Abstract：
:Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.
journal_name
Metab Engjournal_title
Metabolic engineeringauthors
Cheah YE,Xu Y,Sacco SA,Babele PK,Zheng AO,Johnson CH,Young JDdoi
10.1016/j.ymben.2020.03.007subject
Has Abstractpub_date
2020-07-01 00:00:00pages
56-65eissn
1096-7176issn
1096-7184pii
S1096-7176(20)30062-8journal_volume
60pub_type
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