Dataset Information

Genome mining of 2-phenylethanol biosynthetic genes from Enterobacter sp. CGMCC 5087 and heterologous overproduction in Escherichia coli.

ABSTRACT: Background:2-Phenylethanol (2-PE) is a higher aromatic alcohol that is widely used in the perfumery, cosmetics, and food industries and is also a potentially valuable next-generation biofuel. In our previous study, a new strain Enterobacter sp. CGMCC 5087 was isolated to produce 2-PE from glucose through the phenylpyruvate pathway. Results:In this study, candidate genes for 2-PE biosynthesis were identified from Enterobacter sp. CGMCC 5087 by draft whole-genome sequence, metabolic engineering, and shake flask fermentation. Subsequently, the identified genes encoding the 2-keto acid decarboxylase (Kdc) and alcohol dehydrogenase (Adh) enzymes from Enterobacter sp. CGMCC 5087 were introduced into E. coli BL21(DE3) to construct a high-efficiency microbial cell factory for 2-PE production using the prokaryotic phenylpyruvate pathway. The enzymes Kdc4427 and Adh4428 from Enterobacter sp. CGMCC 5087 showed higher performances than did the corresponding enzymes ARO10 and ADH2 from Saccharomyces cerevisiae, respectively. The E. coli cell factory was further improved by overexpressing two upstream shikimate pathway genes, aroF/aroG/aroH and pheA, to enhance the metabolic flux of the phenylpyruvate pathway, which resulted in 2-PE production of 260 mg/L. The combined overexpression of tktA and ppsA increased the precursor supply of erythrose-4-phosphate and phosphoenolpyruvate, which resulted in 2-PE production of 320 mg/L, with a productivity of 13.3 mg/L/h. Conclusions:The present study achieved the highest titer of de novo 2-PE production of in a recombinant E. coli system. This study describes a new, efficient 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its derivatives in the future.

SUBMITTER: Liu C

PROVIDER: S-EPMC6223000 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Genome mining of 2-phenylethanol biosynthetic genes from <i>Enterobacter</i> sp. CGMCC 5087 and heterologous overproduction in <i>Escherichia coli</i>.

Liu Changqing C Zhang Kai K Cao Wenyan W Zhang Ge G Chen Guoqiang G Yang Haiyan H Wang Qian Q Liu Haobao H Xian Mo M Zhang Haibo H

Biotechnology for biofuels 20181108

<h4>Background</h4>2-Phenylethanol (2-PE) is a higher aromatic alcohol that is widely used in the perfumery, cosmetics, and food industries and is also a potentially valuable next-generation biofuel. In our previous study, a new strain <i>Enterobacter</i> sp. CGMCC 5087 was isolated to produce 2-PE from glucose through the phenylpyruvate pathway.<h4>Results</h4>In this study, candidate genes for 2-PE biosynthesis were identified from <i>Enterobacter</i> sp. CGMCC 5087 by draft whole-genome seque ...[more]

PMID: 30455734

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Project description:Phenylpyruvate 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.

Dataset Information

Genome mining of 2-phenylethanol biosynthetic genes from Enterobacter sp. CGMCC 5087 and heterologous overproduction in Escherichia coli.

Publications

Genome mining of 2-phenylethanol biosynthetic genes from <i>Enterobacter</i> sp. CGMCC 5087 and heterologous overproduction in <i>Escherichia coli</i>.

Similar Datasets

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