A novel oleaginous yeast Saccharomyces cerevisiae CU-TPD4 for lipid and biodiesel production.

This study reports on the novel Saccharomyces cerevisiae CU-TPD4 that was isolated from coconut waste residues obtained from a coconut factory in Thailand. The CU-TPD4 isolate was confirmed to be a S. cerevisiae by molecular analysis and to be an oleaginous yeast with more than 20% (w/w) of the cell dry weight (CDW) present in the form of lipids. The lipid content and lipid yield of CU-TPD4 (52.96 ± 1.15% of CDW and 1.78 ± 0.06 g/L, respectively) under optimized growth conditions were much higher than those under normal growth conditions (22.65 ± 1.32% of CDW and 1.24 ± 0.12 g/L, respectively). The major fatty acids produced by CU-TPD4 were oleic (C18:1), palmitoleic (C16:1), stearic (C18:0), and palmitic (C16:0) acids. Mathematical estimation of the physical properties of the biodiesel obtained by transesterification of the extracted lipid suggested it was suitable as biodiesel with respect to the ASTM D6751 and EN 14214 international standards. Consequently, S. cerevisiae CU-TPD4 is expected to emerge as a promising alternative for biodiesel production.

Dataset of blood cockle (Anadara granosa) microbiota from coastal areas and earthen-pond farms around the upper Gulf of Thailand.

The blood cockle (Anadara granosa), a bivalve mollusc, is a unique seafood item in Southeast Asia. Bivalve molluscs are filter feeders upon plankton, and so they may bioaccumulate microbes and heavy metals in their tissues. Bacteria survival can be enhanced by living inside the shell and they can subsequently infect humans and higher vertebrates after ingestion of the bivalve. This study presented a metagenomics analysis of the bacteria associated with A. granosa from six farms around the Gulf of Thailand. Three farms were located on the coast and the other three were from earthen ponds. Genomic DNA was extracted from the samples and analysed via sequencing of the V3-V4 region of the 16S rRNA gene, and then using a 97% DNA sequence similarity cut-off for designation of the operational taxonomic units. The environmental parameters, including temperature, pH, salinity, dissolved oxygen, and the concentration of three heavy metals (Cu, Cr, and Hg) and one metalloid (As) were investigated. The raw sequence data is available at the NCBI Sequence Read Archive accession number PRJNA592226. The Proteobacteria, Bacteroidetes and Cyanobacteria were common components of the microbiota in all six habitats and together comprised more than 77% of the relative abundance in all the samples. This is the first report on the microbiome in blood cockles in Thailand by a culture independent method. The data can be applied for efficiently controlling and improving seafood safety management.

Draft Genome Sequence and Annotation of Paracoccus versutus MAL 1HM19, a Nitrate-Reducing, Sulfide-Oxidizing Bacterium.

Paracoccus versutus MAL 1HM19 is a mixotrophic nitrate-reducing sulfide-oxidizing bacterium which plays a crucial role in hydrogen sulfide (H2S) and nitrate (NO3 -) removal. In this study, we report the draft genome sequence of P. versutus MAL 1HM19.

High-Level Heterologous Expression of Endo-1,4-ß-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression.

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0-9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s-1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

Efficient expression and secretion of endo-1,4-ß-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides.

Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55 °C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40 °C for 6 h. With birchwood xylan as substrate, the enzyme showed a Km of 5.2 mg/mL, and kcat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.

Comparison of sulphide and nitrate removal from synthetic wastewater by pure and mixed cultures of nitrate-reducing, sulphide-oxidizing bacteria.

In this study, the activities of hydrogen sulphide (H2S) oxidation and nitrate (N-NO3-) reduction by three pure and mixed strains of nitrate-reducing, sulphide oxidizing bacteria (NR-SOB) were determined. Batch experiments were performed at 35 °C and pH 7.0-8.0 with initial H2S concentrations of 650-900 ppmv and N-NO3- concentrations of ∼120 mg/L. The strains MAL 1HM19, TPN 1HM1 and TPN 3HM1 were capable of removing 100% gas-phase H2S. The co-cultures showed better performance for H2S and N-NO3- removal. The mixed NR-SOB strains showed a higher H2S oxidation rate (143 ±â€¯18 ppmv/h), while the highest N-NO3- removal rate (5.5 ±â€¯0 and 5.1 ±â€¯0.6 N-NO3- mg/L·h) was obtained by a mixture of two NR-SOB strains. The 16S rDNA sequence analysis revealed that all strains belonged to the sub-class Alphaproteobacteria and are closely related to Paracoccus sp. (>99%).

Laccases from Marine Organisms and Their Applications in the Biodegradation of Toxic and Environmental Pollutants: a Review.

The discharge of industrial effluent creates environmental problems around the world and so necessitates the need for the economically expensive and sometimes technically problematic treatment of the wastewater. Laccases have enormous potential for the oxidative bioremediation of toxic xenobiotic compounds using only molecular oxygen as the sole cofactor for their reaction, and their application is regarded as environmentally friendly. Due to the low substrate specificity of laccases, they can oxidize a variety of substrates. Moreover, by using appropriate mediators, laccases can degrade a wide range of substrates, including those with structural complexity. Thus, laccases are an attractive alternative for wastewater treatment. Marine environments are rich in microorganisms that are exposed to extreme conditions, such as salinity, temperature, and pressure. Laccases from these microorganisms potentially have suitable properties that might be adaptive to bioremediation processes. This review provides the latest information on laccases from marine environments, their sources, biochemical properties, media composition for laccase production, and their applications in the bioremediation of industrial waste, especially focusing on dye decolorization.

