Broadening Aromatic Compound Degradation in Acinetobacter baylyi Using Synthetic Metabolic Pathways
Alyssa Baugh1* (firstname.lastname@example.org), Melissa Tumen-Velasquez,2 Ramesh K. Jha,3 and Ellen L. Neidle1
1University of Georgia; 2Oak Ridge National Laboratory; and 3Los Alamos National Laboratory
This project expands the ability of the soil bacterium Acinetobacter baylyi ADP1 to degrade aromatic compounds. Applications range from lignin valorization to bioremediation of recalcitrant environmental pollutants. Biotechnology applications will benefit from new methods to design and evolve efficient pathways for the metabolism of specific compounds. Pathways targeting the degradation of aromatic compounds were constructed and evolved in ADP1, an ideal model organism for synthetic metabolic pathways. To enable consumption of target compounds, researchers are combining parts of characterized foreign pathways. For compounds without known metabolic routes, including pyrogallol and syringol, the synthetic pathways incorporate modified, as well as foreign, enzymes. Pathways are improved via laboratory evolution, targeted gene amplification, and serial transfer, which enable growth-based selection. Novel metabolic functions can be demonstrated in ADP1 and exported to other microbes.
Lignin is a vastly underutilized, energy-rich renewable resource. Initial processing yields heterogenous mixtures of aromatic compounds that are naturally degraded slowly by microbial consortia. Industrial applications could benefit from combining the metabolic abilities of these bacteria into a single laboratory strain. Carbon funneled through central metabolism could then be used to synthesize a product. Researchers use A. baylyi ADP1, a genetically malleable bacterium in which chromosomal changes can be introduced by simply mixing cells and linear DNA fragments (Bedore et al. 2023). During adaptive evolution, genetic regions can be duplicated, facilitating the accumulation of beneficial mutations (Pardo et al. 2023). Together, these methods offer a generalizable system to create catabolic modules that can be mixed, matched, and applied to diverse aromatic compounds.
As the goal is the metabolism of compounds with unknown natural degradative routes, the team designed a pathway for one such compound, pyrogallol. Extradiol cleavage enzymes that use a structurally similar substrate, catechol, have been reported to cleave pyrogallol, albeit poorly. Bacteria have been discovered to use pyrogallol as a carbon source, but the natural catabolic pathway is unclear. As a first step in the synthetic pathway, researchers inserted genes from Pseudomonas putida into the A. baylyi chromosome. These genes encode extradiol catechol dioxygenases, enzymes that might be modified for pyrogallol cleavage. Two different enzymes, XylE and TodE, replaced a native catechol dioxygenase, which, unlike each foreign enzyme, catalyzes intradiol ring cleavage. Thus, the ring-cleavage products generated by the foreign enzymes are not normally encountered by A. baylyi. A second module is needed to route the non-native ring-cleavage intermediates to central metabolism. This second module consists of a foreign catabolic pathway encoded by multiple pra genes. This foreign pra-gene pathway was previously expressed in A. baylyi, and its functionality was achieved during laboratory evolution. The pra-encoded enzymes can route the products of both catechol and pyrogallol cleavage to central metabolism.
There are several challenges to this approach. Enzymes often initially function sub-optimally in new hosts. Therefore, functionality of XylE or TodE is not guaranteed, even for cleaving the natural substrate, catechol. Additionally, in using catechol or pyrogallol as the carbon source, the catechol dioxygenase must work in conjunction with the pra-encoded enzymes. This pathway comes from a Paenibacillus species with a different natural substrate (protocatechuate) and regulatory context. Growth requires balanced metabolism of many chemical intermediates, several of which are toxic. As a final hurdle, the function of a non-native dioxygenase must be honed and altered to improve activity on pyrogallol, rather than catechol.
Strategies to engineer pyrogallol degradation exploit growth-based laboratory evolution and methods to alter gene dosage. Genes encoding a catechol dioxygenase and the Pra pathway are first integrated in different positions in the A. baylyi chromosome. Independent gene amplification of either module can facilitate selection of functional strains (Tumen-Velasquez et al. 2018). Fluorescent biosensors can detect pathway intermediates to assist strain selection (Jha et al. 2015). After strains with novel capabilities are isolated, the contribution of mutations to the resulting phenotype, identified via whole genome sequencing, can be determined using transformation methods (Bedore et al. 2023).
In this project researchers evolve and select for A. baylyi strains with foreign extradiol catechol dioxygenases capable of cleaving aromatic growth substrates. In these strains, growth using catechol, anthranilate, and benzoate, each as a sole carbon source, required interdependent functions of a foreign catechol dioxygenase and pra-encoded enzymes. The current focus is altering substrate specificity of the dioxygenase to allow use of pyrogallol as the carbon source. Through strategic design of synthetic pathways, researchers are developing A. baylyi ADP1 for biotechnology applications. The unique genetic system of this strain allows researchers to rapidly engineer strains, interchanging different metabolic and regulatory modules.
Bedore, S. R., et al. 2023. Methods in Microbiology. 52:3.
Pardo, I, et al. 2023. Methods in Microbiology. 52:3.
Tumen-Velasquez, M, et al. 2018. “Accelerating Pathway Evolution by Increasing the Gene Dosage of Chromosomal Segments.” The Proceedings of the National Academy of Sciences U.S. 115(27), 7105–10.
Jha, R. K., et al. 2015. “Rosetta Comparative Modeling for Library Design: Engineering Alternative Inducer Specificity in a Transcription Factor.” Proteins: Structure, Function, and Bioinformatics 83(7), 1327–40.
This research was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0022220. Los Alamos National Laboratory is operated by Triad National Security, LLC, for the National Nuclear Security Administration of U.S. Department of Energy (Contract No. 89233218CNA000001). This program is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) under FWP LANLF32A. A.B. received support for some preliminary work from the DOE Office of Science Graduate Student Research (SCGSR) program.