Synthesis Of 2-phenylethanol Research Articles

Advances in synthesis of 2-phenylethanol

2-phenylethanol (2-PE), an aromatic alcohol with a rose fragrance, is the second most widely used flavoring substance in the world. It is widely used in the cosmetic, food, and pharmaceutical industries. This paper introduces the chemical synthesis methods of 2-PE and the synthetic pathways in plants and microorganisms, summarizes the strategies to improve the microbial synthesis of 2-PE, reviews the research progress in de novo synthesis of 2-PE in microorganisms, and makes an outlook on the research prospects, aiming to provide a theoretical basis for the industrial production of 2-PE.

Effects of Environmental Stresses on Synthesis of 2-Phenylethanol and IAA by Enterobacter sp. CGMCC 5087.

2-Phenylethanol (2-PE) and indole-3-acetic acid (IAA) are important secondary metabolites produced by microorganisms, and their production are closely linked to the growth state of microorganisms and environmental factors. Enterobacter CGMCC 5087 can produce both 2-PE and IAA depending on α-ketoacid decarboxylase KDC4427. This study aimed to investigate the effects of different environment factors including osmotic pressure, temperature, and pH on the synthesis of 2-PE and IAA in Enterobacter sp. CGMCC 5087. The bacteria exhibited an enhanced capacity for 2-PE synthesis while not affecting IAA synthesis under 5% NaCl and pH 4.5 stress conditions. In an environment with pH 9.5, the synthesis capacity of 2-PE remained unchanged while the synthesis capacity of IAA decreased. The synthesis ability of 2-PE was enhanced with an increase in temperature within the range of 25 °C to 37 °C, while the synthesis capacity of IAA was not affected significantly. Additionally, the expression of KDC4427 varied under stress conditions. Under 5% NaCl stress and decreased temperature, expression of the KDC4427 gene was increased. However, altering pH did not result in significant differences in gene expression levels, while elevated temperature caused a decrease in gene expression. Furthermore, molecular docking and molecular dynamics simulations suggested that these conditions may induce fluctuation in the geometry shape of binding cavity, binding energy, and especially the dαC-C- value, which played key roles in affecting the enzyme activity. These results provide insights and strategies for the synthesis of metabolic products 2-PE and IAA in bacterial fermentation, even under unfavorable conditions.

Multi-mode enhancement and Co-fermentation mechanism of 2-phenylethanol production by Kluyveromyces marxianus

As a valuable aromatic alcohol, 2-phenylethanol has been widely used in the fields of food, medicine and chemistry, but its production has become a key factor limiting microbial industrialisation. Therefore, atmospheric and room temperature plasma (ARTP) mutagenesis was applied to improve host cell tolerance of 2-phenylethanol, and 2-phenylethanol production was increased from 2.23 g/L to 2.51 g/L in Kluyveromyces marxianus. Fermentation conditions of K. marxianus and Pediococcus lactis co-culture were optimised and production of 2-phenylethanol was further increased by 42.63%, reaching 3.58 g/L. Metabonomics analysis revealed that the cell wall of P. lactis could effectively activate Ehrlich pathway-related metabolism and upregulate arginine synthesis pathways to enhance the cellular robustness of K. marxianus, thereby improving the efficiency of 2-phenylethanol synthesis, and providing a novel strategy for cost-effective 2-phenylethanol production.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76±0.02g/L after 72h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75±0.08g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36±0.06g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28±0.20g/L of 2-PE with a yield of 0.076g/g glucose and productivity of 0.048g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15g/L after 32h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

The De Novo Synthesis of 2-Phenylethanol from Glucose by the Synthetic Microbial Consortium Composed of Engineered Escherichia coli and Meyerozyma guilliermondii.

Synthetic microbial consortia show promising applications for fine chemical production, especially with long metabolic pathways. In this study, a synthetic microbial consortium consisting of Escherichia coli YLC20 and Meyerozyma guilliermondii MG57 was successfully constructed, which could achieve efficient de novo 2-phenylethanol (2-PE) production from glucose. A tyrosine-deficient E.coli YLC20 overexpressing genes of aroF and pheA was first constructed, which could accumulate 29.5 g/L of l-phenylalanine (l-Phe) within 96 h from glucose accompanied by the coproduction of acetate and α-ketoglutarate (α-KG). Furthermore, the engineered M.guilliermondii MG57 was constructed through the stepwise metabolic engineering strategy, which could facilitate the 2-PE synthesis from l-Phe. Moreover, the cosubstrate and material intervention strategies were applied to improve the stability of the microbial consortium and 2-PE production. Finally, the synthetic microbial consortium could de novo synthesize 3.77 g/L of 2-PE from 80 g/L of glucose, providing a reference for the de novo synthesis of fine chemicals with long metabolic pathways.

