Metabolic engineering of Clostridium ljungdahlii for the production of butanol and hexanol

Lauer, Ira; Blank, Lars M. (Thesis advisor); Büchs, Jochen (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


Acetogenic microorganisms possess the remarkable feature of metabolizing C1 gases (CO and CO2) to biomass and various products, mainly acetate. Since CO2 in particular plays a major role as a greenhouse gas and its concentration in the atmosphere is steadily increasing, acetogens could be used to reduce CO2 emissions from process gases. Although a few pilot plants are already producing ethanol from steel mill waste gases using Clostridium species, some further development of the microorganisms is still needed, especially in the field of metabolic engineering, to enable a wide range of products. With this goal in mind, this work investigates the utilization of syngas (CO, CO2, and H2) with the anaerobic acetogenic bacterium Clostridium ljungdahlii to produce the platform chemicals butanol and hexanol. In this work, a construct for the formation of butanol and hexanol was integrated into the genome of C. ljungdahlii for stable expression, thereby increasing the product titers, compared to a strain which expressed the corresponding genes plasmid-based. In a fermentation with continuous gas supply, 451 mg L-1 butanol and 122 mg L-1 hexanol were produced. Potential limitations of protein expression in the biosynthetic pathway were identified compared to the plasmid-based strain using LC-MS/MS-based targeted proteomics. After a promoter exchange in the following resulted in only a slight increase in C4 products (butanol and butyrate) normalized to biomass, an additional biosynthetic operon from the natural producer Clostridium carboxidivorans was introduced and also integrated into the genome, resulting in a twofold genomic integration. This caused a 17-fold increase in hexanol formation compared to the plasmid-based background strain in small-scale cultivations and provided strong evidence that chain elongation of butyryl-CoA with acetyl-CoA to the C6 molecule catalyzed by a thiolase was the limiting step in the hexanol biosynthetic pathway in the previous production strains. The ratio of alcohols produced in this strain shifted significantly toward the longer-chain alcohol hexanol with 393 mg L-1 hexanol and 109 mg L-1 butanol in a fermentation with continuous gas supply. Furthermore, a gas composition of 20% CO2, 80% H2 was identified as favorable for butanol and hexanol formation. The influence of different media additions was investigated, but no increase in product concentrations could be achieved with the tested parameters. In contrast, an increase in hexanol concentration could be obtained by changing process parameters such as reducing the cultivation temperature from 37 °C to 30 °C. Finally, it was shown that selected impurities present in industrial exhaust gas had no negative influence on growth and product formation in the production strain studied.