The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain.

Jonathan Lo,Joshua Jack,Wei Xiong,Zhiyong Jason Ren,Lauren Magnusson,Pin-Ching Maness,Yang Gu,Chris Urban,Jonathan R Humphreys

doi:10.3389/fbioe.2020.560726

Abstract

The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO2, waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

Highlights

Acetogenic bacteria utilize the Wood-Ljungdahl pathway (WLP) to non-photosynthetically fix inorganic carbon into acetyl-CoA, which is converted into products, normally acetate and to a lesser extent, ethanol
Acetate formation is a key characteristic of acetogens, which utilize the WLP to fix CO2 and produce primarily acetate
Under a wide variety of conditions in C. ljungdahlii, Pta is unnecessary for acetate formation, despite being normally understood as a critical enzyme for ATP production and acetate formation

Summary

Introduction

Acetogenic bacteria utilize the Wood-Ljungdahl pathway (WLP) to non-photosynthetically fix inorganic carbon into acetyl-CoA, which is converted into products, normally acetate and to a lesser extent, ethanol. In Clostridium ljungdahlii, a model acetogen, acetate formation primarily occurs sequentially with the conversion of acetyl-CoA to acetyl-P via phosphotransacetylase (PTA) and from. Acetate generation is important for ATP formation, as acetogens have low ATP budgets because they survive on the “thermodynamic limit of life”, on a small free energy change (Schuchmann and Müller, 2014). Based on genome information and published data, C. ljungdahlii is believed to make ethanol from acetylCoA via two established mechanisms: aldehyde ferredoxin oxidoreductase (AOR) and bifunctional aldehyde/alcohol dehydrogenase (AdhE) (Köpke et al, 2010; Leang et al, 2013). AOR catalyzes the reversible conversion of acetate to acetaldehyde, with ferredoxin as the electron acceptor/donor. AdhE is a bifunctional enzyme, catalyzing the reversible conversion of acetyl-CoA to acetaldehyde, to ethanol, using NAD(P)H as the electron donors

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Results

Conclusion