The Characterization of an Efficient Phenylpyruvate Decarboxylase KDC4427, Involved in 2-Phenylethanol and IAA Production from Bacterial Enterobacter sp. CGMCC 5087.

ABSTRACTPhenylpyruvate decarboxylase (PPDC) is a crucial enzyme that plays important roles in 2-phenylethanol (2-PE) biosynthesis. In our previous study, we screened a highly efficient PPDC KDC4427 from the novel 2-PE-producing strain Enterobacter sp. CGMCC 5087. Meanwhile, its decarboxylation activity of indolylpyruvate (IPyA) was also higher than other indolylpyruvate decarboxylases (IPDCs) reported so far. In this study, KDC4427 protein was purified and characterized, and its catalytic mechanisms were analyzed by biological methods. The optimum pH and temperature of KDC4427 was pH 6.5 and 35°C, respectively. The enzyme activity was relatively stable between pH 6 and 8 and over the range of temperatures from 25°C to 45°C. KDC4427 showed the highest catalytic efficiency on phenylpyruvic acid (PPA); meanwhile, it also showed high activity for IPyA and 2-ketobutanoic acid, and it was found that KDC4427 belongs to IPDCs by phylogenetic tree analysis. The coverage of the three-dimensional structure of KDC4427 and EcIPDC from Enterobacter cloacae was 96%. Leucine 542, one of the residues in the substrate-binding pocket, is replaced by isoleucine in KDC4427 compared with EcIPDC. Site-directed mutagenesis showed that the transition from leucine to isoleucine was unlikely to make KDC4427 have high catalytic activity for PPA and IPyA; the mutants at glutamate 468 almost completely lost catalytic activities for both PPA and IPyA, indicating that this glutamate was essential for the catalytic activity. Additionally, alanine 387 plays an important role in the substrate selectivity of KDC4427.IMPORTANCE Compared with the chemical synthesis of 2-phenylethanol (2-PE) by condensation of ethylene oxide and benzene, the biological synthesis of 2-PE is a potential method to replace the traditional process. This makes biotransformation gradually become the main way to produce high-quality 2-PE. Phenylpyruvate decarboxylase (PPDC) is the critical enzyme in 2-PE biosynthesis, and it is a momentous point of penetration to increase the production of 2-PE. In this regard, KDC4427 can catalyze phenylpyruvic acid (PPA) to phenylacetaldehyde more efficiently than any other PPDC previously reported. Moreover, it has high activity of indolepyruvate decarboxylases (IPDCs), which will be a great breakthrough in the synthesis of indole-3-acetic acid (IAA). With this study, we offer insights into the KDC4427 catalytic mechanism and significantly expand the toolbox of available α-ketoacid decarboxylases for application in biosynthesis.

Efficient synthesis of 2-phenylethanol from L-phenylalanine by engineered Bacillus licheniformis using molasses as carbon source.

2-Phenylethanol is a valuable flavoring agent with many applications. Although the bioproduction of 2-phenylethanol has been achieved by microbial fermentation, the low titer and high cost hinder its industrial-scale production. The goal of this study is to develop an efficient process for high-level production of 2-phenylethanol from L-phenylalanine. Firstly, candidate hosts for 2-phenylethanol synthesis were screened by evaluating their tolerance to 2-phenylethanol, and Bacillus licheniformis DW2 was proven to be a promising strain for 2-phenylethanol production. Subsequently, phenylpyruvate decarboxylase and alcohol dehydrogenase from different hosts were screened, and the combination of KivD from Lactococcus lactis and YqhD from Escherichia coli owned the best performance on 2-phenylethanol synthesis, and the attained strain DE4 produced 3.04g/L 2-phenylethanol from 5.00g/L L-phenylalanine using glucose as carbon source. Furthermore, the fermentation process was optimized using molasses as carbon source, and 2-phenylethanol titer was increased to 4.41g/L. In fed-batch fermentation, the maximum 2-phenylethanol titer reached 5.16g/L, with a yield of 0.65g/g on L-phenylalanine and productivity of 0.12g/(L.h), which was the highest 2-phenylethnol titer reported to date when molasses was used as carbon source. Collectively, this study develops a robust strain as well as the cost-efficient process for 2-phenylethanol production, which lays a substantial foundation for industrial production of 2-phenylethanol. Key points •Bacillus licheniformis is an excellent 2-PE stress-tolerant strain. •Coexpressed kivD and yqhD is most suitable for 2-PE production in B. licheniformis. •High-level production of 2-PE (5.16g/L) was obtained by engineered strain DE4.

