Kazuhiko Kurosawa

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.

Rhodostreptomycins, antibiotics biosynthesized following horizontal gene transfer from Streptomyces padanus to Rhodococcus fascians.

Two antibiotics have been isolated from a strain of Rhodococcus fascians that emerged from a competitive co-culture with Streptomyces padanus. The structures of these compounds were elucidated by d...

Bacterial Carbon Storage to Value Added Products

Microorganisms have evolved different systems for storing carbon during times of stress. In the cell’s natural environment, the stored carbon can then be utilized for growth when other nutrients are in better supply. Storage of carbon and other nutrients is ubiquitous throughout the prokaryotic and eukaryotic domains of life. These carbon storage molecules have great industrial importance. They can be useful as value-added products, as either biopolymers or biofuels, and cells are grown in large quantities and these compounds are harvested, usually as a replacement for a petroleum-based product. Nowadays, entire industries have been generated based on the production and utilization of these compounds. We focus on two bacteria that could be considered paradigms of their particular carbon storage strategy: Ralstonia eutropha and Rhodococcus opacus . R. eutropha has been well-studied as a polyhydroxyalkanoate (bioplastic) producer and R. opacus is a model bacterium for high yield triacylglycerol (TAG) production for biofuels. Both species produce carbon storage molecules that can potentially diminish our reliance on fossil-based petroleum. However, in both cases, there are challenges that must be overcome before profitable production schemes are established using these organisms. We explore the previous and current works to address these challenges in this review.

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5–100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA–DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.

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 μL 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.

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.

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

BackgroundLignocellulosic 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.ResultsWe 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.ConclusionThis 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.

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.

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.