Draft Genome Sequence of Halomonas sp. Strain SL1, a Putative Polyhydroxyalkanoate-Producing Halophile.

Halomonas sp. strain SL1, a halophilic gammaproteobacterium, was isolated from samples from the Great Salt Lake in Utah. We report here the draft genome sequence of SL1, which has an estimated total sequence length of 3.6 Mb.

Lignocellulose-derived inhibitors improve lipid extraction from wet Rhodococcus opacus cells.

Extracting lipids from oleaginous microbial cells in a cost effective and environmentally compatible manner remains a critical challenge in developing manufacturing paradigms for advanced liquid biofuels. In this study, a new approach using microbial growth inhibitors from lignocellulose-derived feedstocks was used to extract lipids efficiently from wet cell mass of the oleaginous bacterium Rhodococcus opacus MITXM-61. Nine common lignocellulose-derived inhibitors for treatment of cells prior to solvent extraction were used and evaluated for their efficiency of lipid extraction from the cells. When the inhibitors were individually examined, formic acid and furfural showed the highest extraction efficiency of lipids from wet cell mass. Multiple extractions of lipids with methanol from wet cell mass pretreated with combined common inhibitors or hardwood hydrolysate comprising lignocellulose-derived inhibitors resulted in lipid recovery of greater than 85% of total lipids, a 1.7-fold increase of lipid extraction as compared to those in the absence of the inhibitors.

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-61(SHL33) 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-61(SHL33) 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.

Engineering L-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production.

Advanced biofuels from lignocellulosic biomass have been considered as a potential solution for the issues of energy sustainability and environmental protection. Triacylglycerols (TAGs) are potential precursors for the production of lipid-based liquid biofuels. Rhodococcus opacus PD630 can accumulate large amounts of TAGs when grown under physiological conditions of high carbon and low nitrogen. However, R. opacus PD630 does not utilize the sugar L-arabinose present in lignocellulosic hydrolysates. Here, we report the engineering of R. opacus to produce TAGs on L-arabinose. We constructed a plasmid (pASC8057) harboring araB, araD and araA genes derived from a Streptomyces bacterium, and introduced the genes into R. opacus PD630. One of the engineered strains, MITAE-348, was capable of growing on high concentrations (up to 100 g/L) of L-arabinose. MITAE-348 was grown in a defined medium containing 16 g/L L-arabinose or a mixture of 8 g/L L-arabinose and 8 g/L D-glucose. In a stationary phase occurring 3 days post-inoculation, the strain was able to completely utilize the sugar, and yielded 2.0 g/L for L-arabinose and 2.2 g/L for L-arabinose/D-glucose of TAGs, corresponding to 39.7% or 42.0%, respectively, of the cell dry weight.

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(-1) glycerol, produced 2.3 g L(-1) of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L(-1)) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L(-1) glycerol, 40 g L(-1) glucose, and 40 g L(-1) xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L(-1) of TAGs, representing 51.2% of the CDM. In addition, when 20 g L(-1) glycerol was pulse-loaded into the culture with 40 g L(-1) glucose and 40 g L(-1) xylose at the stationary growth phase, MITGM-173 produced 14.3 g L(-1) of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L(-1) glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-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.

Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production.

BACKGROUND: There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. RESULTS: We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. CONCLUSION: We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates that the engineered strain possesses a key trait of converters for lipid-based fuels production from lignocellulosic biomass.

High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production.

Biodiesel, monoalkyl esters of long-chain fatty acids with short-chain alcohols derived from triacylglycerols (TAGs), can be produced from renewable biomass sources. Recently, there has been interest in producing microbial oils from oleaginous microorganisms. Rhodococcus opacus PD630 is known to accumulate large amounts of TAGs. Following on these earlier works we demonstrate that R. opacus PD630 has the uncommon capacity to grow in defined media supplemented with glucose at a concentration of 300 g l(-1) during batch-culture fermentations. We found that we could significantly increase concentrations of both glucose and (NH4)2SO4 in the production medium resulting in a dramatic increase in fatty acid production when pH was controlled. We describe the experimental design protocol used to achieve the culture conditions necessary to obtain both high-cell-density and TAG accumulation; specifically, we describe the importance of the C/N ratio of the medium composition. Our bioprocess results demonstrate that R. opacus PD630 grown in batch-culture with an optimal production medium containing 240 g l(-1) glucose and 13.45 g l(-1) (NH4)2SO4 (C/N of 17.8) yields 77.6 g l(-1) of cell dry weight composed of approximately 38% TAGs indicating that this strain holds great potential as a future source of industrial biodiesel on starchy cellulosic feedstocks that are glucose polymers.

