Co-feeding strategies for muconic acid and adipic acid production by Acinetobacter baylyi ADP1

Abstract

The escalating environmental challenges linked to climate change, fossil oil depletion, and fossil fuel consumption are heightening the demand for sustainable alternatives. A promising solution is the biological production of valuable chemicals using renewable and abundant raw materials such as lignin. While microbes are versatile and hold vast potential, challenges in production often arise from the delicate balance between microbial growth and product formation. Microbes prioritize optimizing growth and essential cellular functions, limiting various microbial processes by avoiding the overproduction of specific compounds. Therefore, it becomes crucial to implement necessary strategies such as decoupling growth and production, co-feeding, and process optimization, to maintain a balanced flux of carbon and other nutrients for cellular functions.

This study aims to evaluate the capabilities of metabolically engineered Acinetobacter baylyi ADP1 strains in producing two important chemicals—adipic acid and its precursor muconic acid— utilizing carbon sources derived from lignin. A. baylyi ADP1 emerges as a potential host due to its nutritional versatility and exceptional competencies for natural transformation and homogenous recombination. Moreover, its native aromatics degradation pathways enable the utilization of lignin derived molecules as carbon sources. This research studies the effects of decoupling growth and product formation, along with different co-feeding strategies, on yields in these bioprocesses.

The experimental work of this thesis involved cultivating genetically engineered A. baylyi strains in batch processes and bioreactor processes, with a primary focus on muconate synthesis. Special emphasis was placed on exploring the impact of introducing a carbon source mixture of ferulate and coumarate, two model compounds abundant in lignin, on the muconate-producing process. Results indicate that the engineered A. baylyi ASA917 strain successfully produced muconic acid from a mixture of ferulate and coumarate. Utilizing a two-phase cultivation approach, which separates biomass accumulation and product formation into distinct sub-processes, a molar yield of over 80% was achieved. These results are excellent and compete well with results obtained in previous studies with other microbe strains. The outcomes suggest the potential for more sustainable muconic acid and, with a single conversion step, adipic acid production in the future by leveraging diverse lignin streams and utilizing ADP1 strains.

In direct adipic acid production experiments of this study, adipic acid was produced, but only in trace concentrations. The efficiency of this engineered production pathway was limited, with the bottleneck identified between the last conversion steps from substrate to adipic acid. Despite an intermediate compound being produced in significant quantities with a molar yield of 60% from 4-hydroxybenzoate in the A. baylyi ASA932 strain, further research is required to fully demonstrate the potential of these strains in direct adipic acid production. The substantial amount of an intermediate obtained in the cultivations also presents a promising starting point for future research.