Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Modulation of nitrosative stress via glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase.

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.

Biochemical characterization of a functional recombinant aryl-alcohol dehydrogenase from Taiwanofungus camphorata.

BACKGROUND: Aryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds. RESULTS: One TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied. CONCLUSIONS: The coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.

Expression, localization and function of a cis-prenyltransferase in the tapetum and microspores of lily anthers.

The cis-prenyltransferase gene LLA66 (Lilium longiflorum anther-66), the first prenyltransferase to be identified in the tapetum and microspores, was selected from a suppression subtractive cDNA library during microspore development in the anther of L. longiflorum. The LLA66 cDNA encodes a polypeptide of 308 amino acids with a calculated molecular mass of 35.7 kDa. Thermal asymmetric interlaced-PCR was employed to obtain the 5'-regulatory region of LLA66. Sequence alignment revealed that the LLA66 protein shares 30-41% identity with cis-prenyltransferases of various broad-spectrum species and is phylogenetically distinct from other monocot cis-prenyltransferases. Based on critical regulatory domains in cis-prenyltransferase, LLA66 was concluded to catalyze the production of long-chain polyprenyl products. RNA blot analysis indicated that the LLA66 gene is anther specific and differentially expressed during microspore development in the anther. In situ hybridization with the digoxigenin-labeled antisense riboprobe of LLA66 showed strong signals at the tapetal layer of the anther wall. The LLA66 mRNA was also coordinately detected in the microspores. Furthermore, gibberellin inhibitor analysis indicated that the LLA66 gene is endogenously induced by gibberellin, but its induction is independent of ethylene regulation. Reverse transcription-PCR analysis indicated that gene expression of LLA66 both in the microspore and in the anther wall increased to the maximum level, at which stage the tapetum became highly active and secretory. The enzyme activity of prenyltransferases in various stages of microspore development correlated with tapetal growth and disintegration. LLA66 was introduced into Saccharomyces cerevisiae, and the His-tagged LLA66 protein was affinity purified using Ni(2+)-nitrilotriacetic acid-agarose. The involvement of cis-prenyltransferase in the anther in the synthesis of dolichols and polyprenols is discussed.

An arsenate reductase homologue possessing phosphatase activity from sweet potato (Ipomoea batatas [L.] Lam): kinetic studies and characterization.

A cDNA encoding a putative arsenate reductase homologue (IbArsR) was cloned from sweet potato (Ib). The deduced protein showed a high level of sequence homology (16-66%) with ArsRs from other organisms. A 3-D homology structure was created based on AtArsR (PDB code 1T3K ) from Arabidopsis thaliana. The putative active site of protein tyrosine phosphatase (HC(X)(5)R) is conserved in all reported ArsRs. IbArsR was overexpressed and purified. The monomeric nature of the enzyme was confirmed by 15% SDS-PAGE and molecular mass determination of the native enzyme via ESI Q-TOF. The IbArsR lacks arsenate reductase activity but possesses phosphatase activity. The Michaelis constant (K(M)) value for p-nitrophenyl phosphate (pNPP) was 11.11 mM. The phosphatase activity was inhibited by 0.5 mM sodium arsenate [As(V)]. The protein's half-life of deactivation at 25 °C was 6.1 min, and its inactivation rate constant K(d) was 1.1 × 10(-1) min(-1). The enzyme was active in a broad pH range from 4.0 to 11.0 with optimum activity at pH 10.0. Phosphatase would remove phosphate group from nucleic acid or dephosphorylation of other enzymes as regulation signaling.

Cloning, expression, and purification of a functional glutathione reductase from sweet potato (Ipomoea batatas [L.] Lam): kinetic studies and characterization.

A cDNA encoding a putative glutathione reductase (GR) was cloned from sweet potato (Ib). The deduced protein showed high level of sequence homology with GRs from other plants (79-38%). A three-dimensional (3-D) homology structure was created. The active site Cys residues are conserved in all reported GR. Functional IbGR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 10% native polyacrylamide gel electrophoresis (PAGE). The monomeric nature of the enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular mass determination of the native enzyme. The Michaelis constant (K(m)) values for GSSG (glutathione disulfide) and NADPH (ß-nicotinamide adenine dinucleotide phosphate, reduced form) were 0.114 and 0.056 mM, respectively. The enzyme activity was inhibited by Cu(2+) and Zn(2+), but not by Ca(2+). The protein's half-life of deactivation at 70 °C was 3.3 min, and its thermal inactivation rate constant K(d) was 3.48 × 10(-1) min(-1). The enzyme was active in a broad pH range from 6.0 to 11.0 and in the presence of imidazole up to 0.8 M. The native enzyme appeared to be resistant to digestion by trypsin or chymotrypsin.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets.

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

Production of 10-hydroxy-8(E)-octadecenoic acid from oleic acid conversion by strains of Pseudomonas aeruginosa.

Eighteen Pseudomonas aeruginosa strains were examined for their ability to convert oleic acid to produce 10-hydroxy-8(E)-octadecenoic acid (HOD), which was structurally confirmed by GC-MS, NMR, and FTIR. There were no substantial amounts of other new compounds found in the fermentation broths in addition to HOD and 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). The results demonstrated that P. aeruginosa strains possessed varying levels of activity for producing HOD. Under the experimental conditions, strain NRRL B-14938 isolated from sheep manure was the best HOD producer exhibiting the highest HOD to DOD product ratio in the medium most suitable for purifying HOD. Using strain B-14938 as a model system for further characterization, optimum conditions for producing HOD were found to be at 26 degrees C and pH 7.0 after 60 h of reaction time using a medium containing EDTA as a chelating agent. This study has identified a high-yielding P. aeruginosa strain and provided the reaction characteristics needed to develop a scale-up production process of HOD for testing its properties and potential new uses.

Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs.

Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.

Biochemical characterization of a novel 2-Cys peroxiredoxin from Antrodia camphorata.

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A gene encoding a novel 2-Cys Prx was identified based on sequence homology in an expressed sequence tag database of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. The 2-Cys Prx cDNA (940 bp) encodes a protein of 188 amino acid residues with calculated molecular mass of 20,965 Da and a pI of 5.89. The coding region was subcloned into pAVD10, transformed into Escherichia coli, and expressed as a His-tagged fusion protein. The purified enzyme was characterized under various conditions. The Prx retained 68% activity after being heated at 60 degrees C for 2 min. It was stable under a broad pH range from 5 to 11. The enzyme activity was slightly decreased in the presence of 1% sodium dodecyl sulfate. The enzyme was somewhat susceptible to chymotrypsin treatment but resistant to digestion by trypsin.

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability.

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.

Molecular cloning of bovine eIF5A and deoxyhypusine synthase cDNA.

Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.

Higher activity of recombinant bovine deoxyhypusine synthase vs. human deoxyhypusine synthase.

Mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nepsilon-(4-amino-2(R)-hydroxybutyl)lysine). The synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eIF5A. We have subcloned the cDNA encoding bovine and human deoxyhypusine synthase into a pET-11a expression vector, separately. T7-tagged bovine and human deoxyhypusine synthase have been overexpressed in Escherichia coli and purified to homogeneity using T7 antibody affinity chromatography. Activities of the enzyme from both human and bovine have been measured by their ability to convert the eIF5A precursor protein to the intermediate, deoxyhypusine form of eIF5A. Our results have shown that bovine deoxyhypusine synthase has considerably higher activity than human deoxyhypusine synthase in catalyzing the synthesis of deoxyhypusine.