Deep-sea Yeast Research Articles

Bioconversion of non-food corn biomass to polyol esters of fatty acid and single-cell oils

BackgroundLignocellulose is a valuable carbon source for the production of biofuels and biochemicals, thus having the potential to substitute fossil resources. Consolidated bio-saccharification (CBS) is a whole-cell-based catalytic technology previously developed to produce fermentable sugars from lignocellulosic agricultural wastes. The deep-sea yeast strain Rhodotorulapaludigena P4R5 can produce extracellular polyol esters of fatty acids (PEFA) and intracellular single-cell oils (SCO) simultaneously. Therefore, the integration of CBS and P4R5 fermentation processes would achieve high-value-added conversion of lignocellulosic biomass.ResultsThe strain P4R5 could co-utilize glucose and xylose, the main monosaccharides from lignocellulose, and also use fructose and arabinose for PEFA and SCO production at high levels. By regulating the sugar metabolism pathways for different monosaccharides, the strain could produce PEFA with a single type of polyol head. The potential use of PEFA as functional micelles was also determined. Most importantly, when sugar-rich CBS hydrolysates derived from corn stover or corncob residues were used to replace grain-derived pure sugars for P4R5 fermentation, similar PEFA and SCO productions were obtained, indicating the robust conversion of non-food corn plant wastes to high-value-added glycolipids and lipids. Since the produced PEFA could be easily collected from the culture via short-time standing, we further developed a semi-continuous process for PEFA production from corncob residue-derived CBS hydrolysate, and the PEFA titer and productivity were enhanced up to 41.1 g/L and 8.22 g/L/day, respectively.ConclusionsHere, we integrated the CBS process and the P4R5 fermentation for the robust production of high-value-added PEFA and SCO from non-food corn plant wastes. Therefore, this study suggests a feasible way for lignocellulosic agro-waste utilization and the potential application of P4R5 in industrial PEFA production.

Efficient simultaneous production of extracellular polyol esters of fatty acids and intracellular lipids from inulin by a deep-sea yeast Rhodotorula paludigena P4R5

BackgroundPolyol esters of fatty acids (PEFA) are a kind of promising biosurfactants and mainly secreted by Rhodotorula strains. In addition, some strains of Rhodotorula are reliable producers of microbial lipid. Therefore, it is feasible to establish a one step fermentation process for efficient simultaneous production of PEFA and microbial lipids by a suitable Rhodotorula strain.ResultsA newly isolated deep-sea yeast, Rhodotorula paludigena P4R5, was shown to simultaneously produce high level of intracellular lipid and extracellular PEFA. Under the optimized conditions, it could yield 48.5 g/L of PEFA and 16.9 g/L of intracellular lipid within 156 h from inulin during 10-L batch fermentation. The PEFA consisting of a mixture of mannitol esters of 3-hydroxy C14, C16 and C18 fatty acids with variable acetylation showed outstanding surface activity and emulsifying activity, while the fatty acids of the intracellular lipid were mainly C16 and C18 and could be high-quality feedstock for biodiesel production.ConclusionThe deep-sea yeast strain R. paludigena P4R5 was an excellent candidate for efficient simultaneous of biosurfactants and biodiesel from inulin. Our results also suggested that the establishment of fermentation systems with multiple metabolites production was an effective approach to improve the profitability.

Transformation of dimethyl phthalate esters (DMPEs) by a marine red yeast Rhodotorula mucilaginosa isolated from deep sea sediments of the Atlantic Ocean

A marine red yeast capable of degrading dimethyl phthalate esters (DMPEs) was isolated from deep-sea sediments of the Atlantic Ocean using enrichment culture technique. The yeast was identified as Rhodotorula mucilaginosa Mar-Y3 based on internal transcribed spacer (ITS) gene sequence analysis. The biochemical degradation pathways of three DMPE isomers, namely dimethyl phthalate (DMP), dimethyl isophthalate (DMI), and dimethyl terephthalate (DMT), were investigated using this yeast. The yeast cannot completely mineralize DMPEs, but can transform them to respective phthalate monoester or phthalic acid. R. mucilaginosa Mar-Y3 also degraded different DMPE isomers through varied metabolism pathways and biodegradation rates. The yeast performed one-step ester hydrolysis with a relatively low degradation rate to transform DMP and DMI to the respective monoester; however, monomethyl phthalate (MMP) and monomethyl isophthalate (MMI) were the dead-end products and further metabolism did not proceed. The yeast carried out sequential ester hydrolysis with a relatively high degradation rate to transform DMT to terephthalic acid (TA) via monomethyl terephthalate (MMT), but TA was resistant to further metabolism. These results suggested that phthalate esterases produced by the deep-sea yeast R. mucilaginosa Mar-Y3 had a very high substrate specificity for different DMPE isomers, and that the degradation of DMPEs by this yeast involved distinct esterases responsible for the hydrolysis of two identical carboxylic ester bonds.