Unveiling the Multipath Biosynthesis Mechanism of 2-Phenylethanol in Proteus mirabilis.

Proteus mirabilis could convert l-phenylalanine into 2-phenylethanol (2-PE) via the Ehrlich pathway, the amino acid deaminase pathway, and the aromatic amino acid decarboxylase pathway. The aromatic amino acid decarboxylase pathway was proved for the first time in P. mirabilis. In this pathway, l-aromatic amino acid transferase demonstrated a unique catalytic property, transforming 2-penylethylamine into phenylacetaldehyde. Eleven enzymes were supposed to involve in 2-phenylethanol synthesis. The mRNA expression levels of 11 genes were assessed over time by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in vivo. As a result, the expression of 11 genes was significantly increased, suggesting that P. mirabilis could transform l-phenylalanine into 2-phenylethanol via three pathways under aerobic conditions; nine genes were significantly overexpressed, suggesting that P. mirabilis could synthesize 2-phenylethanol via the Ehrlich pathway under anaerobic conditions. This study reveals the multipath synthetic metabolism for 2-phenylethanol in P. mirabilis and will enrich the new ideas for natural (2-PE) synthesis.

Cloning and Functional Verification of Genes Related to 2-Phenylethanol Biosynthesis in Rosa rugosa.

In China, Rosa rugosa is cultivated as a source of natural perfumes. Rose essential oil is known as “liquid gold”, given its high economic and health value. 2-phenylethanol accounts for more than 10% of the total mass fraction of the essential oil derived from R. rugosa. The regulatory mechanisms underlying 2-phenylethanol metabolism in R. rugosa, however, remain unclear. In this study, RrAAAT and RrPPDC1, two genes related to 2-phenylethanol synthesis, were cloned from R. rugosa. Expression analysis revealed that RrAAAT and RrPPDC1 were highly expressed in rose flowers in the full opening and withering stages, and in calyxes. The overexpression vectors of RrAADC, RrAAAT, and RrPPDC1 were established and transformed into Petunia hybrida via Agrobacterium-mediated genetic transformation. Results demonstrated that the overexpression of RrAADC and RrAAAT increased the 2-phenylethanol content of transgenic petunia flowers. The results of this study provide a basis for the introduction of genes related to 2-phenylethanol synthesis into roses to increase the 2-phenylethanol content of rose essential oil.

Genetic engineering of Ehrlich pathway modulates production of higher alcohols in engineered Yarrowia lipolytica.

Microbial cells can produce a vast spectrum of chemical compounds, including those most desired by the global chemical market, for example, higher alcohols, which are promising alternative fuels and chemical feedstock. In the current research, we investigated the effects of the Ehrlich pathway genetic engineering on higher alcohols production in Yarrowialipolytica, which directly follows our previous findings concerning elucidation of putative molecular identities involved in this pathway. To this end, we constructed two alternative expression cassettes composed of previously identified genes, putatively involved in the Ehrlich pathway in Y. lipolytica, and cloned them under the control of constitutive pTEF promoter, and by this released them from extensive native regulation. The effects of the pathway engineering were investigated upon provision of different Ehrlich pathway-inducing amino acids (L-Phe, L-Leu, L-Ile and L-Val). In general, amplification of the Ehrlich pathway in many cases led to increased formation of a respective higher alcohol from its precursor. We observed interesting effects of aminotransferase BAT2 deletion on synthesis of 2-phenylethanol and its acetate ester, significant relationship between L-Val and L-Phe catabolic pathways and extensive 'cross-induction' of the derivative compounds synthesis by non-direct precursors.

