Systematic identification and elimination of flux bottlenecks in the aldehyde production pathway of Synechococcus elongatus PCC 7942

• Published in: Metabolic engineering Vol. 60; no. C; pp. 56 - 65
• Main Authors: Cheah, Yi Ern, Xu, Yao, Sacco, Sarah A., Babele, Piyoosh K., Zheng, Amy O., Johnson, Carl Hirschie, Young, Jamey D.
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.07.2020; Elsevier
• Subjects: Acetolactate Synthase - metabolism; Aldehydes - metabolism; Amino Acids - metabolism; Cyanobacteria; Isobutyraldehyde; Metabolic Engineering; Metabolic Flux Analysis; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxylase - metabolism; Photoautotrophic metabolism; Pyruvate; Pyruvate Dehydrogenase Complex - metabolism; Pyruvate Kinase - metabolism; Pyruvates - metabolism; RNA, Bacterial - biosynthesis; RNA, Bacterial - genetics; Stable isotope; Synechococcus - metabolism; Stable isotope; Isobutyraldehyde; Photoautotrophic metabolism; Metabolic flux analysis; Cyanobacteria; Pyruvate; Phosphoenolpyruvate
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2020.03.007

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. •INST-MFA was used to identify gene targets for increasing aldehyde production.•PDH and PPC fluxes were inversely correlated with productivity.•Knockdown of PDH increased aldehyde productivity by ~50%.•Overexpression of PEPCK increased titer by ~60% but inhibited growth.•Flux analysis provides a systematic approach to guide iterative strain improvement.