Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

Paxilline is an indole-diterpene produced by Penicillium paxilli. Six genes (paxB, C, G, M, P, and Q) in paxilline biosynthetic gene cluster were previously shown to be responsible for paxilline biosynthesis. In this study, we have characterized paxD, which is located next to paxQ and has weak similarities to fungal dimethylallyl tryptophan synthase genes. PaxD was overexpressed in Escherichia coli and the purified enzyme was used for in vitro analysis. When paxilline and dimethylallyl diphosphate were used as substrates, one major and one minor product, which were identified as di-prenyl paxilline and mono-prenyl paxilline by liquid chromatography–mass spectrometry analysis, were formed. The structure of the major product was determined to be 21,22-diprenylated paxilline, showing that PaxD catalyzed the successive di-prenylation. Traces of both products were detected in culture broth of P. paxilli by liquid chromatography–mass spectrometry analysis. The enzyme is likely to be a dimer and required no divalent cations. The optimum pH and temperature were 8.0 and 37 °C, respectively. The Km values were calculated as 106.4?±?5.4 μM for paxilline and 0.57?±?0.02 μM for DMAPP with a kcat of 0.97?±?0.01/s.     

Functional analysis of eubacterial ent-copalyl diphosphate synthase and pimara-9(11),15-diene synthase with unique primary sequences

We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35 degrees C. The Km value was calculated to be 13.7 +/- 1.0 microM for GGDP and the kcat value was 3.3 x 10(-2)/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30 degrees C. The Km value for ent-CDP was estimated to be 2.6 +/- 0.2 microM and the kcat value was 1.4 x 10(-3)/sec.     

A novel enzyme, <Emphasis Type="SmallCaps">d</Emphasis>-3-hydroxyaspartate aldolase from<Emphasis Type="Italic"> Paracoccus denitrificans</Emphasis> IFO 13301: purification,characterization, and gene cloning

A novel enzyme, D-3-hydroxyaspartate aldolase (D-HAA), catalyzing the conversion of D-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301. D-HAA is strictly D-specific as to the alpha-position, whereas the enzyme does not distinguish between threo and erythro forms at the beta-position. In addition to D-3-hydroxyaspartate, the enzyme also acts on d-threonine, D-3-3,4-dihydroxyphenylserine, D-3-3,4-methylenedioxyphenylserine, and D-3-phenylserine. The D-HAA gene was cloned and sequenced. The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues. The predicted amino acid sequence displayed 35% and 22% identity with that of the D-threonine aldolase of Arthrobacter sp. DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively. This is the first paper reporting both a purified enzyme with D-3-hydroxyaspartate aldolase activity and also its gene cloning.     

Heterologous mevalonate production in Streptomyces lividans TK23

Mevalonate is a ubiquitous biosynthetic intermediate of terpenoids and is used as a moisturizer in cosmetics and a chemical for biochemical research. In this study, we have achieved a heterologous production of this useful compound by expression in Streptomyces lividans TK23 of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase genes, which were cloned from Streptomyces sp. strain CL190.     

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.     

Asthma and COPD Overlap Syndrome (ACOS): A Systematic Review and Meta Analysis

Background

The combination of asthma and chronic obstructive pulmonary disease (COPD), or ACOS is a recently defined syndrome. The epidemiology of the condition is poorly described and previous research has suggested ACOS is associated with worse outcomes than either condition alone. We therefore decided to complete a systematic review of the published literature.

Methods

This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta- Analyses guidelines. A structured search was performed in the PubMed, Embase, and Medline databases up to Feb 2015 to identify studies reporting incidence, prevalence, health care utilization, morbidity, or mortality in COPD and asthma.

Results

A total of 19 studies were included in the present study. The pooled prevalence of overlap among COPD was 27% (95% CI: 0.16–0.38, p<0.0001) and 28% (95% CI: 0.09–0.47, p = 0.0032) in the population and hospital-based studies, respectively. We found no significant difference between ACOS and COPD in terms of gender, smoking status, lung function and 6mWD. However, in comparison to subject with only COPD, ACOS subjects were significantly younger, had higher BMI, healthcare utilization, and lower HRQoL.

Conclusion

ACOS is a common condition that exists in a substantial proportion of subjects with COPD. ACOS represents a distinct clinical phenotype with more frequent exacerbations, hospitalization, worse health-related quality of life, and higher healthcare costs than either disease alone. There is a critical need to better define the management and treatment of this syndrome.     

Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol

Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereoselectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride (m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production.