Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile <Emphasis Type="Italic">Metallosphaera sedula</Emphasis>

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu²⁺ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.     

Production of acrylic acid and propionic acid by constructing a portion of the 3-hydroxypropionate/4-hydroxybutyrate cycle from <Emphasis Type="Italic">Metallosphaera sedula</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Acrylic acid and propionic acid are important chemicals requiring affordable, renewable production solutions. Here, we metabolically engineered Escherichia coli with genes encoding components of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula for conversion of glucose to acrylic and propionic acids. To construct an acrylic acid-producing pathway in E. coli, heterologous expression of malonyl-CoA reductase (MCR), malonate semialdehyde reductase (MSR), 3-hydroxypropionyl-CoA synthetase (3HPCS), and 3-hydroxypropionyl-CoA dehydratase (3HPCD) from M. sedula was accompanied by overexpression of succinyl-CoA synthetase (SCS) from E. coli. The engineered strain produced 13.28 ± 0.12 mg/L of acrylic acid. To construct a propionic acid-producing pathway, the same five genes were expressed, with the addition of M. sedula acryloyl-CoA reductase (ACR). The engineered strain produced 1430 ± 30 mg/L of propionic acid. This approach can be expanded to synthesize many important organic chemicals, creating new opportunities for the production of chemicals by carbon dioxide fixation.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Recombinant phospholipase A<Subscript>1</Subscript> of the outer membrane of psychrotrophic <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>: Expression,purification, and characterization

The pldA gene encoding membrane-bound phospholipase A₁ of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A₁ (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A₁ was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A₁ were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A₁ from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

The <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> methionine sulfoxide reductase gene family

The methionine sulfoxide reductases (MSRs) are a group of thiol-dependent enzymes able to catalyze the conversion of methionine sulfoxide to methionine. Although some plant MSRs are known to act as protectants against various abiotic stresses, their activity in the model grass species Brachypodium distachyon has not been characterized as yet. Here, six B. distachyon MSR (BdMSR) genes have been isolated; they generate eight distinct cDNAs, since two of them (BdMSRB1 and -B5) produce a pair of alternatively spliced messages. The genes were transcribed in the root, culm, leaf and during various stages of caryopsis development. Those induced by abiotic stress (salinity, drought, low temperature, CdCl₂, H₂O₂ and abscisic acid) harbored known stress-responsive cis elements in their promoter sequences. The heterologous expression of five of the BdMSRs (-A2, -A4, -B1.1, -B3 and -B5.1) in yeast revealed that their products gave a measure of protection against salinity, mannitol and oxidative stress. Substrate specificity analysis revealed that BdMSRB1.1 could reduce free Met-R-SO to Met. The enzymatic activities of BdMSRA4, -B1.1 and -B5.1 in transformed yeast under salt treatment have checked and increased obviously resulting in reducing more Met-SO to Met including the peptide and the free types under salt stress than those in control.     

Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K _m, 20 µM) than for putrescine (K _m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

Testing the cost of reproduction with the rotifer <Emphasis Type="Italic">Brachionus</Emphasis><Emphasis Type="Italic">patulus</Emphasis> at different pH levels

In the present work, we tested the hypothesis that the cost of reproduction was evident under stressful conditions with the rotifer Brachionus patulus at different pH levels (5–10 at 1 unit intervals). We used sublethal pH levels (pH 5, 9, and 10) to simulate stressful conditions. We analyzed the correlations between age-specific fecundity (m ₁, m ₂, m ₃, …) versus future survival (l _x + 1, l _x + 2, l _x + 3, … for the entire lifespan) (survival costs) and future expectation of reproduction (\( V_{ 1}^{*} , \, V_{ 2}^{*} , \, V_{ 3}^{*} , \ldots \) for the entire lifespan) (reproductive costs), using the data obtained from life table demographic studies of B. patulus under stressful and favorable (pH 6, 7, and 8) pH levels. The results showed that significant negative correlations were observed between age-specific fecundity and future survival and future expectation of reproduction at all tested pH levels, indicating that costs of reproduction exist in the rotifer B. patulus under stressful and favorable pH conditions. However, the percentage of statistically significant negative correlations from total correlations of survival and reproductive costs differed greatly, depending on the tested pH conditions. The percentage of significant negative correlation of reproductive costs is significantly higher under stressful pH conditions (pH 5, 9, and 10) than favorable pH conditions (pH 6, 7, and 8). For survival costs, the same trends are also observed, suggesting that the costs of reproduction were more obvious under stressful pH than favorable pH.     