Isolation of 2-phenylethanol biosynthesis related genes and their relationship with 2-phenylethanol accumulation in Rosa rugosa

Rosa rugosa is a famous traditional flower in China used not only as a landscape plant but also as a time-honored and valuable aromatic plant. The natural aromatic substance 2-phenylethanol is the major indispensable ingredient of rose flower and rose essential oil. This study adopted R. rugosa ‘Tanghong’ as a test material to isolate 2-phenylethanol biosynthesis related genes RrAADC and RrPAR. The temporal and spatial expression patterns of the two genes in different flower developmental stages and floral organ parts were measured, and the relationship of 2-phenylethanol accumulation to RrAADC and RrPAR expression in R. rugosa was determined. The content of 2-phenylethanol in R. rugosa gradually increased with the degree of flower opening and peaked at the withering stage. The expression level of RrAADC gradually decreased with the degree of flower opening. Meanwhile, the expression level of RrPAR gradually increased from the budding stage to the half opening stage, rapidly decreased at the full opening stage, and slightly increased again toward the withering stage. The content of 2-phenylethanol was the highest in the petals, followed by that in the stamens and pistils. However, this compound was not detected in other parts of the fully opened flower. The expression level of RrAADC peaked in the stamens but was relatively low in the other parts. The expression level of RrPAR was the highest in the stamens and calyxes, followed by that in the pistils and receptacles, and the lowest in the petals and stalks. These results suggest that RrAADC and RrPAR coordinately regulate the biological synthesis of 2-phenylethanol in R. rugosa.

L-Phenylalanine catabolism and 2-phenylethanol synthesis in Yarrowia lipolytica--mapping molecular identities through whole-proteome quantitative mass spectrometry analysis.

A world-wide effort is now being pursued towards the development of flavors and fragrances (F&F) production independently from traditional sources, as well as autonomously from depleting fossil fuel supplies. Biotechnological production of F&F by microbes has emerged as a vivid solution to the current market limitations. Amongst a wide variety of fragrant chemicals, 2-PE is of significant interest to both scientific and industrial community. Although the general overview of the 2-PE synthesis pathway is commonly known, involvement of particular molecular identities in this pathway has not been elucidated in Yarrowia lipolytica to date. The aim of this study was mapping molecular identities involved in 2-PE synthesis in Y. lipolytica. To acquire a comprehensive landscape of the proteins that are directly and indirectly involved in L-Phe degradation and 2-PE synthesis, we took advantage of comprehensibility and sensitivity of high-throughput LC-MS/MS-quantitative analysis. Amongst a number of proteins involved in amino acid turnover and the central carbon metabolism, enzymes involved in L-Phe conversion to 2-PE have been identified. Results on yeast-to-hyphae transition in relation to the character of the provided nitrogen source have been presented.

De-novo synthesis of 2-phenylethanol by Enterobacter sp. CGMCC 5087.

Background2-phenylethanl (2-PE) and its derivatives are important chemicals, which are widely used in food materials and fine chemical industries and polymers and it’s also a potentially valuable alcohol for next-generation biofuel. However, the biosynthesis of 2-PE are mainly biotransformed from phenylalanine, the price of which barred the production. Therefore, it is necessary to seek more sustainable technologies for 2-PE production.ResultsA new strain which produces 2-PE through the phenylpyruvate pathway was isolated and identified as Enterobacter sp. CGMCC 5087. The strain is able to use renewable monosaccharide as the carbon source and NH4Cl as the nitrogen source to produce 2-PE. Two genes of rate-limiting enzymes, chorismate mutase p-prephenate dehydratase (PheA) and 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (DAHP), were cloned from Escherichia coli and overexpressed in E. sp. CGMCC 5087. The engineered E. sp. CGMCC 5087 produces 334.9 mg L-1 2-PE in 12 h, which is 3.26 times as high as the wild strain.ConclusionsThe phenylpyruvate pathway and the substrate specificity of 2-keto-acid decarboxylase towards phenylpyruvate were found in E. sp. CGMCC 5087. Combined with the low-cost monosaccharide as the substrate, the finding provides a novel and potential way for 2-PE production.

Palladium supported on chitosan as a recyclable and selective catalyst for the synthesis of 2-phenyl ethanol

Two different chitosan supported palladium based catalysts were prepared, wherein dispersed palladium nanoparticles were obtained via chemical reduction supported on chitosan (Pd/CTS) and amine functionalized modified chitosan (Pd/AFCTS). The catalytic activity of the Pd-based catalysts, Pd/CTS and Pd/AFCTS, were assessed in the hydrogenation of styrene oxide to 2-phenyl ethanol. Both Pd-based catalysts enhanced the formation of the desired 2-phenyl ethanol in contrast to a conventional Pd/C catalyst without the assistance of inorganic or organic base. A considerable influence on the conversion and selectivity was observed in the case of Pd/AFCTS, consisting of palladium nanoparticles stabilized and dispersed on amine-functionalized chitosan matrix, affording complete conversion of styrene oxide with 98% selectivity to 2-phenyl ethanol. The catalyst Pd/AFCTS has also been recycled without significant loss of activity and selectivity.

