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

Multichromatic Optogenetic Control of Microbial Co-Culture Populations for Chemical Production

Authors:

Jaewan Jang1,2* (jaewanj@princeton.edu), José L. Avalos1,2,3,4

Institutions:

1Department of Chemical and Biological Engineering, Princeton University; 2Omenn-Darling Bioengineering Institute, Princeton University; 3Department of Molecular Biology, Princeton University; 4The Andlinger Center for Energy and the Environment, Princeton University

Goals

The goal of this project is to develop and apply multichromatic optogenetic tools in bacteria and yeast to control co-culture populations. Researchers have developed ways to alter microbial growth rates of different strains using blue, dark, red, or near-IR light by controlling the expression of an essential gene or toxin/antitoxin systems in yeast or bacteria, respectively (Wegner et al 2022; Lalwani et al 2021). Testing different light duty cycles and wavelengths allow for the exploration of optimal microbial population ratios when combined with computational methods for real time feedback. This work demonstrates the first example of polychromatic control in microbial co-cultures to maximize the production of valuable commodity chemicals and biofuels.

Abstract

Metabolic engineering enables the sustainable production of valuable chemicals, drugs, or biofuels from low-cost renewable substrates by re-wiring microbial metabolism. However, growth defects caused by excessive metabolic burden, suboptimal expression/activity of heterologous enzymes, and endogenous regulatory mechanisms often limit microbial productivities (Wegner et al 2022). These challenges can be addressed by dividing the labor among different microbes in synthetic microbial communities (consortia or co-cultures). Fragmenting biosynthetic pathways among different strains of bacteria or yeast, each producing unique intermediates, significantly reduces the metabolic burden, while harnessing special capabilities of different microbial species. This strategy also helps optimize each metabolic module in separate strains, override endogenous regulatory mechanisms, and avoid competing pathways to maximize flux through the biosynthetic pathway of interest. However, stabilizing and controlling the composition of microbial consortia is a formidable challenge (Duncker et al 2021).  While some strains grow quickly, others lag–allowing the fast-growing members to take over the culture. Researchers apply optogenetics, where cellular processes are optically controlled using photoswitchable proteins that change shape and function in response to light, to maintain population ratios in co-cultures.

Light as gene inducers is nontoxic, tunable, and inexpensive, unlike chemical inducers. However, optogenetic control of microbial populations has only been demonstrated with blue light and only to control the growth rate of one strain in a two-member consortium, in which the optically controlled member grows significantly faster than the uncontrolled (blind) strain. Thus, an optogenetic tool other than blue light is in need for metabolic engineering applications. Researchers established red/near-IR systems, which enables the control of more complex microbial communities, including ones containing members of comparable growth rates. Combining this system with blue light circuits provide a multichromatic control over bacteria and/or yeast consortia populations (Wegner et al 2022). In principle, four strains (bacteria/yeast) under different optogenetic circuits (blue, darkness, red, near-IR) can be combined to afford complex, multichromatic microbial consortia. This allows the engineering of microbial community members to cooperatively produce various commodity chemicals and biofuels, such as isobutanol, possibly maximizing their titers with various light schedules. This work will significantly advance the use of optogenetic control of microbial communities, which is a new paradigm with enormous potential to not only improve the basic understanding of microbial community interactions, but also to overcome the obstacles that have stifled the use of synthetic microbial consortia for biotechnological applications. These multichromatic co-culture methods are generalizable and can easily be commercialized when significant yield of any important fine chemicals is achieved.

Image

Multichromatic microbial fermentation

References

Duncker, K. E., et al. 2021. “Engineered Microbial Consortia: Strategies and Applications,” Microbial Cell Factories 20(211).

Lalwani, M. A., et al. 2021. “Optogenetic Control of Microbial Consortia Populations for Chemical Production,” ACS Synthetic Biology 10(8), 2015–29.

Wegner, S. A., et al. 2022. “The Bright Frontiers of Microbial Metabolic Optogenetics,” Current Opinion in Chemical Biology 71, 102207.

Funding Information

This research was supported by the DOE Office of Science, BER Program, (Award Numbers DE-SC0019363 and DE-SC0022155).