期刊
JOURNAL OF BIOTECHNOLOGY
卷 186, 期 -, 页码 91-97出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2014.06.033
关键词
L-Phenylglycine; L-4-Hydroxyphenylglycine aminotransferase; Amycolatopsis orientalis; Streptomyces coelicolor
资金
- '863' Program [2012AA021201]
- Program for New Century Excellent Talents in University [NCET-11-0665]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Innovative Program for Graduate Student of Jiangsu Province [CXLX12_0733]
- Fundamental Research Funds for the Central Universities [JUDCF12016]
- Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China [KLIB-KF201102]
The aproteinogenic amino acid L-phenylglycine (L-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis L-Phg from glucose, an engineered Escherichia coli containing L-Phg synthetic genes was firstly developed by an L-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (L-4-hydroxymandelate synthase), Hmo (L-4-hydroxymandelate oxidase) and HpgT (L-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on L-Phg formation. HpgT conversing phenylglyoxylate to L-Phg uses an unusual amino donor L-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of L-Phg, the effects of attenuating L-phenylalanine transamination was investigated. After deletion of tyrB and aspC, L-Phg yield increased by 12.6-fold. The limiting step in the L-Phg biosynthesis was also studied; the L-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of L-phenylalanine transamination, the L-Phg yield was increased by 224-fold comparing with the original strain. (C) 2014 Elsevier B.V. All rights reserved.
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