Biomanufacturing depends on microbial platforms. For efficiency’s sake, these single-celled production engines must maximize output while minimizing inputs, including energy inputs, which ultimately take the form of adenosine-5′-triphosphate (ATP). But how, exactly, should biomanufacturing’s pit crews keep microbial platforms well-tuned?

To answer this question, scientists at Washington University in St. Louis (WashU) looked under the hood of microbial metabolism. Specifically, they employed a genetically encoded ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. This work allowed the scientists to elucidate the relationship between ATP dynamics and bioproduction, and also to generate suggestions for enhancing bioproduction in various microbial species.

Details appeared recently in Nature Communications, in an article titled, “ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction.”

“We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively,” the article’s authors wrote. “We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction.”

Because ATP levels vary across different growth phases and carbon sources in multiple microbial strains, it is critical to map the connections between ATP levels and microbial growth and nutrient quality and how ATP levels affect yields of the microbial products. And yet these connections have remained largely unexplored.

Going where few have gone before, Fuzhong Zhang, PhD, the Francis Ahmann Professor in the McKelvey School of Engineering and co-director of the Synthetic Biology Manufacturing of Advanced Materials Research Center (SMARC), led the WashU effort. Zhang and colleagues studied ATP dynamics in various fermentation conditions and developed a cost-effective approach to enhance bioproduction through supplementation of ATP-promoting carbon sources.

“This study has broad implications for understanding microbial energy homeostasis, optimizing bioproduction processes, and identifying sources of metabolic burden,” said Xinyue Mu, a PhD student in Zhang’s laboratory and the first author of the paper.

Zhang and colleagues found that feeding microbes with different carbon sources results in very different ATP dynamics. “Normally you wouldn’t think acetate is a good carbon source for E. coli,” said Mu, who noted that acetate is considered a byproduct of glucose metabolism, something E. coli excretes when eating glucose. “Actually, by feeding it acetate, we see a higher ATP level associated with an enhanced yield of target products,” she added.

This is an encouraging result for those who are interested in using acetate as a feedstock. It is especially for the McKelvey researchers because they are already working on methods that can convert carbon dioxide to acetate.

For P. putida, the preferred feedstock consisted of fatty acids. They substantially enhanced PHA content, yields, and productivity.

In addition to finding beneficial carbon sources for fermentation, the study also demonstrated that the ATP biosensor could be used to shed light on complicated metabolic processes. The process considered in the study involved the synthesis of limonene, which generally delivers low yields. Also, this process is notorious for sucking up ATP and reducing cell growth.

“In the production of limonene by E. coli, heterologous enzyme expression significantly reduced ATP levels, impacting limonene yields without substantially affecting cell growth,” the article’s authors reported. “Similar energy burdens are likely to present in the production of other chemicals from ATP-demanding pathways. Consequently, heterologous protein expression must be carefully tuned to maintain ATP balance, with the ATP sensor serving as a powerful tool for identifying proteins and reactions that impose energy burdens on host cells.”

“This work,” Zhang concluded, “not only elucidates the relationship between ATP dynamics and bioproduction, but also offers a simple and effective strategy to enhance bioproduction by choosing an ATP-beneficial feedstock. It is useful to various biomanufacturing systems.”

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