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Title: Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Here, 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.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Vanderbilt Univ., Nashville, TN (United States)
Publication Date:
Grant/Contract Number:
SC0008118; AC05-06OR23100; P200A090323
Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 42; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Research Org:
Vanderbilt Univ., Nashville, TN (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Synechococcus elongatus PCC7942; Isobutyraldehyde; Photoautotrophic metabolism; Nonstationary 13C metabolic flux analysis; 13C INST-MFA; Pyruvate kinase
OSTI Identifier:
1462034
Alternate Identifier(s):
OSTI ID: 1397026