Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Metabolic Remodeling: Stylish Options for Bacterial Interior Design

Authors:

Ellen L. Neidle* (eneidle@uga.edu), Alyssa C. Baugh, Justin B. Defalco, Isabella R. Zempel, Frank M. Harris

Institutions:

University of Georgia–Athens

Goals

This project seeks to expand the metabolic capabilities of a genetically malleable soil bacterium, Acinetobacter baylyi ADP1. This strain naturally degrades a wide variety of plant-derived aromatic compounds. Augmentation and alteration of these natural capabilities have the exciting potential to improve biotechnology applications ranging from lignin valorization to biomanufacturing. This team’s specific aims are to create novel pathways for the catabolism of syringol and pyrogallol and to develop new methods for large-scale genomic remodeling.

Abstract

Advances in bacterial metabolic engineering and synthetic biology enable comprehensive genomic change. Altered aromatic compound metabolism in Acinetobacter baylyi ADP1 is facilitated by its exceptionally efficient natural transformation system. In this project, success was achieved by combining multiple approaches including enzyme design and the construction of modular synthetic pathways. Nevertheless, the biological consequences of such manipulation are often unpredictable. To achieve desired results, a growth-based adaptive evolution method was developed called Evolution by Amplification and Synthetic biology (EASy) (Tumen-Velasquez 2018). With this method, the targeted amplification of chromosomal regions serves as a rudimentary form of regulation to balance expression of different pathway segments.

Researchers focused on creating a pathway for syringol (2,6-dimethoxyphenol) degradation. This compound arises during lignin pyrolysis from the decomposition of sinapyl alcohol moieties. When converting lignin-derived mixtures to valuable products by microbes, syringol can be problematic both as an inhibitory compound and as an underutilized substrate. Since there is no characterized pathway for syringol consumption, researchers designed one to be expressed from the A. baylyi chromosome. In general, aromatic compound catabolism can be considered modular. The first module involves reactions to generate one of a limited number of aromatic substrates of ring-cleavage enzymes. The next key step is ring-cleavage itself, accomplished aerobically using ortho (intradiol) or meta (extradiol) dioxygenases. Finally, a multistep “lower” pathway typically feeds metabolites to central metabolism.

Researchers sought to convert syringol to pyrogallol, a potential ring-cleavage target. Pyrogallol cleavage can be mediated by some ortho– and meta-catechol dioxygenases, although specific protein sequences and responsible enzymes remain unknown. Strains were constructed to express combinations of different catechol dioxygenases and a guaiacol demethylase (GcoAB) variant, which converts syringol to pyrogallol (Machovina et al. 2019). The team’s initial attempts failed to express two separate enzymes capable of producing and cleaving pyrogallol from syringol. In contrast, colorometric assays suggested that the rational design of a novel chimeric enzyme successfully led to the in vivo metabolism of syringol and to the cleavage of pyrogallol by A. baylyi cells. The design of this chimeric enzyme was based on a similar enzyme that emerged from EASy experiments using guaiacol as a growth substrate (Tumen-Velasquez 2018). Key to success was the choice of sequence encoding an ortho-cleaving catechol dioxygenase with augmented activity on pyrogallol compared to the native version of this enzyme (CatA). Thus, these steps, mediated by a fabricated enzyme, represent two modules of a synthetic pathway for syringol degradation. As the final module to enable syringol to be used as growth substrate, the team incorporated a foreign pathway that is not native to A. baylyi for protocatechuate metabolism. Researchers mixed and matched these modules for functionality using a variety of different aromatic growth substrates. Growth on aromatic substrates using these non-native pathways involved targeted gene amplification and combinations of mutations selected during laboratory evolution. Collectively, these results highlight the feasibility of large-scale genomic remodeling for biotechnology. ADP1 offers exciting potential for further development as a synthetic biology chassis.

References

Machovina M. M, et al. 2019. “Enabling Microbial Syringol Conversion Through Structure-Guided Protein Engineering,” Proceedings of the National Academy of Sciences of the U.S.A.116,13970–76. DOI:10.1073/pnas.1820001116.

Tumen-Velasquez, M., et al. 2018. “Accelerating Pathway Evolution by Increasing the Gene Dosage of Chromosomal Segments,” Proceedings of the National Academy of Sciences of the U.S.A. 115, 7105–10. DOI:10.1073/pnas.1803745115.

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE- SC0022220. A.C.B. received support for some preliminary work from the DOE Office of Science Graduate Student Research (SCGSR) program.