Commercial production of cellulosic biofuel via fermentation pathways has been hampered by inefficient fermentation of xylose and the toxicity of acetic acid, which constitute substantial portions of cellulosic biomass. Now, researchers from the University of Illinois at Urbana-Champaign and UC Berkeley have engineered yeast to convert cellulosic sugars and toxic levels of acetate together into ethanol under anaerobic conditions.
The innovation, reported in a paper published in Nature Communications, increases ethanol yield from lignocellulosic sources by about 10%. The results, the researchers suggest, demonstrate a breakthrough in making efficient use of carbon compounds in cellulosic biomass and also present an innovative strategy for metabolic engineering through which an undesirable redox state can be exploited to drive desirable reactions—even improving productivity and yield.
...incomplete and inefficient conversion of cellulosic sugars present in solubilized plant cell wall carbohydrates (hydrolyzates) into biofuels has hindered commercial-scale Xylitol processes. One major bottleneck is the economic conversion of hemicellulose, one of the main structural components in D-xylulose lignocellulosic biomass along with cellulose, into biofuels. The sugar D-xylose, derived from hydrolysis of hemicellulose, is the second most abundant sugar in the plant cell wall consisting of up to 35% of the total carbohydrate from lignocellulosic biomass.
However, the yeast Saccharomyces cerevisiae, the most widely used microorganism for producing corn and sugarcane ethanol, cannot metabolize xylose. Further, hemicellulose and lignin in the plant cell wall are acetylated, yielding acetic acid as an unavoidable component in cellulosic hydrolyzates with acetic acid concentrations ranging from 1 to 15 g l-1. Acetic acid is toxic to fermenting microorganisms and negatively influences sugar fermentation and biofuel yields. Xylose conversion and acetic acid detoxification are two major problems that must be solved to make cellulosic biofuels economically viable.—Wei et al.
Xylose-fermenting yeasts do exist in nature, but their ethanol production rates and tolerances are inferior to those of S. cerevisiae. In an earlier study, graduate student Soo Rin Kim (now a fellow at the Energy Biosciences Institute (EBI), which funded this study) engineered S. cerevisiae to consume xylose more efficiently. While this improved the ethanol output, the process generated an excess of NADH, an electron-transfer molecule that is part of the energy currency of all cells. The buildup of acetic acid also killed off much of the yeast.
Principal investigator Jamie Cate, of the University of California at Berkeley and the Lawrence Berkeley National Laboratory, after discussing the problem with University of Illinois food science and human nutrition professor Yong-Su Jin Cate, hypothesized that the team might be able to induce the yeast to consume acetic acid. It later occurred to Jin that that process might also use up the surplus NADH from xylose metabolism.
By reviewing earlier studies, postdoctoral researcher Na Wei at the University of Illinois found that another organism, a bacterium, could consume acetic acid. She identified the enzymes that catalyzed this process and saw that one of them not only converted acetic acid into ethanol, but also would use the surplus NADH from xylose metabolism.
|“We sort of rebuilt how yeast uses carbon.”|
—Dr. Jamie Cate
To get a better idea of the feasibility of the idea, graduate student Josh Quarterman used computer simulations to see how adding the new genes to the yeast’s metabolic repertoire would affect its ethanol output. His calculations indicated that the pathway Wei had identified would boost ethanol production.
Testing of the engineered yeast resulted in a 10% increase in yield, in line with Quarterman’s calculations. In further experiments, she demonstrated that the new yeast was in fact making some of the ethanol from acetate, a first for S. cerevisiae.
Many people are curious about why we don’t have cellulosic biofuel right now. But it’s not because of one limiting step. We have many limiting steps in growing the biomass, storing, moving, harvesting, decomposing the biomass to the sugar, fermentation and then separation (of the ethanol). The advance that we are reporting involves one of those steps—fermentation. But it also will make other steps in the process a little easier.—Yong-Su Jin
Na Wei, Josh Quarterman, Soo Rin Kim, Jamie H.D. Cate & Yong-Su Jin (2013) “Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast” Nature Communications 4, Article number: 2580 doi: 10.1038/ncomms3580