Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis

Journal Article · 08 September 2023 · Metabolic Engineering

DOI: https://doi.org/10.1016/j.ymben.2023.09.002 · OSTI ID:2004929

Wang, Bo  ^[1]; Zuniga, Cristal ^[2]; Guarnieri, Michael T.  ^[3]; Zengler, Karsten ^[2]; Betenbaugh, Michael ^[4]; Young, Jamey D.  ^[1]

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1. Vanderbilt University, Nashville, TN (United States)
2. University of California, San Diego, CA (United States)
3. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
4. Johns Hopkins University, Baltimore, MD (United States)

The capability of cyanobacteria to produce sucrose from CO₂ and light has a remarkable societal and biotechnological impact since sucrose can serve as a carbon and energy source for a variety of heterotrophic organisms and can be converted into value-added products. However, most metabolic engineering efforts have focused on understanding local pathway alterations that drive sucrose biosynthesis and secretion in cyanobacteria rather than analyzing the global flux re-routing that occurs following induction of sucrose production by salt stress. Here, we investigated global metabolic flux alterations in a sucrose-secreting (cscB-overexpressing) strain relative to its wild-type Synechococcus elongatus 7942 parental strain. Here we used targeted metabolomics, ¹³C metabolic flux analysis (MFA), and genome-scale modeling (GSM) as complementary approaches to elucidate differences in cellular resource allocation by quantifying metabolic profiles of three cyanobacterial cultures - wild-type S. elongatus 7942 without salt stress (WT), wild-type with salt stress (WT/NaCl), and the cscB-overexpressing strain with salt stress (cscB/NaCl) - all under photoautotrophic conditions. We quantified the substantial rewiring of metabolic fluxes in WT/NaCl and cscB/NaCl cultures relative to WT and identified a metabolic bottleneck limiting carbon fixation and sucrose biosynthesis. This bottleneck was subsequently mitigated through heterologous overexpression of glyceraldehyde-3-phosphate dehydrogenase in an engineered sucrose-secreting strain. Our study also demonstrates that combining ¹³C-MFA and GSM is a useful strategy to both extend the coverage of MFA beyond central metabolism and to improve the accuracy of flux predictions provided by GSM.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC36-08GO28308; SC0019404; SC0018344
• OSTI ID: 2004929
• Alternate ID(s): OSTI ID: 2369681
• Report Number(s): NREL/JA--2700-87582; {"Journal ID: ISSN 1096-7176",UUID:410b90a7-bf7d-4b1d-933c-0759143ba980,MainAdminID:70691,MainId:88357}
• Journal Information: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 80; ISSN 1096-7176
• Publisher: ElsevierCopyright Statement
• Country of Publication: United States
• Language: English