Isotopically nonstationary C flux analysis of cyanobacterial isobutyraldehyde production.

Jazmin LJ, Xu Y, Cheah YE, Adebiyi AO, Johnson CH, Young JD
Metab Eng. 2017 42: 9-18

PMID: 28479191 · PMCID: PMC5660605 · DOI:10.1016/j.ymben.2017.05.001

We applied isotopically nonstationary C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.

Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

MeSH Terms (8)

Aldehydes Bacterial Proteins Carbon Isotopes Malate Dehydrogenase Phosphoenolpyruvate Carboxylase Pyruvate Kinase Staining and Labeling Synechococcus

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