR8, and PARL. Whole-cell biocatalytic conversion of 1 g/L phenylacetaldehyde by these strains gave 2-PE titers of 762.71, 703.62, 759.37, 786.58, 435.61, 437.33, 399.11, 501.10, and 379.28 mg/L, which the highest 2-PE titer was produced by PAR4.

    Additionally, we also investigate the performances of alcohol dehydrogenases in Y. lipolytica. All alcohol dehydrogenases annotated by Genbank (https://www.ncbi.nlm.nih.gov/genbank/) in Y. lipolytica were respectively cloned into plasmid pYLXP’, and yield plasmids pYLXP’-ADH1 (YALI0D25630g, BBa\_K4297034), pYLXP’-ADH2 (YALI0E17787g, BBa\_K4297035), pYLXP’-ADH3 (YALI0A16379g, BBa\_K4297036), pYLXP’-ADH4 (YALI0A15147g, BBa\_K4297037), pYLXP’-ADH5 (YALI0E07766g, BBa\_K4297038), pYLXP’-ADH6 (YALI0E15818g, BBa\_K4297039), pYLXP’-ADH7 (YALI0D02167g, BBa\_K4297040), pYLXP’-ADH8 (YALI1C17782g, BBa\_K4297041) and pYLXP’-ADH9 (SFA1, BBa\_K4297062). Each plasmid was then introduced into po1g, and obtained stains (ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8 and ADH9) were performed the whole-cell biocatalytic conversion. Unexpectedly, suboptimal results of 2-PE titers were detected in these strains compared to that of overexpressing putative phenylacetaldehydes (Fig. 4d), and the highest titer of 2-PE just reached 723.74 mg/L produced by strain ADH2.

![](https://static.igem.wiki/teams/4297/wiki/results/results1.jpg)

Fig. 1 Identifying the optimal phenylacetaldehyde reductase/alcohol dehydrogenase
a) The morphology analysis of engineering strains; b) Performing the whole-cell biocatalytic conversion; c) The 2-PE titer of the whole-cell biocatalytic conversion

Table 1 Plasmids constructed for identifying the optimal phenylacetaldehyde reductase/alcohol dehydrogenase

![](https://static.igem.wiki/teams/4297/wiki/results/table-1.jpg)

2. Overexpressing the Ehrlich pathway

#### What work we have done:

    Constructing the expression vectors, including pYLXP’-ylARO10, pYLXP’-ylARO8, pYLXP’-ylARO9, pYLXP’-ylARO10-ylARO8, pYLXP’-ylARO10-ylARO9, and pYLXP’-ylARO10-ylARO9-PAR4 (please see the part of engineering success).

    Obtaining a remarkable increase of 2-PE titer by overexpressing the Ehrlich pathway, which reached 1086.86 mg/L and is the 1.17-fold of the control (please see the part of engineering success).

#### Results:

    To get an efficient Ehrlich pathway, we adopted the stepwise pathway engineering. Firstly, the recognized genes ylARO8, ylARO9 and ylARO10 in Ehrlich pathway were overexpressed in Y. lipolytica po1g under the control of strong constitutive promoter pTEP with intron via plasmid pYLXP’, respectively. Shaking flask results (Fig. 2d, e, f) showed that overexpression of genes ylARO8 and ylARO9 (strain po1gP1 and po1gP2) has no effect on the 2-PE production. However, overexpression of gene ylARO10 (strain po1gP3) has a remarkable increase of 2-PE titer, which reached 922.86 mg/L and is the 1.33-fold of the control. It strongly suggested that the reaction catalyzed by phenylpyruvate decarboxylase ylARO10 is a limiting-step in Ehrlich pathway.

