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Understanding the performance of key metabolic enzymes is critical to metabolic engineering. It is important to know the kinetic parameters of both native enzymes and heterologously expressed enzymes that play key roles in pathway performance (Zeldes et al., Front Microbiol 6:1209, 2015; Keller et al., Metab Eng 27:101-106, 2015). This step cannot be overlooked as gene expression is not always a good indicator of the production of fully active enzymes, especially those requiring cofactor assembly and processing (Zeldes et al., Front Microbiol 6:1209, 2015; Chandrayan et al., J Biol Chem 287:3257-3264, 2012; Basen et al., MBio 3:e00053-e00012, 2012). Additionally, knowing kinetic parameters and having accurate and reproducible assays allows for the use of powerful computational and in vitro pathway optimization tools that can inform metabolic engineering efforts that in turn can lead to improvements in pathway performance (Keller et al., Metab Eng 27:101-106, 2015; Copeland et al., Metab Eng 14:270-280, 2012). To take full advantage of these tools, understanding the roles of both enzymes directly involved in a pathway of interest, together with those in related pathways that may syphon off key intermediates, is ideal (Keller et al., Metab Eng 27:101-106, 2015; Thorgersen et al., Metab Eng 22:83-88; Lin et al., Metab Engi 31:44-52, 2015). |
