Line 326: | Line 326: | ||
<div id="2-PE"> <h2> The pathway of production</h2> </div> | <div id="2-PE"> <h2> The pathway of production</h2> </div> | ||
<p > A heterologous Ehrlich pathway was constructed for 2-PE bio-synthesis in <span style="font-style:italic;">E. coli</span> by co-overexpressing the aromatic transaminase from <span style="font-style:italic;">E. coli</span> (TyrB, WP032305522.1), phenylpyruvate decarboxylase from <span style="font-style:italic;">S. cerevisiae</span> (Aro10, NP 010668.3) and dehydrogenation of reductase from <span style="font-style:italic;">Rose</span> (PAR, BAG 13450.2). </br> | <p > A heterologous Ehrlich pathway was constructed for 2-PE bio-synthesis in <span style="font-style:italic;">E. coli</span> by co-overexpressing the aromatic transaminase from <span style="font-style:italic;">E. coli</span> (TyrB, WP032305522.1), phenylpyruvate decarboxylase from <span style="font-style:italic;">S. cerevisiae</span> (Aro10, NP 010668.3) and dehydrogenation of reductase from <span style="font-style:italic;">Rose</span> (PAR, BAG 13450.2). </br> | ||
− | + | 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.</br> | |
The overall stoichio-metric equation of the reactions in this biosynthetic system is:</br> | The overall stoichio-metric equation of the reactions in this biosynthetic system is:</br> | ||
<span style="font-weight:bold;">L-Phe+2-OG+NAD(P)H+H+→2-PE+L-Glu+NAD(P)++CO2</span> | <span style="font-weight:bold;">L-Phe+2-OG+NAD(P)H+H+→2-PE+L-Glu+NAD(P)++CO2</span> |
Revision as of 17:23, 16 October 2018
Description
Phenyllactic acid
Phenylllisted acid (PLA)[1], also known as 3-phenyllactic acid or β-PLA, whose system called 2-hydroxy-3-phenylpropanoic acid. Phenyllactic acid (PLA) is widely found in cheese, honey and other foods, and it is not toxic for human and animal cells [2]. And it is a very stable and important natural small molecule organic acid, whose molecular formula is C9H10O3[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.
![](https://static.igem.org/mediawiki/2018/a/a2/T--FJNU-China--D-_PLA_.png)
Fig.1 Chemical structure of PLA
Application
PLA has high safety and is non-toxic for human and animal cells. As a new natural antibacterial substance and preservative, it can inhibit a series of gram-negative, gram-positive bacteria and fungi. And as a clinical hemostatic drug, PLA can be used to prevent platelet aggregation and coronary artery expansion. In addition, phenyllactic acid can also be used as a skin protectant to prevent dry skin.
PLA is a new type of natural antibacterial substance and preservative.
PLA can be used as a hemostatic drug And skin protectant
PLA produced by synthetic biology and it has less pollutants.
The pathway of production
Phenylalanine is converted to phenylpyruvate by the action of an aminotransferase (Tyrb) from Escherichia coli 21B, which is then dehydrogenated by 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 transaminase and glutamate dehydrogenase are very low. In order to achieve higher D-PLA production, we must solve the balance expression of the D-lactate dehydrogenase, transaminase and glutamate dehydrogenase in the metabolic pathway. Therefore, we reduced the expression level of D-lactate dehydrogenase by introducing of the rare arginine codons in the D-lactate dehydrogenase gene, and it has been proved by experiments that the D-PLA has the highest production after 4 Arg rare codons were introduced.
![](https://static.igem.org/mediawiki/2018/7/70/T--FJNU-China--PLA_circulation_short.png)
Fig.2 Chemical structure of PLA
Since D - lactate dehydrogenase is a NADH - dependent oxidoreductase in the metabolic pathway, theoretically the cofactor NADH produced by Escherichia coli cells cannot meet the redox reaction requirements of the over-expressed 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-lactic acid dehydrogenase, resulting in the more efficient conversion of phenylpyruvic acid into D-PLA.
![](https://static.igem.org/mediawiki/2018/9/94/T--FJNU-China--PLA_circulation_long_.png)
Fig.3 Metabolic engineering for bioconversion of L-Phe to produce PLA
The D-lactic dehydrogenase used in this project was derived from Lactobacillus bulgaricus ATCC 11842 D-ldh (BBa_K2570012), and the 52nd 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 acid dehydrogenase to 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 for improvement of the D-PLA production. Then the improved NADH regeneration can be achieved when the co-expression of rocG , D-lactate dehydrogenase D-ldh and phenylalanine transaminase Tyrb were realized. Finally, the conditions of whole-cell transformation were optimized and the D-PLA production by the recombinant engineered E. coli was further improved.
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.
![](https://static.igem.org/mediawiki/2018/e/ec/T--FJNU-China--Description-2pe-chemi.png)
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
2-PE is traditionally extracted from rose, the yield is typically low. Nowadays, 2-PE is mostly produced by chemical synthesis, which is environmentally unfriendly and produces unwanted by-products. On the other hand, bio-technologically produced flavors are currently considered as natural by European and U.S. food agencies. Additionally, bio-technological 2-PE production is highly desirable and holds promise to be the most commercially viable route to produce 2-PE. Biosynthesis 2-PE The main method is to synthesize 2-PE by Ehrlich pathway using L-phenylalanine (L-Phe) as a substrate.
It can be used as a drug which has funtion of anti-inflammatory, anti-viral, anti-tumor, anti-oxidation, immune regulation, enhance memory, etc.
It can effectively inhibit many diseases and can be used as antibacterial substances to replace synthetic fungicides and disinfectants.
People prefer to use 2-PE from a "natural" source, which is much more expensive than chemical synthesis.
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.[5] 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.
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
![](https://static.igem.org/mediawiki/2018/b/b8/T--FJNU-China--2_PE_long_passway.png)
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.
Reference
[1]Dieuleveux V, Van DPD, Chataud J, et al. Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum[J]. Applied & Environmental Microbiology. 1998, 64(2):800. [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).