代谢工程大肠杆菌过量生产番茄红素
Overproduction of Lycopene by Metabolic Engineering Escherichia coli

作者: 翁志明 , 王 玥 , 刘建忠 :中山大学生命科学学院生物工程研究中心,广州 ;

关键词: 番茄红素大肠杆菌代谢工程启动子置换组成型质粒Lycopene Escherichia coli Metabolic Engineering Promoter Replacement Constitutive Plasmid

摘要: 番茄红素是一种高效抗氧化剂和潜在抗癌药物。gdhAaceEfdhF基因敲除促进了重组大肠杆菌番茄红素的合成,其中gdhAaceE双基因敲除和gdhAaceEfdhF三基因敲除具有相似的作用。在gdhAaceE双基因敲除的基础上,dxs基因天然启动子被T5启动子置换后,重组大肠杆菌番茄红素的产量提高了103%。为了避免采用诱导剂进行基因表达,构建了一系列组成型质粒,最终构建的代谢工程大肠杆菌BW25113(rgdhAraceE, PT5-dxspAC316-WZM4R)在不需诱导条件下摇床发酵可产番茄红素15.6 mg/g DCW

Abstract: Lycopene is an effective antioxidant and a potential pharmaceutical drug with anticancer. The knockout of gdhA, aceE or fdhF was beneficial to lycopene production in engineered E. coli. The double (gdhA and aceE) gene knockouts showed a similar effect to the triple (gdhA, aceE and fdhF) on lycopene production. Replacement of native promoter of dxs gene in the double gene knockout strain resulted in 103% increase in lycopene content. In order to avoid application of expensive inducer, we also constructed some constitutive plasmids containing heterologous carotenoid genes. The final engineered E. coli BW25113 (rgdhAraceE, PT5-dxs, pAC316-WZM4R) produced lycopene of 15.6 mg/g DCW without inducer in a batch shake flask.

文章引用: 翁志明 , 王 玥 , 刘建忠 (2012) 代谢工程大肠杆菌过量生产番茄红素。 生物过程, 2, 51-57. doi: 10.12677/bp.2012.22009

参考文献

[1] H. Alper, G. Stephanopoulos. Uncovering the gene knockout landscape for improved lycopene production in E. coli. Applied Microbiology and Biotechnology, 2008, 78(5): 801-810.

[2] H. Alper, Y. S. Jin, J. F. Moxley, et al. Identifing gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metabolic Engineering, 2005, 7(3): 155-164.

[3] Y. S. Jin, G. Stephanoupoulos. Multi-dimensional gene target search for improving locopene biosynthesis in Escherichia coli. Metabolic Engineering, 2007, 9(4): 337-347.

[4] W. R. Farmer, J. C. Liao. Precursor balancing for metabolic engineering of lycopene production in Escherichia coli. Biotechnology Progress, 2001, 17(1): 57-61.

[5] M. J. Kang, S. H. Yoon, Y. M. Lee, S. H. Lee, J. E. Kim, K. H. Jung, Y. C. Shin and S. W. Kim. Enhancement of lycopene production Escherichia coli by optimization of the lycopene synthetic pathway. Journal of Microbiology and Bio-technology, 2005, 15(4): 880-886.

[6] S. H. Yoon, Y. M. Lee, J. E. Kim, S. H. Lee, J. H. Lee, J. Y. Kim, K. H. Jung, Y. C. Shin, J. D. Keasling and S. W. Kim. Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate. Biotechnology and Bioengineering, 2006, 94(6): 1025-1032.

[7] L. Z. Yuan, P. E. Rouviére, R. A. LaRossa and W. Suh. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metabolic Engineering, 2006, 8(1): 79-90.

[8] H. Alper, C. Fischer, E. Nevoigt and G. Stephanopoulos. Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the USA, 2005, 102(36): 12678-12683.

[9] H. Alper, G. Stephanopoulos. Global transcription machinery engineering: A new approach for improving cellular phenotype. Metabolic Engineering, 2007, 9(3): 258-267.

[10] C. J. Chiang, P. T. Chen and Y. P. Chao. Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli. Biotechnology and Bioengineering, 2008, 101(5): 985-995.

[11] K. E. J. Tyo, P. K. Ajikumar and G. Stephanopoulos. Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nature Biotechnology, 2009, 27: 760-765.

[12] F. X. Cunningham, Z. Sun, D. Chamovitz, J. Hirschberg and E. Gantt. Molecular structure and enzyme function of lycopene cyclase from the Cyanobacterium Synechococcus sp. Strain PCC7942. The Plant Cell, 1994, 6(8): 1107-1121.

[13] K. A. Datsenko, B. L. Wanner. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy Sciences of the USA, 2000, 97(12): 6640-6645.

[14] L. M. Guzman, D. Belin, M. J. Carson and J. Beckwith. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 1995, 177(14): 4121-4130.

[15] D. Langley, J. R. Guest. Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: Genetic characterization and regulatory properties of deletion mutants. Journal of General Microbiology, 1978, 106(1): 103-117.

[16] M. O’Connor, M. Peifer and W. Bender. Construction of large DNA segments in Escherichia coli. Science, 1989, 244(4910): 1307-1312.

[17] D. Noack, M. Roth, R. Geuther, G. Müller, K. Undisz, C. Hoffmeier and S. Gáspár. Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat. Molecular and General Genetics, 1981, 184(1): 121-124.

[18] T. Nishizaki, K. Tsuge, M. Itaya, N. Doi and H. Yanagawa. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Applied and Environmental Microbiology, 2007, 73(4): 1355-1361.

分享
Top