Organisms engineered to churn out valuable chemicals tend to slack off when they should be working most productively, after they’ve been introduced to large-scale fermentation vessels. But why? Some feel that large-scale vessels are plagued with physical inefficiencies. Some feel that in large populations of microbes, there are bound to be a few bad apples, phenotypic variants that feel they’ve got the right to coast. Eventually, these variants spoil the whole barrel, err…bioreactor.

Another productivity-degrading mechanism, however, may be just as relevant. It’s called evolution. According to scientists at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark (TUM), evolution that occurs within bioreactors or fermentation vessels may constrain bioproduction. To reach this conclusion, the scientists used ultra-deep time-lapse DNA sequencing and population-level bioinformatics. Not only did the scientists confirm their hunch that evolution matters even over the short time courses of industrial-scale production, they also developed an approach that could help constrain evolution, and thus boost productivity.

The ultra-deep sequencing and bioinformatics work appeared February 20 in the journal Nature Communications, in an article entitled “Diverse Genetic Error Modes Constrain Large-Scale Bio-Based Production.” The article describes how the TUM scientists experimentally simulate large-scale fermentation with mevalonic acid-producing Escherichia coli.

“By tracking growth rate and production, we uncover how populations fully sacrifice production to gain fitness within 70 generations,” the article’s authors wrote. “…we identify multiple recurring intra-pathway genetic error modes. This genetic heterogeneity is only detected using deep-sequencing and new population-level bioinformatics, suggesting that the problem is underestimated.”

Results hint that engineered production genes in chemical producing bacteria were mainly mutated by nonexpected disruptions and genetic rearrangements, rather than the slower, classical point mutations known as single-nucleotide polymorphisms (SNPs). The mutations make the nonproducing cells more fit in the competition for nutrients of a fermentation tank.

“We discovered that a wide diversity of genetic disruptions turned producing cells into nonproducing cells when we deep-sequenced thousands of production organisms over time. Cells have many built-in ways to remove unneeded genes, and it turned out the most important is overlooked in standard analysis tools,” said Peter Rugbjerg, Ph.D., a postdoc at the Novo Nordisk Foundation Center for Biosustainability.

The evolutionary mutants that eventually led to complete loss of production could be detected very early at frequencies below 0.1%, which may permit future identification of failing fermentations earlier.

Residing at the ultra-low frequency when grown in the laboratory, the mutants played no negative role in production in shake flasks and were traditionally difficult to detect. However, the advantage of lost production means these early mutations had already determined the magnitude of production decline that happens as the process is scaled up to industrial growth durations.

“Based on these findings I would encourage companies running industrial-scale fermentations to deploy deep sequencing of the fermentation populations to assess the extent of detrimental evolution,” said Morten Sommer, Ph.D., noted professor and scientific director of the Bacterial Synthetic Biology section at the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark.

The scientists also suggested that biomanufacturers consider strategies for postponing genetically driven production declines. One such strategy was detailed in another study published by the TUM team.

This study appeared February 20 in the Proceedings of the National Academy of Sciences, in an article entitled “Synthetic Addiction Extends the Productive Life Time of Engineered Escherichia coli Populations.” This article describes how the TUM team postponed detrimental evolution by synthetically addicting production cells to production. The TUM scientists carefully linked signals of product presence to expression of nonconditionally essential genes.

“We addict Escherichia coli cells to their engineered biosynthesis of mevalonic acid by fine-tuned control of essential genes using a product-responsive transcription factor,” the article’s authors reported. “Over the course of a long-term fermentation equivalent to industrial 200-m3 bioreactors such addicted cells remained productive, unlike the control, in which evolution fully terminated production.”

The key, the scientists noted, is to rewire production cells to grow only when they contain high product concentration. Thus, evolution can be circumvented and cells will be able to produce the biochemicals within an industrial timescale.

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