Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors

Background: Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. A significant technical challenge to using lignocellulose is the presence of microbial growth inhibitors generated during pretreatment processes. Triacylglycerols (TAGs) are potential precursors for lipid-based biofuel production. Rhodococcus opacus MITXM-61 is an oleaginous bacterium capable of producing large amounts of TAGs on high concentrations of glucose and xylose present in lignocellulosic hydrolysates. However, this strain is sensitive to ligonocellulose-derived inhibitors. To understand the toxic effects of the inhibitors in lignocellulosic hydrolysates, strain MITXM-61 was examined for tolerance toward the potential inhibitors and was subjected to adaptive evolution for the resistance to the inhibitors. Results: We investigated growth-inhibitory effects by potential lignocellulose-derived inhibitors of phenols (lignin, vanillin, 4-hydroxybenzaldehyde (4-HB), syringaldehyde), furans (furfural and 5-hydroxymethyl-2-furaldehyde), and organic acids (levulinic acid, formic acid, and acetic acid) on the growth and TAG production of strain MITXM-61. Phenols and furans exhibited potent inhibitory effects at a concentration of 1 g L−1, while organic acids had insignificant impacts at concentrations of up to 2 g L−1. In an attempt to improve the inhibitor tolerance of strain MITXM-61, we evaluated the adaptation of this strain to the potential inhibitors. Adapted mutants were generated on defined agar media containing lignin, 4-HB, and syringaldehyde. Strain MITXM-61SHL33 with improved multiple resistance of lignin, 4-HB, and syringaldehyde was constructed through adaptive evolution-based strategies. The evolved strain exhibited a two- to threefold increase in resistance to lignin, 4-HB, and syringaldehyde at 50% growth-inhibitory concentrations, compared to the parental strain. When grown in genuine lignocellulosic hydrolysates of corn stover, wheat straw, and hardwood containing growth inhibitors, strain MITXM-61SHL33 exhibited a markedly shortened lag phase in comparison with that of strain MITXM-61. Conclusion: This study provides important clues to overcome the negative effects of inhibitors in lignocellulosic hydrolysates on TAG production of R. opacus cells. The findings can contribute to significant progress in detoxified pretreatment of hydrolysates and development of more efficient strains for industrial TAG fermentations of R. opacus using lignocellulosic biomass

Magnitude of Annual Soil Loss from a Hilly Cultivated Slope in Northern Vietnam and Evaluation of Factors Controlling Water Erosion

A soil erosion experiment was conducted in northern Vietnam over three rainy seasons to clarify the magnitude of soil loss and factors controlling water erosion. The plot had a low (8%) or medium (14.5%) slope with land-cover of cassava or morning glory or being bare. Annual soil loss (177 to 2,361 g/m2) was a tolerable level in all low-slope plots but was not in some medium-slope plots. The effects of slope gradient and seasonal rainfall on the mean daily soil loss of the season were confirmed, but the effect of land-cover was not, owing to the small canopy cover ratio or leaf area index during the season. The very high annual soil loss (>2,200 g/m2) observed in the first year of some medium-slope plots was the site-specific effect from initial land preparation. Since the site-specific effect was large, the preparation must be done carefully on the slope

Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels

Background: Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations. Results: An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L[superscript −1] glycerol, produced 2.3 g L[superscript −1] of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g[superscript −1] of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L[superscript −1]) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L[superscript −1] glycerol, 40 g L[superscript −1] glucose, and 40 g L[superscript −1] xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L[superscript −1] of TAGs, representing 51.2% of the CDM. In addition, when 20 g L[superscript −1] glycerol was pulse-loaded into the culture with 40 g L[superscript −1] glucose and 40 g L[superscript −1] xylose at the stationary growth phase, MITGM-173 produced 14.3 g L[superscript −1] of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L[superscript −1] glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g[superscript −1] on the initial addition and 0.279 g g[superscript −1] on the pulse addition of glycerol. Conclusion: We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.Sweetwater Energy, Inc.MIT Energy Initiativ

Differential roles of prostaglandin E-type receptors in activation of hypoxia-inducible factor 1 by prostaglandin E1 in vascular-derived cells under non-hypoxic conditions

Prostaglandin E1 (PGE1), known pharmaceutically as alprostadil, has vasodilatory properties and is used widely in various clinical settings. In addition to acute vasodilatory properties, PGE1 may exert beneficial effects by altering protein expression of vascular cells. PGE1 is reported to be a potent stimulator of angiogenesis via upregulation of VEGF expression, which is under the control of the transcription factor hypoxia-inducible factor 1 (HIF-1). However, the molecular mechanisms behind the phenomenon are largely unknown. In the present study, we investigated the mechanism by which PGE1 induces HIF-1 activation and VEGF gene expression in human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs), both vascular-derived cells. HUVECs and HASMCs were treated with PGE1 at clinically relevant concentrations under 20% O2 conditions and HIF-1 protein expression was investigated. Expression of HIF- 1α protein and the HIF-1-downstream genes were low under 20% O2 conditions and increased in response to PGE1 treatment in both HUVECs and HASMCs in a dose- and time-dependent manner under 20% O2 conditions as comparable to exposure to 1% O2 conditions. Studies using EP-receptor-specific agonists and antagonists revealed that EP1 and EP3 are critical to PGE1-induced HIF-1 activation. In vitro vascular permeability assays using HUVECs indicated that PGE1 increased vascular permeability in HUVECs. Thus, we demonstrate that PGE1 induces HIF- 1α protein expression and HIF-1 activation under non-hypoxic conditions and also provide evidence that the activity of multiple signal transduction pathways downstream of EP1 and EP3 receptors is required for HIF-1 activation

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences

Triacylglycerol Production from Corn Stover Using a Xylose-Fermenting Rhodococcus opacus Strain for Lignocellulosic Biofuels

Triacylglycerols (TAGs) are in the spotlight as a feasible source of hydrocarbon-based biofuels. Rhodococcus opacus PD630 produces large amounts of intracellular TAGs in cultivations containing high concentrations of glucose, but it does not utilize xylose present in all hydrolysates of lignocellulosic biomass. We constructed a highpotency xylose-fermenting R. opacus strain MITXM-61 that exhibited robust growth and TAG biosynthesis on high concentrations of xylose by activating potential xylose-metabolism genes. MITXM-61 had the uncommon capacity to grow in defined media supplemented with xylose at concentrations of greater than 200 gl-1. MITXM-61 grown in corn stover hydrolysates containing 118 gl-1 of initial total sugars was capable of completely and simultaneously utilizing both xylose and glucose in the genuine lignocellulosic feedstock, and yielded 15.9 gl-1 of TAGs, corresponding to 54% of the cell dry weight. The oleaginous bacterium R. opacus strain proved useful for developing a new manufacturing paradigm to generate advanced lignocellulosic biofuels.United States. Defense Advanced Research Projects Agency (Grant HR0011-10-C-0187)Logos Technologies (Firm)MIT Energy InitiativeSweetwater Energy, Inc