Convergent preparation of 2-phenylethanol

The convergent synthesis of 2-phenylethanol was executed with bromination of benzene in the presence of pyridine to give bromobenzene which was reacted with magnesium metal to give phenylmagnesium bromide. Ethene was generated from ethanol and converted to 1,2-dibromoethane which in turn was reacted with the Grignard in an inverse manner to give 2-phenylbromoethane. Reaction of 2-phenylbromoethane and aqueous sodium hydroxide, in acetone as solvent, yielded 2-phenylethanol. Key words: 2-Phenylethanol, 1,2-dibromoethane, 2-phenylbromoethane

In Silico Study of Aro Genes Involved in the Ehrlich Pathway: Comparison between Saccharomyces Cerevisiae and Kluyveromyces Lactis

ABSTRACTAt the present state of the art, genome sequencing techniques are advancing with a faster pace than the experimental proof of predicted genes and their functions. Many approaches for clustering annotated genes based on the degree of their sequence homology have emerged during the last decade. In the current comparative genomic study a parallel has been drawn between a set of ARO genes involved in the aromatic alcohol synthesis in the model yeast organism Saccharomyces cerevisiae and their orthologs in Klyuveromyces lactis. Through a combination of sequence comparison, online resources for homology and topology-based clustering, and a tool for prediction of the cellular localisation of proteins, it has been shown that the genes involved in the first two steps of the 2-phenylethanol synthesis in K. lactis are generally conserved when compared to their S. cerevisiae orthologs. They show 40–62% identity of the encoded protein as well as conserved synteny and cellular localisation. It is also likely that these species utilise the same type of regulation mechanisms for the process. The presence of a second ARO8 ortholog in K. lactis suggests a more environmentallyflexible and also effective production of 2-phenylethanol. This comparison aims to facilitate the design of further experiments that can reveal the regulation of fusel alcohol formation in K. lactis and improve their production.

Tomato phenylacetaldehyde reductases catalyze the last step in the synthesis of the aroma volatile 2-phenylethanol

The volatile compounds, 2-phenylacetaldehyde and 2-phenylethanol, are important for the aroma and flavor of many foods, such as ripe tomato fruits, and are also major constituents of scent of many flowers, most notably roses. While much work has gone into elucidating the pathway for 2-phenylethanol synthesis in bacteria and yeast, the pathways for synthesis in plants are not well characterized. We have identified two tomato enzymes (LePAR1 and LePAR2) that catalyze the conversion of 2-phenylacetaldehyde to 2-phenylethanol: LePAR1, a member of the large and diverse short-chain dehydrogenase/reductase family, strongly prefers 2-phenylacetaldehyde to its shorter and longer homologues (benzaldehyde and cinnamaldehyde, respectively) and does not catalyze the reverse reaction at a measurable rate; LePAR2, however, has similar affinity for 2-phenylacetaldehyde, benzaldehyde and cinnamaldehyde. To confirm the activity of these enzymes in vivo, LePAR1 and LePAR2 cDNAs were individually expressed constitutively in petunia. While wild type petunia flowers emit relatively high levels of 2-phenylacetaldehyde and lower levels of 2-phenylethanol, flowers from the transgenic plants expressing LePAR1 or LePAR2 had significantly higher levels of 2-phenylethanol and lower levels of 2-phenylacetaldehyde. The in vivo alteration of volatile emissions is an important step toward altering aroma volatiles in plants.

Direct synthesis of 2-phenylethanol by hydrogenation of methyl phenylacetate using homogeneous ruthenium-phosphine catalysis under low hydrogen pressure

Methyl phenylacetate could be efficiently hydrogenated to yield 2-phenylethanol under lower hydrogen pressure (<10 atm) by using ruthenium-phosphine catalysis composed of Ru(acac) 3, P( n-C 8H 17) 3 in the presence of Zn. Effect of both phosphine and Zn plays an crucial role in order for this hydrogenation to proceed under mild conditions.