Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

Journal Article · Fri Jun 16 00:00:00 EDT 2017 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.00996-17· OSTI ID:1395092

Guarnieri, Michael T.  ^[1]; Chou, Yat -Chen ^[1]; Salvachua, Davinia Rodriquez  ^[1]; Mohagheghi, Ali ^[1]; St. John, Peter C.  ^[1]; Peterson, Darren J. ^[1]; Bomble, Yannick J. ^[1]; Beckham, Gregg T. ^[1]; Atomi, Haruyuki ^[2]

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1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
2. Kyoto Univ. (Japan)

Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO₂ into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO₂ to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.

• Research Organization: National Renewable Energy Lab. (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
• Grant/Contract Number: AC36-08GO28308
• OSTI ID: 1395092
• Report Number(s): NREL/JA-5100-68889
• Journal Information: Applied and Environmental Microbiology, Vol. 83, Issue 17; ISSN 0099-2240
• Publisher: American Society for Microbiology
• Country of Publication: United States
• Language: English

Related Subjects

09 BIOMASS FUELS
succinic acid
genetic modifications
biorefineries