Electron availability in CO₂, CO and H₂ mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii

Abstract

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO 2 footprint by converting industrial emissions containing CO 2 , CO and H 2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO 2 + H 2 , CO and CO + CO 2 + H 2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood-Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD + provider for CO 2 + H 2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd red formation when CO is used as substrate, whereas Fd red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO 2 + H 2. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

“…Details of strain construction are described elsewhere Weitz et al (2021) . Medium and preculture seed train was described earlier ( Hermann et al, 2020 ). The last pre-culture step was based on syngas, characterized by the same gas composition as the bioreactor substrate.…”

“…Anaerobic syngas-based batch cultivations were performed in a fully controlled 3-L stirred-tank bioreactor (Bioengineering, Wald, Switzerland) with an operational volume of 1.5 L. The detailed reactor equipment was previously described in Hermann et al (2020) . Temperature and pH were kept constant at 37°C and 5.9, respectively.…”

“…The extracellular formation of ethanol, acetate, 2,3-butanediol, lactate, and isobutanol was observed using an isocratic high-performance liquid chromatography (HPLC) equipped with a RI detector and a Rezex ROA-Organic Acid H + column. Measuring parameters and sample preparation are described in Hermann et al (2020) .…”

“…Cell density was determined offline via a UV/Visible spectrophotometer at 600 nm. A detailed description is found in Hermann et al (2020). FIGURE 1 | Wood-Ljungdahl pathway and product formation of C. ljungdahlii.…”

“…Consequently, Fd red availability tightly links energy management, substrate composition, and product formation in C. ljungdahlii . In this context, we identified syngas as a suitable substrate to produce reduced alcohols, presenting the highest 2,3-butanediol formation using a batch process with C. ljungdahlii described so far ( Hermann et al, 2020 ). Furthermore, C. ljungdahlii is genetically accessible enabling the optimized formation of natural and recombinant products via metabolic engineering ( Huang et al, 2019 ; Molitor et al, 2016 ; Woolston et al, 2018 ).…”