Title: Thermodynamic and Kinetic Modeling Directs Pathway Optimization for Isopropanol Production in a Gas-Fermenting Bacterium

Abstract

Rational engineering of gas-fermenting bacteria for high yields of bioproducts is vital for a sustainable bioeconomy. It will allow the microbial chassis to renewably valorize natural resources from carbon oxides, hydrogen, and/or lignocellulosic feedstocks more efficiently. To date, rational design of gas-fermenting bacteria such as changing the expression levels of individual enzymes to obtain the desired pathway flux is challenging, because pathway design must follow a verifiable metabolic blueprint indicating where interventions should be executed. Based on recent advances in constraint-based thermodynamic and kinetic models, we identify key enzymes in the gas-fermenting acetogen Clostridium ljungdahlii that correlate with the production of isopropanol. To this extent, we integrated a metabolic model in comparison with proteomics measurements and quantified the uncertainty for a variety of pathway targets needed to improve the bioproduction of isopropanol. Based on in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, we identified the top two significant flux control sites, i.e., acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC), overexpression of which could lead to increased isopropanol production. Our predictions directed iterative pathway construction, which enabled a 2.8-fold increase in isopropanol production compared to the initial version. The engineered strain was further testedmore » under gas-fermenting mixotrophic conditions, where more than 4 g/L isopropanol was produced when CO, CO₂, and fructose were provided as the substrates. In a bioreactor environment sparging with CO, CO₂, and H₂ only, the strain produced 2.4 g/L isopropanol. Our work highlighted that the gas-fermenting chasses can be fine-tuned for high-yield bioproduction by directed and elaborative pathway engineering. IMPORTANCE Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering. Here, we provide a case study by engineering isopropanol production in gas-fermenting Clostridium ljungdahlii. We demonstrate that a modeling approach based on the thermodynamic and kinetic analysis at the pathway level can provide actionable insights into strain engineering for optimal bioproduction. This approach may pave the way for iterative microbe redesign for the conversion of renewable gaseous feedstocks« less

Authors:

Lo, Jonathan ^[1]; Wu, Chao ^[1]; Humphreys, Jonathan R. ^[1]; Yang, Bin ^[1]; Jiang, Zhenxiong ^[2]; Wang, Xin ^[2]; Maness, PinChing ^[1]; Tsesmetzis, Nicolas ^[3]; Xiong, Wei ^[1]; Bucci, ed., Vanni

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+ Show Author Affiliations

1. Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, USA
2. Department of Microbiology, Miami University, Oxford, Ohio, USA
3. Shell International Exploration and Production, Inc., Houston, Texas, USA

Publication Date:

Thu Apr 27 00:00:00 EDT 2023

Research Org.:

National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Org.:

USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); Shell International Exploration and Production; Miami University

OSTI Identifier:

1963526

Alternate Identifier(s):

OSTI ID: 1969535

Report Number(s):

NREL/JA-2700-82557
Journal ID: ISSN 2379-5077; e01274-22

Grant/Contract Number:  

AC36-08GO28308

Resource Type:

Published Article

Journal Name:

mSystems

Additional Journal Information:

Journal Name: mSystems Journal Volume: 8 Journal Issue: 2; Journal ID: ISSN 2379-5077

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Clostridium ljungdahlii; gas fermentation; isopropanol; metabolic robustness analysis; thermodynamics analysis