Design
2-Phenylethanol (2-PE), an aliphatic alcohol with a pleasant rose aroma is considered to be an important fragrance ingredient used in the food and cosmetic industries due to its mild, warm, and rose-honey like odor (Wang et al., 2019). It also can be used as the substrate for synthesizing other flavors or pharmaceutical compounds, such as 2-phenylethylacetate (2-PEAc) and phenylacetaldehyde (PA). Currently, the global market output of 2-PE is in excess of 10,000 tons annually. In nature, 2-PE is mainly extracted from the essential oil of flowers and plant tissues, such as rose, jasmine, tomato, and buckwheat (Qian et al., 2018). However, the extraction process is very complicated and costly, because the 2-PE concentration in these plants is very low. Besides, the harvest of flowers is greatly influenced by the weather, plant diseases, and trade restrictions, which lead to the short supply and exorbitant cost of natural 2-PE. In addition, 2-PE can be obtained by chemically synthesized through ethylene oxidation of benzene or reduction of styrene oxide. However, chemical synthesis processes are generally operated under harsh conditions, such as high temperature, high pressure, and strong acid or alkali environments, causing many undesirable by-products, such as ethylbenzene and styrene, which not only increase the downstream costs, but also seriously debase the grade of 2-PE.
Fig. 1 Using roses to make perfume
The increasing demand for environmental friendly processes and the preference for “natural” products for consumers have considerably stimulated the development of biological production processes for flavors and fragrances (Noda & Kondo, 2017). Therefore, attention has turned to the bioproduction of 2-PE. Specifically, a number of microorganisms synthesize 2-PE naturally at low concentration as a communication molecule. In wetlab, we attempted to engineer the nonconventional oleaginous yeast Yarrowia 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.
Herein, Y. lipolytica was chosen as the host strain because of its strong acetyl-CoA flux and high TCA metabolic activity, as demonstrated by its superior performance for production of advanced biofuels and oleochemicals. In addition, Y. lipolytica is also a “Generally Regarded As Safe” (GRAS) organism in the food and nutraceutical industry (Larroude et al., 2018). Its genome has been sequenced and annotated, metabolic pathways have been extensively studied, and many genetic engineering tools have been developed. Moreover, Y. lipolytica can utilize a variety of inexpensive renewable substrates as carbon sources and can accommodate high flux of acetyl-CoA (Markham & Alper, 2018). These characteristics make Y. lipolytica a remarkable industrial host for the production of many products, such as α-farnesene, carotenoids, linalool, and others.
Fig. 2 Macroscopic appearance of Y. lipolytica colonies Colonies of Y. lipolytica Po1f strain grown on (A) yeast-peptone-glucose (YPD) solid medium at 30 °C for 4 days, showing the convoluted white surface, (B) YPD at 30 °C for 2 days, showing the pale matte surface (C) YPD at 30 °C for 2 days then kept at 4 °C for 1 week, showing the smooth surface.
The E. coli DH5α, lacking the immune mechanism, is usually used to geneticly construct and enrich recombinant plasmids. Therefore, we selected E. coli DH5α to construct recombinant plasmids, which is one of the most widely used genetic engineering bacteria (Wong et al., 2017). Specifically, E. coli DH5α high efficiency competent cells were obtained from NEB.
The plasmid pYLXP’ (BBa_K4297032) was selected as the expression vector for genetic expression of genes in our project, which is a shuttle plasmid between E. coli and Y. lipolytica. The process of plasmid constructions also has been reported8. For example, recombinant plasmids of pYLXP’-ylARO8 (BBa_K4297051) were built by Golden Gate assembly of linearized pYLXP’ and the appropriate DNA fragments PCR-amplified from the genome of Y. lipolytica. Moreover, multi-genes expression plasmids were constructed based on subcloning with using isocaudamers AvrII and NheI. All genes were respectively expressed by the TEF promoter (BBa_K4297021) with intron sequence and XPR2 terminator (BBa_K4297022), and the modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China).
Fig. 3 The map of plasmid pYLXP'
In Y. lipolytica, production of 2-PE is mainly obtained by two routes, namely, the de novo pathway from glucose and bioconversion from L-phenylalanine by the Ehrlich pathway. Owing to the hard-wired, tightly complex feedback regulation33 and lengthy reaction steps (>20 steps) of the de novo pathway, bioconversion by the Ehrlich pathway is considered as the preferred biological route to synthesize 2-PE.
