(671d) Engineering of Oxidative Stress-Tolerant Yeast to Support Continuous Biomanufacturing of Chemicals from Acetate

There are multiple advantages of continuous process over batch operation. First, it minimizes equipment downtime hence increases volumetric productivity; as the consequence, the plant can be built with smaller footprint hence reduced capital investment. Additionally, it saves money, time and energy through reduction of inventory and transportation costs; it reduces the overall waste; and it reduces the risk of human error because the whole process can be automated and/or there will be fewer people involved in the production pipeline.

While there are many examples of continuous chemical plants, continuous biomanufacturing plants (where the product synthesis is conducted by cell factories) are rare. In fact, today’s biomanufacturing is dominated by batch and fed-batch processes, even though it is continuous fermentation that allows for growth-coupled biochemical syntheses to occur at their highest possible rate.

Strain stability, which in the context of cell biofactories refers to its ability to produce the target product under a constant productivity level over time, is arguably the limiting factor. Under various stressful conditions typical of industrial fermentation, it is expected that cells will accumulate cellular damages faster compared to non-stressed cells. Damaged cells will obviously exhibit reduced productivity. At a constant dilution rate, over time they will accumulate in the fermenter, biasing the population towards the non-productive sub-group, thus reducing productivity of the whole population.

Among various stresses, oxidative stress due to self-produced reactive oxygen species (ROS) is arguably the one with the widest effect: ROS directly compromises the integrity of genome, proteome, and membrane system. In the context of yeast biofactories, elevated ROS level can happen when the cell overproduces secreted-, disulfide-bond-containing, or metal-cofactor-containing protein like antibody, hemoglobin, or cytochrome P450 monooxygenase (P450). The latter is especially overproduced in strains dedicated for natural products synthesis, and its catalytic activity itself generates ROS.

The use of acetate as carbon source in fermentation has been considered as a better approach to biorefinery because this carbon source can be generated in a cheaper, more sustainable way, without a need to compete with food. It can renewably be generated through pyrolysis of lignocellulosic biomass, anaerobic fermentation of municipal solid waste, and syngas fermentation by acetogenic bacteria. However, its widespread utilization has been hampered by its toxicity in yeast. Years of study reveals that, when toxicity due to medium acidification is ruled out, acetate promotes cell death through elevation of aerobic respiration, which in turn elevates ROS production.

Based on those observations, we hypothesized that engineering of oxidative stress-tolerant yeast can support not only continuous biomanufacturing by yeast biofactories in general but also a widespread utilization of acetate as carbon source for biorefinery. Specifically, we hypothesized that yeast’s native response to oxidative stress can be enhanced through overexpression of the right combination of chaperones. Chaperones are long known to be involved in various cellular response toward environmental stresses, and we hypothesized that their upregulated level, if it is dictated solely by native stress response pathway, might not be optimal. To test the hypothesis, we first created a library of strains overexpressing chaperones in various combinations. We then subjected the library to an elevated oxidative stress to identify chaperone combinations that effectively attenuate the stress’ negative effect. This information was then used to construct a yeast background with higher tolerance to oxidative stress. As a proof of concept, here we are reporting performance of such engineered physiological background in continuous production of a fungal pigment using acetate as the sole carbon source.