Liu Z.L.,US BioEnergy
Applied Microbiology and Biotechnology | Year: 2011
Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications. © 2011 Springer-Verlag (outside the USA).
Hamdan L.J.,U.S. Navy |
Fulmer P.A.,US BioEnergy
Aquatic Microbial Ecology | Year: 2011
Hydrocarbon-degrading bacteria are important for controlling the fate of natural and anthropogenic hydrocarbons in the marine environment. In the wake of the Deepwater Horizon spill in the Gulf of Mexico, microbial communities will be important for the natural attenuation of the effects of the spill. The chemical dispersant COREXIT® EC9500A was widely deployed during the response to the Deepwater Horizon incident. Although toxicity tests confirm that COREXIT® EC9500A does not pose a significant threat to invertebrate and adult fish populations, there is limited information on its effect on microbial communities. We determined the composition of the microbial community in oil that had been freshly deposited on a beach in Louisiana, USA, as a result of the Deepwater Horizon spill. The metabolic activity and viability in cultures obtained from oil samples were determined in the absence and presence of COREXIT® EC9500A at concentrations ranging from 0.001 to 100 mg ml-1. In length heterogeneity PCR (LH-PCR) fingerprints of oil samples, the most abundant isolates were those of Vibrio, followed by hydrocarbon-degrading isolates affiliated with Acine - tobacter and Marinobacter. We observed significant reductions in production and viability of Acineto - bacter and Marinobacter in the presence of the dispersant compared to controls. Of the organisms examined, Marinobacter appears to be the most sensitive to the dispersant, with nearly 100% reduction in viability and production as a result of exposure to concentrations of the dispersant likely to be encountered during the response to the spill (1 to 10 mg ml-1). Significantly, at the same concen tration of dispersant, the non-hydrocarbon-degrading Vibrio isolates proliferated. These data suggest that hydro-carbon-degrading bacteria are inhibited by chemical dispersants, and that the use of dispersants has the potential to diminish the capacity of the environment to bioremediate spills. © Inter-Research 2011.
Mertens J.A.,US BioEnergy
Applied Biochemistry and Biotechnology | Year: 2013
The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a K m of 55 μM and k cat of 10.3 s-1 while RPG16 was shown to have greater affinity for (GalpA)2 with a K m of 16 μM, but lesser catalytic activity with a k cat of 3.9 s-1. Both enzymes were inhibited by the product, galacturonic acid, with app K i values of 886 and 501 μM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA) 3 with a K m of 9.2 μM and a similar k cat at 10.7 s-1 relative to (GalpA)2. Catalytic constants for RPG16 hydrolyzing (GalpA)3 could not be determined; however, single-injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)3 relative to (GalpA)2. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes. © 2013 Springer Science+Business Media New York (outside the USA).
Ma M.,US BioEnergy |
Ma M.,Sichuan Agricultural University |
Liu Z.L.,US BioEnergy |
Moon J.,US BioEnergy
Bioenergy Research | Year: 2012
For economical lignocellulose-to-ethanol production, a desirable biocatalyst should tolerate inhibitors derived from preteatment of lignocellulose and be able to utilize heterogeneous biomass sugars of hexoses and pentoses. Previously, we developed an inhibitor-tolerant Saccharomyces cerevisiae strain NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF). In this study, we genetically engineered Y-50049 to enable and enhance its xylose utilization capability. A codon-optimized xylose isomerase gene for yeast (YXI) was synthesized and introduced into a defined chromosomal locus of Y-50049. Two newly identified xylose transport related genes XUT4 and XUT6, and previously reported xylulokinase gene (XKS1), and xylitol dehydrogenase gene (XYL2) from Scheffersomyces stipitis were also engineered into the yeast resulting in strain NRRL Y-50463. The engineered strain was able to grow on xylose as sole carbon source and a minimum ethanol production of 38.6 g l -1 was obtained in an anaerobic fermentation on mixed sugars of glucose and xylose in the presence of furfural and HMF. © 2012 Springer Science+Business Media, LLC. (outside the USA).
Jordan D.B.,US BioEnergy |
Braker J.D.,US BioEnergy
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2011
Conformational inversion occurs 7-8 kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA) > 4-nitrophenyl-β-d-xylopyranoside (4NPX) > xylobiose (X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite - 1 residues decrease the ratio kcat 4NPX/4NPA, suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite + 1 and the subsite - 1/+1 interface residues increase the ratio kcat 4NPX/4NPA, suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite - 1 residues raise kcat X2/4NPX and alanine mutations of subsite + 1 and interface residues lower kcat X2/4NPX. We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite - 1. Other factors leading to the transition state are supported entirely by native residues of subsite + 1 and interface residues. © 2011 Elsevier B.V. All Rights Reserved.