Cloning, expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris.

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35-37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg2+, Mn2+, Li+, Ca2+, Ni2+, CHAPS, DTT, and EDTA and inhibited by Hg2+, Ag+, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.

Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.

The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.

The potential of cellulosic ethanol production from grasses in Thailand.

The grasses in Thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process. The average percentage composition of cellulose, hemicellulose, and lignin in the samples of 18 types of grasses from various provinces was determined as 31.85-38.51, 31.13-42.61, and 3.10-5.64, respectively. The samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification. The total reducing sugars in most grasses ranging from 500-600 mg/g grasses (70-80% yield) were obtained. Subsequently, 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation (SSCF). The enzymes, cellulase and xylanase, were utilized for hydrolysis and the yeasts, Saccharomyces cerevisiae and Pichia stipitis, were applied for cofermentation at 35 °C for 7 days. From the results, the highest yield of ethanol, 1.14 g/L or 0.14 g/g substrate equivalent to 32.72% of the theoretical values was obtained from Sri Lanka ecotype vetiver grass. When the yields of dry matter were included in the calculations, Sri Lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 L/ha/year, whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 L/ha/year (0.98 g/L or 0.12 g/g substrate equivalent to 30.60% of the theoretical values).

Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica.

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 µmol/(min mg protein), respectively, with p-nitrophenyl-ß-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha.

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.

Butanol and ethanol production from tapioca starch wastewater by Clostridium spp.

Total (TWW) and tapioca starch wash wastewater (TSWW) from a cassava processing plant in Thailand were analyzed for their composition with a view to evaluate their potential as substrates for solvent production by ABE fermentation with Clostridium spp. Starch was detected at a 1.63-fold higher level in the TWW than that in the TSWW (24.4% and 15.0% (w/w), respectively). The chemical oxygen demand (COD) was broadly similar (20,093 and 20,433 mg/L), but the biological oxygen demand (BOD) was 1.84-fold higher (18,000 and 9,750 mg/L) in the TWW than that in the TSWW. Thus, the TSWW was selected as a substrate to evaluate its potential for butanol and ethanol production by two Clostridium spp. The combined ethanol plus butanol production in the TSWW at pH 6.5 was higher than that at pH 4.5, being around 27.8- and 3.4-fold higher in C. butyricum TISTR 1032 and C. acetobutylicum ATCC 824, respectively. In both strains, the butanol (and combined butanol plus ethanol) production level was improved at pH 5.5. The addition of yeast extract increased the bacterial cell production, but did not significantly improve solvent productivity in C. acetobutylicum, and even decreased butanol production by C. butyricum.

NirK and nirS Nitrite reductase genes from non-agricultural forest soil bacteria in Thailand.

The genetic heterogeneity of the nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in a non-agricultural forest soil in Thailand was investigated using soil samples from the Plant Germplasm-Royal Initiation Project area in Kanchanaburi Province, Thailand. Soil bacteria were screened for denitrification activity and 13 (from 211) positive isolates were obtained and further evaluated for their ability to reduce nitrate and to accumulate or reduce nitrite. Three species with potentially previously unreported denitrifying activities were recorded. Analysis of the partial nirK and nirS sequences of these 13 strains revealed a diverse sequence heterogeneity in these two genes within the same environment and even potentially within the same host species, the potential existence of lateral gene transfer and the first record of both nirK and nirS homologues in one bacterial species. Finally, isolates of two species of bacteria (Corynebacterium propinquum and Micrococcus lylae) are recorded as denitrifiers for the first time.

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid.

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha.

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 degrees C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l(-1). Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.

Solventogenic-cellulolytic clostridia from 4-step-screening process in agricultural waste and cow intestinal tract.

A clostridial bacterium is accepted to be one of the important and efficient microorganisms for the application in fuel fermentation process. However, the lack of cellulolytic activity of cellulosome in this organism appears to be one of the main important problems for efficient production of the fuel. It is therefore interesting to search for the genetic resource of natural clostridial bacteria for the application in bioengineering. Presently, Clostridium species selection and identification are based on various physiological properties tests. This article developed the way for a 4-step screening process via mainly three criteria and 16S rDNA identification. In this study, solvent-producing clostridial bacteria were successfully isolated from decomposed sources, cow feaces, and dry grass in Thailand. Anaerobes were screened by cellulolytic activity and butanol tolerance in selective media that composed of basal media supplemented with 2% cellulose and 5% butanol. Thirty isolates of cellulolytic and butanol-tolerant anaerobic bacteria were obtained from screening in this medium. Fifteen isolates were rapidly classified as in the class Clostridia by three selected criteria (endospore formation, sulfite-reducing ability, and metabolic products). Secondary metabolites of the bacteria such as acetone, butanol and ethanol were varied depending on the process. Clostridial differential medium was used as a genus identification tool. Finally, PCR-amplified gene fragments coding for 16S rDNA were analyzed as a key to identify bacteria species. This process can be used to screen and identify Clostridium species in short period. Cellulosome and non-cellulosome cellulases productivity were analyzed. The results revealed that the selected cellulolytic strains (such as Fea-PA) exhibited EngD non-cellulosome cellulase activity especially endoglucanase activity on carboxymethyl cellulose. The selective system in this research was appropriate for the screening of Clostridiaceae in a similarity range between 83% and 100%.

Search for a dextransucrase minimal motif involved in dextran binding.

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K(d) of 2.8x10(-9)M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl S300HR for rapid purification was evaluated and is discussed.