The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro 2 of one subunit (Met 1 is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the re- The sensing of dissolved gas molecules by proteins in biology has recently attracted considerable biochemical interest. The role of nitric oxide in a variety of important biochemical processes (1, 2), and its receptor, soluble guanylyl cyclase (sGC) 1 have been well documented in eukaryotic systems (3, 4). FixL, which modulates the expression of genes responsible for nitrogen fixation in rhizobia, is an example of an oxygen sensor (5, 6). An oxygen sensor in Escherichia coli, termed DOS ("direct oxygen sensor"), has been reported although its physiological role remains undefined (7). Finally, for carbon monoxide (CO), CooA, the CO-oxidation activator protein, modulates the expression of genes required for the utilization of CO as a sole energy source in the photosynthetic bacterium Rhodospirillum rubrum (8). All of the above mentioned proteins have in common a heme prosthetic group to which their respective gas molecules bind. The binding event is then followed by a conformational change in the protein that effects activity.Numerous studies have clearly demonstrated the physiological importance of CO in a wide variety of processes (9 -11), and although sGC has been implicated in sensing CO (12-14), direct evidence of a CO-receptor in eukaryotic signal transduction systems is lacking. CooA senses CO through a heme moiety and represents the current model system for biological CO-sensing (19,20). Finally, the ligand that is displaced upon binding CO remains speculative.Recently, the three-dimensional structure of Fe II CooA has been solved by x-ray diffraction techniques (21). This report showed that the general folding topology of CooA was indeed similar to that of CRP (22). In addition to the verification of His 77 as one of the heme-axial ligands in Fe II CooA, inspection of the structure identified the other axial ligand as an Nterminal proline residue (Pro 2 ; Met 1 is removed by processing) from the other subunit of the dimer. This structural environment represents an unprecedented axial ligation arrangement for a heme protein.In a previous study (18), we altered His 77 and found that the UV-visual spectra of these variants was normal in the Fe ʈ To whom correspondence should be addressed. Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts@bact.wisc.edu.1 The abbreviations used are: sGC, soluble guanylyl cyclase; CO, carbon monoxide; CRP, cAMP receptor protein; FixL, oxygen sensor of Rhizobium meliloti; Mb, myoglobin; P-4...

BackgroundInterannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during 3 years with significantly different precipitation profiles, representing a major drought year (2012) and 2 years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strains of Saccharomyces cerevisiae and Zymomonas mobilis. A chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates.ResultsWhile most corn stover and switchgrass hydrolysates were readily fermented, growth of S. cerevisiae was completely inhibited in hydrolysate generated from drought-stressed switchgrass. Based on chemical genomics analysis, yeast strains deficient in genes related to protein trafficking within the cell were significantly more resistant to the drought-year switchgrass hydrolysate. Detailed biomass and hydrolysate characterization revealed that switchgrass accumulated greater concentrations of soluble sugars in response to the drought and these sugars were subsequently degraded to pyrazines and imidazoles during ammonia-based pretreatment. When added ex situ to normal switchgrass hydrolysate, imidazoles and pyrazines caused anaerobic growth inhibition of S. cerevisiae.ConclusionsIn response to the osmotic pressures experienced during drought stress, plants accumulate soluble sugars that are susceptible to degradation during chemical pretreatments. For ammonia-based pretreatment, these sugars degrade to imidazoles and pyrazines. These compounds contribute to S. cerevisiae growth inhibition in drought-year switchgrass hydrolysate. This work discovered that variation in environmental conditions during the growth of bioenergy crops could have significant detrimental effects on fermentation organisms during biofuel production. These findings are relevant to regions where climate change is predicted to cause an increased incidence of drought and to marginal lands with poor water-holding capacity, where fluctuations in soil moisture may trigger frequent drought stress response in lignocellulosic feedstocks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0657-0) contains supplementary material, which is available to authorized users.