Schematic overview of biofuel production through co-utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass by engineered yeast. XR: xylose reductase. XDH: xylitol dehydrogenase. XK: xylulose kinase. ACS: acetyl-CoA synthetase. AADH: acetylating acetaldehyde dehydrogenase. ADH: alcohol dehydrogenase. Credit: ACS, Wei et al. Click to enlarge.

Lignocellulosic biomass, looked to as a prime feedstock for biofuels due to its low cost, large-scale availability and potential for reducing greenhouse gas emissions, is composed of cellulose (40−55%), hemicellulose (25−50%), and lignin (10−40%).

Cellulose is composed of glucose, but hemicellulose consists of both five- and six-carbon sugars (pentose and hexose). Pretreatment and hydrolysis of lignocellulosic biomass thus results in a mixture of sugars as well as substantial amounts of acetic acid due to acetylation of hemicelluloses.

The yeast Saccharomyces cerevisiae has been widely used for sugar fermentation and is a preferred microorganism for metabolic engineering efforts to produce biofuels from cellulosic biomass because of its high carbon fluxes in central metabolic pathways, osmotolerance, and genetic tractability. However, S. cerevisiae cannot naturally ferment xylose, the second most abundant component in lignocellulosic hydrolysates, due to the lack of a functional assimilation pathway.

Extensive studies have focused on engineering S. cerevisiae for efficient fermentation of xylose, but xylose metabolism is highly repressed in the presence of glucose, which makes it difficult to realize continuous fermentation using hydrolysates that contain a xylose and glucose mixture. Meanwhile, the acetic acid ubiquitous in cellulosic hydrolysates can strongly diminish the bioconversion of sugar compounds due to its toxicity to fermenting microorganisms.

… A robust microbial system that efficiently utilizes mixed substrates derived from plant cell wall materials in toxic cellulosic hydrolysates is required for economically feasible production of lignocellulosic biofuels, but such a system has yet to be developed. … the objective of this study was to integrate the heterologous cellobiose assimilation pathway, xylose fermentation pathway, and acetic acid reduction pathway into a single yeast strain and demonstrate a strategy for making complete and efficient use of cellulosic carbons. The study demonstrated for the first time successful integration of the three distinct heterologous pathways into one microbial platform using synthetic biology approaches and the pathway integration brought unique synergistic effects in enhancing biofuel production.

—Wei et al.

The researchers had previously developed a yeast strain that efficiently fermented xylose. To this, they implement an efficient cellobiose fermentation pathway and an acetic acid reduction pathway to achieve co-conversion of the three substrates to ethanol by one strain.

One of the resulting strains (EJ-4) simultaneously consumed cellobiose, xylose, and acetic acid under strictly anaerobic conditions. All the cellobiose added (40 g/L) and nearly 40 g/L xylose were consumed within 120 h and 1.4 g/L acetate was consumed, demonstrating functional coexpression of the three heterologous pathways.

The final ethanol concentration in the fermentation was above 30 g/L; by contrast, the control strain had substantial amounts of cellobiose and xylose remaining even after 120 h and only 25 g/L ethanol was produced. The results, the researchers said, demonstrated that a single engineered yeast platform could integrate the pathways for co-utilization of three important cellulosic biomass components (cellobiose, xylose, and acetic acid) for ethanol production under industrially relevant anaerobic conditions.

The substrate co-utilization approach described here not only increases the ethanol yield and productivity but can also contribute to the development of consolidated bioprocessing (CBP) for cellulosic biofuels. CBP has received increased attention and research effort due to the economic benefits of process integration. Its ultimate goal is to combine all of the processes involved in cellulosic biofuel production into one cost-effective step.

One critical challenge and technical obstacle to process integration is the development of a robust CBP-enabling microorganism, which ideally has inhibitor tolerance, ability to utilize hexose and pentose sugars simultaneously at high efficiency, and ability to depolymerize lignocelluloses. The strain EJ4-a shows several traits that are beneficial for CBP: the simultaneous utilization of various major substrates from lignocellulosic biomass increases productivity and yield and reduces the overall fermentation time; reduction of the fermentation inhibitor acetic acid allows conversion of the previously unused carbon fraction to ethanol as well as in situ detoxification; and the assimilation of cellobiose and intra-cellular hydrolysis using β-glucosidase produced by strain reduces the requirement for exogenous enzymes. Future work will focus on strain optimization to improve the above- mentioned traits and thus advance the development of economically viable cellulosic biofuels.

—wei et al.

Resources

• Na Wei, Joong Oh, Gyver Million, Jamie H. D. Cate, and Yong-Su Jin (2015) “Simultaneous Utilization of Cellobiose, Xylose, and Acetic Acid from Lignocellulosic Biomass for Biofuel Production by an Engineered Yeast Platform” ACS Synthetic Biology doi: 10.1021/sb500364q