Engineering Success

    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.

    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).

    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).

    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.

    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.

    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.

    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.

    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.

    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.

    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.

    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).

    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.

    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.

    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.