Heavy metal tolerance in the psychrotolerant Cryptococcus sp. isolated from deep‐sea sediments of the Central Indian Basin

A deep-sea isolate of the psychrotolerant yeast Cryptococcus sp. (NIOCC#PY13) obtained from polymetallic nodule-bearing sediments of the Central Indian Basin was examined for its capacity to grow in the presence of various concentrations of the heavy metal salts i.e., ZnSO4 , CuSO4 , Pb(CH3 COO)2 and CdCl2 . It demonstrated considerable growth in the presence of 100 mg/l concentrations of the above-mentioned four heavy metal salts both at 30°C and 15°C. This strain tolerated comparatively higher levels of these four metal salts than other deep-sea and terrestrial yeast isolates belonging to Cryptococcus, Rhodotorula, Rhodosporidium and Sporidiobolus spp. Optimum pH for growth of this isolate was in the range of 6-8 in the presence of heavy metal salts at these two temperatures. Scanning electron microscopic (SEM) studies exhibited altered cell surface morphology of the cells under the influence of heavy metals compared to that with control. The adsorption of heavy metals to the cells was demonstrated by FTIR and EDAX analysis. As evidenced by atomic absorption spectrophotometric (AAS) analysis, about 30-90% of the heavy metals were removed from the culture supernatant after 4 days of growth at 30°C. This deep-sea yeast isolate appears to be a potential candidate for bioremediation of metal-contaminated sites. Moreover, its metal tolerance properties provide a significant insight into its ecological role and adaptations to growth in such extreme conditions.

Differentially expressed genes under simulated deep-sea conditions in the psychrotolerant yeast Cryptococcus sp. NIOCC#PY13

Microorganisms exhibit varying degrees of tolerance to extreme conditions of pressure and temperature. In the present study, the psychrotolerant deep-sea yeast, Cryptococcus sp. NIOCC#PY13, was exposed to elevated hydrostatic pressure and low temperature to explore the differentially expressed genes responsible for its survival at such extreme conditions. The suppression subtractive hybridization technique was employed for identification of expressed upregulated genes at extreme conditions of pressure and temperature. The effect of elevated pressure was found to be different than that of combined pressure and temperature exposures. Altogether, 17 and 20 upregulated genes were identified at 50 MPa and 50 MPa/5 °C, respectively. These differentially expressed genes were similar to the NCBI database ESTs (expressed sequence tags), coding for proteins such as arachidonic acid metabolism, amino acid transport and unsaturation of membrane fatty acids, which have been previously demonstrated to assist in the survival of microorganisms under stress conditions. Interestingly, about 50 % of the upregulated genes matched with hypothetical proteins at a percentage similarity of ≤96, suggesting their probability of being novel. Detailed studies of the above genes/proteins from deep-sea microorganisms are suggested for future investigations, which may shed more light on the existence and adaptation mechanisms adopted by these for their survival under such extreme conditions.

Application of Deep Sea Yeast—Production of Biosurfactants—

Yeast is promising microbe to applying for brewing, bread-making, and some fermented foods, and to development recently for producing bio-ethanol. Yeast exists also in the deep-sea and the many species have been described from the deep-sea in previous reports. Most of the deep-sea yeasts are “non-conventional yeasts” including approximately 1000 species excepting well-known Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the best of our knowledge. Yeast isolated from the deep-sea often possesses unique metabolic properties. We recently focused on a biosurfactant producing yeast, strain SY62, which was isolated from at a depth of 1,156 m in Sagami Bay in Japan. Although the yeast indicated little difference from conventional Pseudozyma hubeiensis on taxonomy, the secreted glycolipid-type biosurfactant, mannosylerythritol lipids (MEL), indicated structurally differences from those of conventional strains. The structural differences on MEL produced by SY62 resulted in the good hydrophilicity compared to those produced by conventional strains. The results indicated that the deep-sea bio-resources have great potential for exploring novel useful microorganisms and metabolites. The difference of metabolites seemed to be caused by evolution under high-pressure stress in the deep-sea.

Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 ( Cl-SOD1). In this study, the 1.2 Å resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.

Cloning and functional characterization of the copper/zinc superoxide dismutase gene from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 possesses high superoxide dismutase (SOD) activity and a high tolerance toward metal ions. To clarify the relationship between metal tolerance and SOD activity in this strain, we cloned the Cu/Zn SOD gene. This gene (Cl-SOD1) consists of 471 bp encoding 157 amino acids; the associated protein had 59.9-76.7% identity with Cu/Zn SOD proteins of other yeast species. The highest identity corresponded to Cryptococcus gattii (76.7%). Cl-SOD1 expression in the sod1 mutant of Saccharomyces cerevisiae revealed that this SOD protein was functional in S. cerevisiae. The Cl-SOD1 protein possessed approximately fourfold greater activity than S. cerevisiae SOD1 (Sc-SOD1) at 30 degrees C. The amount of Cl-SOD1 mRNA in strain N6 increased in the presence of copper ion. However, the level of this transcript was not dependent on an increase in copper ion concentration and did not correlate well with changes in the amount of Cu/Zn SOD protein. This result suggests that strain N6 possesses other Cu/Zn SOD genes induced in a manner different from Cl-SOD1 as found in Candida albicans, or that the Cl-SOD1 gene undergoes posttranscriptional regulation upon increase of copper ion.

Phylogenetic analysis of pectin degrading yeasts from deep-sea environments

Deep-sea yeast strains were isolated from mud samples collected in Sagami Bay (1,100-1,400 m) and the Japan Trench (4,500-6,500 m). All of the 46 yeast isolates were capable of growth at 24°C and atmospheric pressure, suggesting that these strains might originate neritic regions or land but survived in deep sea. Based on sequencing of 26S rRNA gene, 15 strains were classified into basidiomycetous yeasts including Cryptococcus liquefaciens, Kondoa aeria, Rhodosporidium diobovatum, Rhodosporidium sphaerocarpum, Rhodotorula mucilaginosa, Rhodotorula dairenensis and Rhodotorula slooffiae, and 8 strains were classified into ascomycetous yeasts including Aureobasidium pullulans, Candida zeylanoides, Kluyveromyces nonfermentans, Metschnikowia bicuspidate and Williopsis saturnus. Screening of the 46 isolates appeared to yield a high frequency of polygalacturonase (PGase) producers capable of degrading pectin. We suggest that deep-sea yeasts are new sources of PGase producers.

Characterization of Cold- and High-Pressure-Active Polygalacturonases from a Deep-Sea Yeast,Cryptococcus liquefaciensStrain N6

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.

Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6

The activity and expression of superoxide dismutase (SOD) was analyzed in a copper-tolerant yeast, Cryptococcus sp. N6. Using cell extracts, two distinct bands exhibiting SOD activity appeared on native PAGE: one band, with higher mobility, appeared when the cells were grown without CuSO4, and the other band appeared when the cells were grown with 10 mM CuSO4. Cells grown with 3 mM CuSO4 produced both SOD isoforms. Western blot analysis, using a monoclonal antibody against human SOD-1, showed that SOD protein was expressed in the absence of CuSO4 and that the expression level increased when the cells were grown with 3 or 10 mM CuSO4. The molecular weight of SOD from strain N6 was approx. 18 kDa. Treatment of the cells with the protein synthesis inhibitor, cycloheximide at 0.5 μg ml−1, did not affect cell growth in the absence of CuSO4 but significantly inhibited growth in the presence of 10 mM CuSO4 and inhibited expression of SOD protein. This suggests that SOD may play a role in cell growth in the presence of high concentrations of CuSO4.

Purification and characterization of novel extracellular endopolygalacturonases from a deep-sea yeast, Cryptococcus sp. N6, isolated from the Japan Trench

Two novel endo-type polygalacturonases (PGase), with molecular weights of 36 kDa and 40 kDa (named p36 and p40, respectively), were purified from the supernatant of the culture medium of a deep-sea yeast, strain N6, isolated from the Japan Trench. The N-terminal 20 amino acids of p36 and p40 were identical, and the sequence homology was 47.4% in comparison with the PGase of Fusarium moniliforme. A treatment of p40 with glycopeptidase F reduced the molecular weight to 36 kDa, suggesting that p40 possessed N-acetylglucosamines on its asparagine residues and p40 might be matured by glycosylation of p36.