Proof of Concept
This year, NJXDF-CHN attempted to engineer the nonconventional oleaginous yeast Yarrowia lipolytica as a competitive platform host to produce 2-PE. To prove the concept and get a reliable product, we a series of experiments to prove the system is practicable. The results have almost fully convinced our concept. See more experiment designs on our Design page, and more results on our Results page.
To engineer Y. lipolytica to produce 2-PE, we first determined the detection method of 2-PE. We found that the concentrations of 2-PE can be 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 (Fig. 1). 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.
Fig. 1 2-PE measurement and quantification
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 reductases (Fig. 2). Additionally, we also investigate the performances of alcohol dehydrogenases in Y. lipolytica, and got nine alcohol dehydrogenases that are annotated by Genbank (https://www.ncbi.nlm.nih.gov/genbank/).
Fig. 2 The results of screening the phenylacetaldehyde reductase in Y. lipolytica
To identify the optimal phenylacetaldehyde reductase/alcohol dehydrogenase, we expressed these genes individually in Y. lipolytica and perform the whole-cell biocatalytic conversion of phenylacetaldehyde. As a result, we constructed 18 plasmid, 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). Furthermore, we transformed these plasmids into Y. lipolytica po1g, and obtained 18 engineering strains (Fig. 3a).
Fig. 3 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.
As a result, 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, 507.32, and 394.50 mg/L, which the highest 2-PE titer was produced by PAR4.
Fig. 4 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
To get an efficient Ehrlich pathway, we adopted the stepwise pathway engineering. Firstly, the recognized genes ylARO8 (BBa_K4297018), ylARO9 (BBa_K4297019) and ylARO10 (BBa_K4297020) 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 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 (BBa_K4297054) and pYLXP’-ylARO10-ylARO9 (BBa_K4297055), 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 (BBa_K4297056) and transformed into Y. lipolytica, obtaining strain po1g6. 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.
Fig. 5 Overexpression of the Ehrlich pathway. a) Constructing multi-genes expression vectors construction; b) The map of plasmids pYLXP’-ylARO10-ylARO8 and pYLXP’-ylARO10-ylARO9; c) The 2-PE titer, yield, and cell growth of strains po1g1, po1g2, and po1g3; d) The 2-PE titer, yield, and cell growth of strains po1g4, po1g5, and po1g6.
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). 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.
Fig. 6 Blocking the pathway of phenylacetate synthesis. 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; c) Deleting genes ALD2 and ALD3 in po1fk; d) The 2-PE titer, yield, 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.
Fig. 7 The 2-PE titer and cell growth of strain po1fk3 under adding the 2-oxoglutarate.
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.