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Title: Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

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 CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 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 productionmore » 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.« less
Authors:
ORCiD logo [1] ;  [1] ; ORCiD logo [1] ;  [1] ; ORCiD logo [1] ;  [1] ;  [1] ;  [1] ;  [2]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Kyoto Univ. (Japan)
Publication Date:
Report Number(s):
NREL/JA-5100-68889
Journal ID: ISSN 0099-2240
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 17; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; succinic acid; genetic modifications; biorefineries; Actinobacillus succinogenes; biochemical; biorefinery; fermentation; metabolic engineering
OSTI Identifier:
1395092