Development of microbial platforms

The number of carboxydotrophic isolates is still limited, and it is a challenge to isolate novel microorganisms able to convert CO at high rates and possibly produce distinct product profiling. Expanding the range of products from CO may encompass genetic engineering of already isolated or novel carboxydotrophs that can be explored for the production of chloromethane.This project focuses on the discovery and development of thermophilic carboxydotrophs for syngas fermentation. Further, it aims to design and build synthetic anaerobic microbial consortia for the tailored production of a broad range of high-value chemicals from CO/syngas. With the innovative synthetic mixed culture approach, the project targets to produce bespoke fashion products that cannot be produced by a single microorganism. 

This workpackage consists of 3 projects
Project 1: Novel biocatalysts for syngas conversion

Isolation of novel carboxydotrophic anaerobes from thermophilic environments 

While employing thermophilic microbes for syngas fermentation offers various benefits, the majority of isolated thermophilic carboxydotrophs tend to predominantly generate hydrogen. This project aims to isolate novel thermophilic carboxydotrophic microbes that can produce high value compounds from CO. The project is sub-divided into three research lines.

The first research line investigates the genomic potential of previously isolated thermophilic carboxydotrophs. Genomes from thermophilic carboxydotrophs were examined for genes related to high-value compound production, such as ethanol and acetone.

The second research line explores the carboxydotrophic potential of microbial communities in diverse hydrothermal environments. Utilizing publicly available metagenomes, the aim is to identify biotechnologically relevant microbial candidates with the capacity to consume CO and generate valuable compounds.

The third research line focuses on isolating novel carboxydotrophic microbes. Microbial enrichments were conducted using thermophilic anaerobic sludge samples. This targeted approach aims to expand the repertoire of thermophilic carboxydotrophs capable of producing high-value compounds from CO.

Metabolic engineering of acetogenic carboxydotrophic microorganisms for the production of volatile compounds 

This project focuses on unlocking the untapped potential of Clostridium autoethanogenum, a syngas-converting microbe, for synthesizing biochemical building blocks not previously achieved from syngas. 

The first focus of the research involves the production of methyl chloride from carbon monoxide. Methyl chloride is a gaseous compound with versatile applications in various industries. The engineered microbial chassis, C. autoethanogenum, was equipped with the necessary enzymatic pathway utilizing methyl halide transferase (MHT) from the saltwort Batis maritima. Despite successful proof of principle, the yield of methyl chloride was low, prompting optimization attempts, including the exploration of different promoters for MHT expression and media optimization for chloride supplementation. Although some improvements were achieved, the overall production remained unsatisfactory.

Due to the challenges encountered with methyl chloride, the project shifted its focus to ethyl acetate, another volatile compound with potential as a biodegradable solvent. The microbial pathway for ethyl acetate production relies on alcohol acyl transferases (AATs), specifically Eat1 and Atf1 from yeast origin. Despite successful proof of principle, final yields of ethyl acetate from syngas were also low. The cause was attributed to a lack of functional enzyme activity, particularly the inactivity of Eat1 in C. autoethanogenum, though the reason for this remains unknown.

In parallel with product-focused research, efforts are dedicated to understanding the central metabolism of acetogens. Explorations of ethanol metabolism via Ald and Adh enzymes unveiled a reversible process, demonstrating the potential for converting acetyl-CoA to ethanol and vice versa. Additionally, the role of the Pta pathway, essential for acetogens, is under scrutiny, as a Pta knockout unexpectedly displayed growth on carbon monoxide. The unexpected growth of Pta knockout on carbon monoxide triggers an in-depth exploration of its implications for acetogenic metabolism.

Project 2: Synthetic microbial consortia for syngas conversion

This research presents an innovative method for the production of polyhydroxy-butyrate (PHB) from syngas. While previous studies focused on utilizing pure CO with genetically modified acetogens or introducing CO and acetate to unmodified Rhodospirillum rubrum, this project pioneers a novel technique employing a synthetic co-culture.  

The system relies on the synergistic collaboration between R. rubrum and an acetogen. R. rubrum is well known to gain ATP and/or reducing equivalents from CO through the biological water gas shift reaction, producing H2 and CO2. The ATP and reducing equivalents are used to assimilate CO2 into biomass, and under certain conditions PHB. To circumvent the ATP-intensive reverse TCA cycle and eliminate the need for acetate addition, an acetogen can be introduced that converts H2 and CO2 to acetate. This strategic integration ensures the conversion of CO into PHB without relying on secondary substrates.

Project 3: Biokinetic modeling for syngas process design

This project aims to comprehensively comprehend the metabolism of microorganisms involved in syngas fermentation, with a particular focus on extrapolating their behavior in industrially relevant settings. Additionally, the objective is to diversify the product range of syngas fermentation coupling it to microbial chain elongation. Thus, the project comprises of two distinct research lines. 

The first line concentrates on the kinetic characterization of syngas fermenting organisms, employing Clostridium autoethanogenum as a model acetogen. Through bioreactor experiments, particularly pulse-feeding strategies in steady-state chemostats, the metabolic strategy of C. autoethanogenum is examined under various conditions. Key kinetic parameters, such as maximum specific substrate uptake rate and maximum growth rate are identified experimentally and theoretically. Beyond microbial rate characterization, the project explores how different cultivation conditions impact the process output, emphasizing the significance of the type of end products (acetate or ethanol) in facilitating the production of longer organic acids through chain elongation.

The second line explores microbial chain elongation in open cultures, addressing co-cultivation challenges. Efforts aim to guide the product spectrum towards medium-chain fatty acids, and a mathematical model is being developed to understand and optimize these organisms. Additionally, efforts are directed towards understanding how to direct the product spectrum of chain elongating organisms towards medium-chain rather than short-chain fatty acids. This interdisciplinary approach seeks to advance understanding and practical application in syngas fermentation and microbial chain elongation.