Phenyllactic acid

    Phenyllactic acid (PLA)[1] (C₉H₁₀O₃), also is known as 3-phenyllactic acid or β-PLA. Phenyllactic acid (PLA) is widely found in cheese, honey and other foods, and it is nontoxic for human and animal cells. PLA is a very stable and important natural small molecule organic acid [2].
    There are two isomers of phenyllactic acid is D- phenyllactic acid (D-PLA) and L- phenyllactic acid (L-PLA), while D- phenyllactic acid (D-PLA) has higher antibacterial activity.

Fig.1 Chemical structure of D-PLA

Application

    PLA is a safe compound and nontoxic for human and animals. As a new natural antibacterial substance and preservative, it can inhibit a series of gram-negative, gram-positive bacteria and fungi. As a clinical hemostatic drug, PLA can be used to prevent platelet aggregation and coronary artery expansion. In addition, PLA can also be used as a skin protectant to keep skin hydrated.

    For the PLA biosynthesis pathway, phenylalanine is converted to phenylpyruvic acid by the phenylalanine aminotransferase (Tyrb) from Escherichia coli 21B, and then phenylpyruvic acid is dehydrogenated by D-lactate dehydrogenase (D-ldh) to form phenyllactic acid (PLA).
    However, high level of D-lactate dehydrogenase expression can be obtained, while the expression levels of the other two enzymes phenylalanine aminotransferase and glutamate dehydrogenase are very low. In order to achieve higher D-PLA production, we must solve the expression balance of the D-lactate dehydrogenase, phenylalanine aminotransferase and glutamate dehydrogenase in the metabolic pathway. Therefore, we reduced the expression level of D-lactate dehydrogenase by introducing the rare arginine codons in the D-lactate dehydrogenase gene, and the results showed that the D-PLA had the highest production after 4 rare codons of Arg were introduced.

Fig.2 Schematic diagram and equation of D-PLA production system

    Since D - lactate dehydrogenase is a NADH - dependent oxidoreductase in the metabolic pathway, theoretically the cofactor NADH produced by E. coli cells cannot meet the redox reaction requirements of the over-expressed D - lactate dehydrogenase. In order to avoid the high cost of adding exogenous NADH, we designed the regeneration system of NADH that was regulated by glutamate dehydrogenase rocG from Bacillus subtilis to produce additional NADH to meet demand of the catalytic reaction of D-lactate dehydrogenase. Higher efficiency of the conversion of phenylpyruvic acid into D-PLA can be achieved by increased availability of NADH.

Fig.3 Schematic diagram and equation of D-PLA production by introducing a self-sufficient system

    The D-lactic dehydrogenase used in this project was derived from Lactobacillus bulgaricus ATCC 11842 D-ldh (BBa_K2570012), and the 52nd （Tyrosine mutates into leucine） D-lactic dehydrogenase was mutated into the hydrophobic amino acid leucine by fusing PCR, resulting in improvement of the enzymatic activity of the D-lactic dehydrogenase. The phenylalanine transaminase TyrB (BBa_K2570002) was derived from Escherichia coli 21B, and it was co-expressed with D-lactic dehydrogenase in the vector （pRB1s), and then the engineered host cells can produce D-PLA using phenylalanine as the substrate. To improve the NADH availablity of the engineered host cells, we introduced a glutamate dehydrogenase rocG (BBa_K2570013) into the cells to enhance the D-lactate dehydrogenase-dependent NADH cofactor regeneration system. Then the improved NADH regeneration can be achieved when the co-expression of rocG , D-lactate dehydrogenase D-ldh and phenylalanine aminotransferase Tyrb were realized.
    Details can be found on our Result page

2-Phenylethanol

    2-Phenylethanol (2-phenylethanol, 2-PE) is one of the most important perfume, which has a rose-like quietly elegant, delicate and persistent aroma. 2-PE was first discovered as a characteristic aroma compound in roses. Among the essential oils of Damascus rose, 2-PE accounts for more than 60% of its total volatile matter content. 2-PE is popular and loved by its aromatic aroma, making 2-PE the most widely used perfume compound in the perfume and cosmetic industries.
    In addition, 2-PE is widely used as a flavor in the food industry, such as beverages, bread, biscuits, chewing gum, and the like. Hence, 2-PE is widely used as a main ingredient in rose scent flavors in the perfume, cosmetics and food industries.

Fig.4 Chemical structure of 2-Phenylethanol

    Simultaneously, due to the long fermentation cycle of the yeast, the substrate conversion rate is low, and 2-PE as a bactericide can inhibit yeast growth, and it is difficult to accumulate a higher concentration of 2-PE in yeast fermentation.^[3]
    Hence, we used E. coli tnaA gene deletion strain as our engineering strain to construct E. coli whole-cell catalyst biosynthesis 2-PE, which can effectively separate cell growth and 2-PE synthesis process by whole cell catalysis. To avoid the inhibition of cell growth by 2-PE, it can effectively avoid the inhibition of cell growth by 2-PE.