    Next, we constructed plasmids pYLXP’-ylARO10-ylARO8 and pYLXP’-ylARO10-ylARO9, and transformed into po1g, obtaining strains po1gP4 and po1gP5. However, shake flask results showed no significant differences in 2-PE production between these two strains, indicating that Y. lipolytica has the obstacle in catalyzing phenylacetaldehyde to 2-PE. Therefore, we further constructed the plasmid pYLXP’-ylARO10-ylARO9-PAR4 and transformed into Y. lipolytica, obtaining strain xx. Shaking flask results showed that overexpression of Ehrlich pathway has an important effect on the 2-PE production, which reached 1086.86 mg/L and is the 1.17-fold of the control.

Table 2 Plasmids constructed for overexpressing the Ehrlich pathway

![](https://static.igem.wiki/teams/4297/wiki/results/table-2.jpg)

![](https://static.igem.wiki/teams/4297/wiki/results/results2.jpg)

Fig. 2 Overexpressing the Ehrlich pathway to increase the production of 2-PE
a) The 2-PE titer, yield, and cell growth of strains po1g1, po1g2, and po1g3; b) The colony of po1g6; c) The 2-PE titer, yield, and cell growth of strains po1g4, po1g5, and po1g6

3. Enhancing the production of 2-PE by blocking competitive pathways

#### What work we have done:

    Constructing gene knockout vectors, including pYXLP’-loxp-uras-ALD2 and pYXLP’-loxp-uras-ALD3 (please see the part of engineering success).

    Deleting gene ALD2 and ALD3 in Y. lipolytica, which increased the 2-PE production (please see the part of engineering success).

#### Results:

    To further improving the production of 2-PE, we turned to remove the competitive metabolic pathways. The main byproduct was phenylacetate, which was synthesized by aldehyde dehydrogenases ALD2 and ALD3 with phenylacetaldehyde as the precursor and generation of one mole NADH. A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported (Lv et al., 2019; Lv et al., 2020). Here, we take the process of constructing the gene knockout vectors pYXLP’-loxp-uras-ALD2 as an example. For performing gene knockout, the upstream and downstream sequences (both 1000 bp) flanking ALD2 were PCR-amplified. These two fragments, the loxP-Ura/Hyr-loxP cassette (digested from plasmid pYLXP’-loxP-Ura/Hyr by AvrII and salI), and the residual plasmid backbone of pYLXP’-loxP-Ura/Hyr were joined by Golden Gate assembly, obtaining the knockout plasmids pYXLP’-loxp-uras-ALD2. The obtained vector was sequenced by Sangon Biotech (Shanghai, China).

Table 3 Plasmids constructed for blocking competitive pathways

![](https://static.igem.wiki/teams/4297/wiki/results/table-3.jpg)

    It should be noted that we orderly knocked out genes ALD2 (YALI0D07942g, BBa\_K4297060) and ALD3 (YALI0F04444g, BBa\_K4297061) in po1fk, obtaining strain po1fk3, and transformed plasmids pYLXP’-ylARO10-ylARO8-PAR4 (BBa\_K4297056), obtaining strain po1fk3. As a result, shaking flask of po1fk3 showed an increased 2-PE titer, which was 1846.49 mg/L, 1.70-fold of the control.

![](https://static.igem.wiki/teams/4297/wiki/results/results3.jpg)

Fig. 3 Enhancing the production of 2-PE by blocking competitive pathways. a) The colony of po1g6; b) The 2-PE titer and cell growth of strain po1fk3

    Moreover, as suggested by the mathematical model, 2-PE yield is more sensitive to the supply of akG. We added the 2-oxoglutarate (5g/L) into the fermentation medium. As a result, the 2-PE titer reached to 2015.57 with the yield of 0.575 g/g.

![](https://static.igem.wiki/teams/4297/wiki/results/results4.jpg)

Fig. 4 The 2-PE titer and cell growth of strain po1fk3 under adding the 2-oxoglutarate

#### References

    Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.

    Lv, Y., Gu, Y., Xu, J., Zhou, J., Xu, P. 2020. Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield. Metab Eng, 61, 79-88.

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