PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production.

Audrey P Gasch,Michael Polymenis,Ellen R Wagner,Joshua J Coon,Kevin S Myers,Nicholas M Riley

doi:10.1371/journal.pone.0212389

Abstract

Lignocellulosic biomass offers a sustainable source for biofuel production that does not compete with food-based cropping systems. Importantly, two critical bottlenecks prevent economic adoption: many industrially relevant microorganisms cannot ferment pentose sugars prevalent in lignocellulosic medium, leaving a significant amount of carbon unutilized. Furthermore, chemical biomass pretreatment required to release fermentable sugars generates a variety of toxins, which inhibit microbial growth and metabolism, specifically limiting pentose utilization in engineered strains. Here we dissected genetic determinants of anaerobic xylose fermentation and stress tolerance in chemically pretreated corn stover biomass, called hydrolysate. We previously revealed that loss-of-function mutations in the stress-responsive MAP kinase HOG1 and negative regulator of the RAS/Protein Kinase A (PKA) pathway, IRA2, enhances anaerobic xylose fermentation. However, these mutations likely reduce cells’ ability to tolerate the toxins present in lignocellulosic hydrolysate, making the strain especially vulnerable to it. We tested the contributions of Hog1 and PKA signaling via IRA2 or PKA negative regulatory subunit BCY1 to metabolism, growth, and stress tolerance in corn stover hydrolysate and laboratory medium with mixed sugars. We found mutations causing upregulated PKA activity increase growth rate and glucose consumption in various media but do not have a specific impact on xylose fermentation. In contrast, mutation of HOG1 specifically increased xylose usage. We hypothesized improving stress tolerance would enhance the rate of xylose consumption in hydrolysate. Surprisingly, increasing stress tolerance did not augment xylose fermentation in lignocellulosic medium in this strain background, suggesting other mechanisms besides cellular stress limit this strain’s ability for anaerobic xylose fermentation in hydrolysate.

Highlights

Lignocellulosic biomass offers a sustainable source for bioenergy
We demonstrate that while upregulating Protein Kinase A (PKA) is important for increased cell growth on glucose, HOG1 deletion benefits xylose utilization, in part by preventing phosphorylation of glycolytic enzymes by Hog1
Our results show perturbing Bcy1 sequence by protein fusion dramatically improved stress tolerance, as seen by increased growth in toxic 9% glucan-loading AFEXpretreated corn stover hydrolysate (ACSH), but xylose fermentation remained blocked, suggesting physiological stress sensitivity is unlikely the cause of halted xylose consumption in concentrated hydrolysate

Summary

Introduction

The use of leftover agriculture byproducts and plants grown on marginal lands for biofuel production reduces waste and removes dependency on food-based cropping systems. There are two major bottlenecks for sustainable biofuel production from lignocellulosic material. Many microbes, including industrially relevant Saccharomyces cerevisiae, cannot innately ferment pentose sugars like xylose, which comprise a significant fraction of the sugars released from deconstructed biomass [1]. The harsh chemical treatment of plant biomass required to release lignocellulosic sugars produces a variety of toxins and stresses that inhibit microbial growth and fermentation [2,3]. A goal for the biofuel industry is to engineer stress-tolerant microbes to convert all available sugars to the desired products by routing cellular resources toward product formation and away from cell growth and other unnecessary physiological responses [4]

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Conclusion