(122c) Engineering C. Glutamicum to Improve b-Ketoadipate Pathway Productivity

With over 50 million tons of lignin generated as a waste product annually, it is seen as the most abundant renewable feedstock for aromatic molecules [1]. Through chemical pretreatment, lignin can be broken down into two major aromatics, p-coumarate and trans-ferulate. Due to its resistance to the toxicity of aromatic molecules, Corynebacterium glutamicum is capable of metabolizing aromatics [2]. Additionally, there are several valuable intermediates within the aromatic catabolism pathways of C. glutamicum including beta-ketoadipate, a precursor for nylon-like polymers [3]. By utilizing the native pathway in C. glutamicum, along with genetic modifications for optimization, we propose that the biomanufacturing of beta-ketoadipate can be enhanced. This project will focus on metabolically engineering C. glutamicum to improve the productivity of the beta-ketoadipate pathway, with the ultimate goal of enhancing the microbial utilization of lignin-derived substrates.

The beta-ketoadipate pathway plays a pivotal role in the microbial metabolism of aromatic compounds in C. glutamicum, serving as a key route for the degradation of lignin-derived aromatic compounds [4]. This pathway is a complex metabolic route responsible for the conversion of aromatic compounds into central metabolites, making it an attractive target for metabolic engineering to enable sustainable bioproduction. Through previous research, we have found that the 4-hydroxybenzoate → protocatechuate step is a major bottleneck in this pathway. Researchers have found an alternative enzyme to pobA, praI [5], that can also convert 4-hydroxybenzoate into protocatechuate. In this project, I compare the catabolic efficiency of two different strains of C. glutamicum. One strain with pobA, one with praI. After creating the strains, I will feed them different substrates including p-coumarate, 4-hydroxybenzoate, and trans-ferulate, and look for other potential bottlenecks in the pathway to improve beta-ketoadipate production.

I have already cloned praI into a production strain, the next step in this project would be to clone and purify the pobA. Once we confirm that we have the correct gene, I will begin to construct a plasmid with pobA. PCR and gel electrophoresis would need to be used in order to verify that our constructed plasmids are correct. The next step would be to transform my plasmids into Escherichia coli. This step is necessary because transforming straight into C. glutamicum would have very low efficiency. After this, I will extract the recombinant plasmid and transform it a second time into a specialized strain of E. coli that will demethylate the plasmid. C. glutamicum will not accept methylated plasmids so transforming into a demethylation strain of E. coli first is required. Next, I will extract the demethylated plasmid and finally transform it into C. glutamicum to make a second production strain. Once I confirm that the C. glutamicum has accepted my plasmid, and they are correct, I can begin to test beta-ketoadipate production.

I will begin by creating cultures with each strain containing my plasmids and feeding them 4-hydroxybenzoate, sampling each culture daily. Once they stop growing I will run the samples I collected on a high-performance liquid chromatography machine. This will show me how much beta-ketoadipate is being produced, and will also allow me to identify if the protocachuate bottleneck was resolved. Pending the results, I would like to feed my cultures other substrates including p-coumarate, trans-ferulate, a mixture of different aromatic substrates, and then lignin-hydrolysate in order to show real-world application. In addition to this, I would like to do a 13C analysis on the pathways in both strains to see whether any of the intermediates are leaking into the central metabolism of C. glutamicum.