Distribution and stress tolerance of <Emphasis Type="Italic">Fimbristylis dichotoma</Emphasis> subsp. <Emphasis Type="Italic">podocarpa</Emphasis> (Cyperaceae) growing in highly acidic solfatara fields

Solfatara fields, areas surrounding fumaroles (volcanic vents) near the hot springs or volcanoes, are characterized by severe environmental conditions such as low soil pH and high aluminum contents. Fimbristylis dichotoma subsp. podocarpa is an endangered plant distributed in solfatara fields of Kyushu, western Japan. This species is known to form stands in sites closed to fumaroles where another solfatara plant Miscanthus sinensis do not colonize. We conducted field survey and culture experiments to test the hypothesis that F. dichotoma has higher tolerance to low pH and high aluminum conditions than M. sinensis, which corresponds to the distribution pattern of the two species. In the study site of Myoban Hot Spring, Oita Prefecture, rhizosphere soils of F. dichotoma showed lower pH and higher aluminum contents than those of M. sinensis. The culture experiment showed that germination of F. dichotoma was not inhibited even at pH 2, whereas significant decline in germination (%) was observed in M. sinensis. However, because of the low germination (%) of F. dichotoma, the values of the two species were similar at pH 2. In the pot culture, seedling growth of M. sinensis was suppressed at the aluminum concentrations tested (400 mg AlCl₃ L^?1). Conversely, no significant decline in the seedling growth was observed in F. dichotoma at the same aluminum levels. Considering soil conditions in the field, we concluded that that the difference in the tolerance to aluminum between the two species played a significant role in determining their distribution pattern.     

Molecular cloning and characterization of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase gene in rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis>)

S-Adenosylmethionine decarboxylase (SAMDC) is a key rate-limiting enzyme involved in polyamines biosynthesis, and it plays important roles in plant growth, development and stresses response. However, no SAMDC gene was reported in rubber tree. Here we report characteristics of an SAMDC gene (HbSAMDC1) in rubber tree. HbSAMDC1 contains a 1080 bp open reading frame (ORF) encoding 359 amino acids. Quantitative real-time PCR analyses revealed that HbSAMDC1 exhibited distinct expression patterns in different tissues and was regulated by various stresses, including drought, cold, salt, wounding, and H₂O₂ treatments. HbSAMDC1 5′ untranslated region (UTR) contains a highly conserved overlapping tiny and small upstream ORFs (uORFs), encoding 2 and 52 amino acid residues, respectively. No introns were located in the main ORF of HbSAMDC1, whereas two introns were found in the 5′ UTR. In transgenic tobaccos, the highly conserved small uORF of HbSAMDC1 is found to be responsible for translational repression of downstream β-glucuronidase reporter. To our knowledge, this is the first report on molecular cloning, expression profiles, and 5′ UTR characteristics of HbSAMDC1. These results lay solid foundation for further elucidating HbSAMDC1 function in rubber tree.     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Cloning and characterization of a novel amidase from <Emphasis Type="Italic">Paracoccus</Emphasis> sp. M-1, showing aryl acylamidase and acyl transferase activities

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0–10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k _cat and K _m were 2.8 s^?1 and 158 μM, respectively, and the catalytic efficiency value (k _cat/K _m) was 0.018 μM^?1 s^?1. In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.     

Phylogenetic relationship and time of divergence of <Emphasis Type="Italic">Mus terricolor</Emphasis> with reference to other <Emphasis Type="Italic">Mus</Emphasis> species

Mitochondrial DNA control region of Mus terricolor, three aboriginal species M. spretus, M. macedonicus, M. spicilegus; the Asian lineage M. caroli, M. cervicolor, M. cookii; and the two house mice, M. musculus domesticus and M. m. castaneus were analysed to estimate the substitution rate, phylogenetic relationship and the probable time of divergence. Results showed that M. spretus, M. caroli and M. terricolor are highly diverged from each other (caroli/terricolor = 0.146, caroli/spretus = 0.147 and terricolor/spretus = 0.122), whereas M. spretus showed less divergence with two house mice species (0.070 and 0.071). Sequence divergence between M. terricolor and the Palearctic group were found to be ranging from 0.121 to 0.134. Phylogenetic analysis by minimum evolution, neighbour-joining, unweighed pair group method with arithmetic mean and maximum parsimony showed almost similar topology. Two major clusters were found, one included the Asian lineage, M. caroli, M. cookii and M. cervicolor and the other included the house mice M. m. domesticus, M. m. castaneus and the aboriginal mice M. macedonicus and M. spicilegus along with M. spretus, forming the Palearctic clade. M. terricolor was positioned between the Palearctic and Asian clades. Results showed that Palearctic-terricolor and the Asian lineages diverged 5.47 million years ago (Mya), while M. terricolor had split around 4.63 Mya from their ancestor. M. cervicolor, M. cookii and M. caroli diverged between 4.70 and 3.36 Mya, which indicates that M. terricolor and the Asian lineages evolved simultaneously. M. spretus is expected to have diverged nearly 2.9 Mya from their most recent common ancestor.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.