Isotopically nonstationary 13 C flux analysis of cyanobacterial isobutyraldehyde production

Metab Eng. 2017 Jul;42:9-18. doi: 10.1016/j.ymben.2017.05.001. Epub 2017 May 4.

Abstract

We applied isotopically nonstationary 13C 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 13C 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.

Keywords: (13)C INST-MFA; Isobutyraldehyde; Nonstationary (13)C metabolic flux analysis; Photoautotrophic metabolism; Pyruvate kinase; Synechococcus elongatus PCC7942.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehydes / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Carbon Isotopes / metabolism*
  • Malate Dehydrogenase / genetics
  • Malate Dehydrogenase / metabolism
  • Phosphoenolpyruvate Carboxylase / genetics
  • Phosphoenolpyruvate Carboxylase / metabolism
  • Pyruvate Kinase / genetics
  • Pyruvate Kinase / metabolism
  • Staining and Labeling / methods*
  • Synechococcus / genetics
  • Synechococcus / metabolism*

Substances

  • Aldehydes
  • Bacterial Proteins
  • Carbon Isotopes
  • isobutyraldehyde
  • Malate Dehydrogenase
  • Pyruvate Kinase
  • Phosphoenolpyruvate Carboxylase