Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement

被引:93
作者
Chung, Bevan K. S. [1 ,2 ]
Selvarasu, Suresh [2 ]
Andrea, Camattari [2 ]
Ryu, Jimyoung [3 ]
Lee, Hyeokweon [3 ]
Ahn, Jungoh [3 ]
Lee, Hongweon [3 ]
Lee, Dong-Yup [1 ,2 ,4 ]
机构
[1] Natl Univ Singapore, NUS Grad Sch Integrat Sci & Engn, Singapore 117456, Singapore
[2] ASTAR, Bioproc Technol Inst, Centros 138668, Singapore
[3] KRIBB, Biotechnol Proc Engn Ctr, Taejon 305806, South Korea
[4] Natl Univ Singapore, Dept Chem & Biomol Engn, Singapore 117576, Singapore
来源
MICROBIAL CELL FACTORIES | 2010年 / 9卷
关键词
RECOMBINANT PROTEIN-PRODUCTION; BIOCATALYTIC KETONE REDUCTION; N-LINKED GLYCOSYLATION; ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; CARBON-SOURCES; SYSTEMS BIOTECHNOLOGY; ELEMENTAL COMPOSITION; CHIRAL ALCOHOLS; POWERFUL TOOL;
D O I
10.1186/1475-2859-9-50
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism. Results: A fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified. Conclusion: The genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.
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页数:15
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