A New Tool in the Fermentation Arsenal: Transcriptomics

Industrial fermentations present unique challenges for microbes.  So the pathways to scaling up industrial fermentations present formidable challenge for strain developers and process engineers. Strains must first be engineered, evolved, and/or discovered to have desirable traits and performance. This is difficult enough but since development work has to be performed at  lab-scale and then has to be transitioned to the production site, it is easy to see why many interesting and arduously developed microbes never make it to commercialization.

Why are industrial fermentations so challenging for microbes?  Microbes are derived from strains that undergo millions of years of evolution in wild environments. Adapting their behavior and metabolic and reproductive processes to cost-effective production timescales and feedstocks is not always apparent or direct.

How does looking at gene expression help to improve fermentation runs? The key is the just-in-time gene expression regulation that all organisms, prokaryotes, archaea, and eukaryotes perform. When an organism is stimulated to perform a particular task, such as the activation of a biosynthesis pathway, many biochemical steps must occur in rapid succession.

First, a sensor-signal activates a signaling cascade leading up to the biosynthesis pathway. Most of the components of a non-constitutive pathway are only produced when the cell needs them, otherwise their production would burden the cell with an unacceptable cost in ATP and amino acids to keep proteins at the ready, synthesized, folded, and trafficked to the proper cellular organelle. The pathway activation signaling typically turns on gene expression for the necessary genes. This action leads to rapid increases in the RNA from those genes which is then translated to proteins. Finally, these enzymes produce the desired molecule to adequate levels and a feedback signal then either (a) activates repressors or (b) discontinues the activating signaling for generation of the protein. Additional layers of regulation exist, but central to the energetic conservation of single-cell organisms is the production of proteins only when necessary. This means that the level of RNA at a particular time is a sensitive and global indicator of the proteins that the pathways in a cell are required by the cell at a specific fermentation phase.

Taking repeated samples in a time-series, Mimetics can construct a moving picture of the highly dynamic status of gene expression. RNA sequencing technology looks at the entire genome and Mimetics technology is a single platform providing a highly sensitive, global picture of what the cells are doing at each step of a fermentation run. Mimetics’ strength is using our technology to filter the thousands of genes dynamically expressed in each fermentation to discover the useful and relevant information for our clients. The clarity of this picture is enhanced by the fact that the cells share the environment inside a fermentation vessel, so that the signaling, gene expression, and cellular performance of the population is highly synchronized, making analysis possible from benchtop to industrial scale.

Mimetics has extensive experience tackling difficult-to-solve fermentation issues. In some cases we have detected that the fermenting microbes suffer from lack of a particular nutrient, inhibiting proliferation. In other situations, time-series analysis of gene expression regulation paired with metadata has shown how low-cost, precision dosing can ameliorate growth defects. We have also observed cases where stress signaling obstructs fermentation goals. This enabled Mimetics to lay out a roadmap for eliminating the negative consequences of that signaling in a microbe. Mimetics technology not only detects the expression of genes central to generating large or small molecules, but also tracks genes involved in translation, trafficking, folding, and transport of the fermentation product.  Finally, Mimetics gene expression analysis observes the transcriptomics of other microbial species that may be present in the vessel or feedstock; Mimetics’ analysis observes and reports both positive and negative interplay between the fermenting organism and the microbiome of the fermentation run.