In the Ehrlich pathway (Fig. 1), L-phenylalanine is converted to 2-PE through three enzymatic dependent processes: (i) L-phenylalanine is transaminated to phenylpyruvate by amino transferase with 2-oxoglutarate (aKG) as the amine-receptor; (ii) phenylpyruvate is further decarboxylated to phenylacetaldehyde by phenylpyruvate decarboxylases; (iii) and finally, phenylacetaldehyde is reduced to 2-PE by phenylacetaldehyde alcohol dehydrogenases with NADH as cofactor. However, in Y. lipolytica, transaminases and the phenylpyruvate decarboxylase are ylARO8 (encoded by gene YALI0E20977g) or ylARO9 (encoded by gene YALI0C05258g) and ylARO10 (encoded by gene YALI0D06930g), respectively. However, the specific phenylacetaldehyde alcohol dehydrogenase has not been characterized. Therefore, we plan to identify the specific phenylacetaldehyde alcohol dehydrogenases in Y. lipolytica, and further improve the performances of 2-PE production produced by Y. lipolytica.
Fig. 4 The synthesis pathway of 2-PE in Y. lipolytica. L-phe, L-phenylalanine; PPY, phenylpyruvate; PAH, phenylacetaldehyde; 2-PE, 2-phenylethanol; ylARO8, the transaminase; ylARO9, the transaminase; ylARO10, the phenylpyruvate decarboxylase.
Our biobricks design is mainly divided into three parts: screening the specific phenylacetaldehyde dehydrogenase, overexpressing Ehrlich pathway, and blocking competing pathways.
Fig. 5 Optimizing the Ehrlich pathway and intracellular metabolic networks to effectively producing 2‑PE. L-phe, L-phenylalanine; PPY, phenylpyruvate; PAH, phenylacetaldehyde; 2-PE, 2-phenylethanol; ylARO8, the transaminase; ylARO9, the transaminase; ylARO10, the phenylpyruvate decarboxylase. PEP, phosphoenolpyruvate; E4P, erythrose-4P; DAHP, 3-deoxy-arabino-heptulonate-7-phosphate.
To refactor the optimal alcohol dehydrogenase (ADH) or phenylacetaldehyde reductase (PAR), we screened the entire genome of Y. lipolytica with putative rose phenylacetaldehyde reductase PARL (BBa_K4297017) as the template, and identified eight PARs with high similarity (>70%), encoded 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), respectively. Similarly, we also investigated the performances of alcohol dehydrogenases (ADHs) in Y. lipolytica. Nine Y. lipolytica ADHs annotated by Genbank (https://www.ncbi.nlm.nih.gov/genbank/) and GRYC (http://gryc.inra.fr/) were tested, including ADH1 (YALI0D25630g, BBa_K4297001), ADH2 (YALI0E17787g, BBa_K4297002), ADH3 (YALI0A16379g, BBa_K4297003), ADH4 (YALI0A15147g, BBa_K4297004), ADH5 (YALI0E07766g, BBa_K4297005), ADH6 (YALI0E15818g, BBa_K4297006), ADH7 (YALI0D02167g, BBa_K4297007), ADH8 (YALI1C17782g, BBa_K4297008), and ADH9 (SNF1, BBa_K4297062). Next, the candidates were tested by the whole-cell bioconversion of phenylacetaldehyde.
Fig. 6 Screening the specific phenylacetaldehyde alcohol dehydrogenase
Our goal is to synthesize 2-PE on large scale. In order to convert L-phenylalanine into 2-PE, we are going to overexpress the Ehrlich pathway to improve 2-PE titer. In Y. lipolytica, transaminases and the phenylpyruvate decarboxylase are ylARO8 (encoded by gene YALI0E20977g, BBa_K4297018) or ylARO9 (encoded by gene YALI0C05258g, BBa_K4297019) and ylARO10 (encoded by gene YALI0D06930g, BBa_K4297020), respectively. After screening the specific phenylacetaldehyde alcohol dehydrogenase, we plan to overexpress the Ehrlich pathway to improve 2-PE titer by overexpressing the Ehrlich pathway using the strongly constitutive promoter pTEF.
Fig. 7 Overexpression of Ehrlich pathway to improve 2-PE titer
To further improve 2-PE titer, we attempted to delete the competing pathways. The major byproduct is phenylacetate, which is the oxidized product of phenylacetaldehyde catalyzed by aldehyde dehydrogenases (ALD2 and ALD3). Thus, we sequentially knocked out both ALD2 (YALI0D07942g, BBa_K4297060) and ALD3 (YALI0F04444g, BBa_K4297061) in po1fk.
Fig. 8 Blocking competing pathways to improve 2-PE titer by the Cre/loxp method
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