Antibiotic biosynthesis following horizontal gene transfer: new milestone for novel natural product discovery?

Bacteria obtain a significant proportion of their genetic diversity via acquisition of DNA from distantly related organisms, a phenomenon known as horizontal gene transfer. The focus of horizontal gene transfer investigations has been primarily on the impact of this phenomenon on the ecological and/or pathogenic characteristics of bacterial species, with very little effort devoted to investigating horizontal gene transfer as a means of drug discovery. Here, we describe a novel approach to harness the power of horizontal gene transfer to produce novel chemotherapeutic molecules, a process that is easily scalable. We describe the state of the art in this field and discuss the current limiting factors associated with this phenomenon. Utilising a horizontal gene transfer method, we have identified and characterised a novel antimicrobial compound. Production of this antibiotic, termed rhodostreptomycin, is associated with the transfer of DNA from a species of Streptomyces to Rhodococcus by an as yet identified mechanism. We believe that horizontal gene transfer may represent the future of natural product discovery and engineering.

Differential gene expression profiles and real-time measurements of growth parameters in Saccharomyces cerevisiae grown in microliter-scale bioreactors equipped with internal stirring.

Combining real-time growth kinetics measurements with global gene expression analysis of microbial cultures is of significant value for high-throughput biological research. We have performed differential gene expression analysis in the eukaryotic model Saccharomyces cerevisiae grown in galactose and glucose media in 150 muL bioreactors equipped with sensors for in situ and real-time measurements of optical density (OD), pH, and dissolved oxygen (DO). The microbioreactors were fabricated from poly(dimethylsiloxane) (PDMS) and poly(methyl methacrylate) (PMMA) and equipped with internal magnetic ministirrers and evaporation compensation by water replacement. In galactose-grown cells, the core genes of the GAL operon GAL2, GAL1, GAL7, and GAL10 were upregulated at least 100-fold relative to glucose-grown cells. These differential gene expression levels were similar to those observed in large-scale culture vessels. The increasing rate at which complete genomic sequences of microorganisms are becoming available offers an unprecedented opportunity for comparative investigations of these organisms. Our results from S. cerevisiae cultures grown in instrumented microbioreactors show that it is possible to integrate high-throughput studies of growth physiology with global gene expression analysis of microorganisms.

Improvement of alpha-amylase production by modulation of ribosomal component protein S12 in Bacillus subtilis 168.

The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances alpha-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing alpha-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg2+ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in alpha-amylase production seen in the affected strain.

Ushikulides A and B, immunosuppressants produced by a strain of Streptomyces sp.

Novel immunosuppressants, ushikulides A and B, were isolated from the culture broth of Streptomyces sp. IUK-102. Ushikulides A and B both have the same molecular formula, determined as C40H68O10. The structures of both compounds were elucidated to be novel 22-membered macrolides. Both compounds showed immunosuppressive activity for murine splenocyte proliferation in vitro.

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis. III. Immunosuppressive efficacy.

Novel immunosuppressants, SNF4435C and D produced by a strain of Streptomyces spectabilis, were examined for their pharmacodynamical profiles. SNF4435C and D suppressed the responses of both murine splenocytes and human peripheral blood lymphocytes in the mixed lymphocyte reaction (MLR) with IC50 values of 0.5 microM and 0.2 microM, respectively. In the mouse MLR experiments, SNF4435C and D did not block the production of interleukin-2 (IL-2) and the compounds-induced suppression was not restored by the addition of exogeneous IL-2. In addition, the significant inhibitory action was still retained even when SNF4435C or D was added after 48 hours from the start of the culture. These results were distinct from the behaviors observed with FK-506. SNF4435C, the major component, suppressed mouse delayed type hypersensitivity (DTH) and prolonged rat skin allograft survival.