Application

    As a rose-like fragrance material, 2-PE is traditionally extracted from roses，but the yield is usually low. Nowadays, 2-PE is produced mainly by chemical synthesis, but it also has many disadvantages such as being environmentally unfriendly and producing unwanted byproducts. To effectively avoid the problems mentioned above, our project is planned to produce 2-PE through biosynthesis. Bio-technologically produced flavors are currently considered as natural by European and U.S. food agencies, which also proves the feasibility and safety of our project.
    Moreover, bio-technological production of 2-PE is highly desired in many fields and holds promise to be the most commercially viable route to produce 2-PE. The main method of biosynthesis 2-PE is to synthesize 2-PE by Ehrlich pathway using L-phenylalanine (L-Phe) as a substrate.

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The pathway of production

    A heterologous Ehrlich pathway was constructed for 2-PE bio-synthesis in E. coli by co-overexpressing the aromatic transaminase from E. coli (TyrB, WP032305522.1), phenylpyruvate decarboxylase from S. cerevisiae (Aro10, NP 010668.3) and dehydrogenation of reductase from Rose (PAR, BAG 13450.2).
  The substrate phenylalanine is converted to phenylpyruvate by transamination of tyrosine aminotransferase (TyrB), followed by decarboxylation of phenylpyruvate decarboxylase (Aro10) to form phenylacetaldehyde, and finally by phenylacetaldehyde. Dehydrogenation of reductase (PAR) produces 2-PE. The synthesis of 2-PE was achieved, and the concentration of L-Phe, 2-PE was measured by HPLC.
    The overall stoichio-metric equation of the reactions in this biosynthetic system is:
        L-Phe+2-OG+NAD(P)H+H+→2-PE+L-Glu+NAD(P)++CO₂

Fig.5 Metabolic engineering for bioconversion of L-Phe to produce 2-PE

    In further experiments, we found that 2-PE, like many alcohols, has a high concentration of 2-PE that is toxic to microbial cells.
    Hence, 2-PE is used in disinfectants and preservatives in the medical field. Therefore, the inhibition of cell growth by high concentration of 2-PE is one of the bottlenecks for the synthesis of 2-PE by microbial biotransformation. In order to cope with the toxicity of 2-PE to cells, we maintained the 2-PE concentration in the fermentation broth by continuously recovering 2-PE in the fermentation medium, thereby releasing the inhibitory effect of 2-PE on the production strain, ie In situ product removal (ISPR) of the product. In our project, microbial intact cells are used as catalysts for biocatalytic reactions. Compared to enzyme catalysis, whole-cell catalysis can use the cell's own reaction to achieve co-factor recycling and regeneration, so there is no need to add expensive cofactors [4]; again, the cell wall is complex and harsh as an enzyme and the outside world. The environmental barrier enhances the stability of the enzyme [5]. Compared to enzyme catalysis, whole cells can achieve multiple cascades of enzymes in the same cell; and because all enzymes are encapsulated in one cell, the spatial distance of each enzyme is closer, allowing multiple cascades of reactions Efficient implementation[6].
    At the same time, the whole cell catalysis method separates the cell growth protein induction phase from the product synthesis process in time and space, avoiding the inhibition of cell growth by the product synthesis process[7].
    On top of glutamate dehydrogenase plays a key role in 2-PE biosynthesis, and the cofactor preference of the reductases was responsible for the difference in 2-PE production. The NADPH-dependent glutamate dehydrogenase in E. coli, which catalyzes the conversion of L-Glu and NADP+ to 2-OG and NADPH, may help to regenerate the cosubstrate and redox equivalents in the 2-PE whole-cell transformation process[8].
    Details can be found on our Result page

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Reference

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[2]Lavermicocca P, Valerio F, Evidente A, et al. Purification and Characterization of Novel Antifungal Compounds from the Sourdough Lactobacillus plantarum Strain 21B[J]. Applied & Environmental Microbiology. 2000, 66(9):4084.
[3]Cao, M., Jiang, X., Zhang, H., Xian, M., & Huang, F. (2012). The Study of Biotechnological Production of 2-Phenylethanol *, 2012(June), 89–97.
[4] Wang, P., Yang, X., Lin, B., Huang, J., & Tao, Y. (2017). Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol. Metabolic Engineering, 44(August), 143–149. https://doi.org/10.1016/j.ymben.2017.09.013.
[5]Hummel W., Groger H. Strategies for regeneration of nicotinamide coenzymes emphasizing self- sufficient closed-loop recycling systems[J]. Journal of Biotechnology, 191, 22-31 (2014).
[6] Wachtmeister J., Rother D. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale[J]. Current Opinion in Biotechnology, 42, 169-177 (2016).
[7] Muschiol J., Peters C., Oberleitner N. et al. Cascade catalysis - strategies and challenges en route to preparative synthetic biology[J]. Chemical Communications, 51, 5798-5811 (2015).
[8] Hwang J. Y., Park J., Seo J. H. et al. Simultaneous synthesis of 2-phenylethanol and L- homophenylalanine using aromatic transaminase with yeast Ehrlich pathway[J]. Biotechnology and Bioengineering, 102, 1323-1329 (2009).