Generation and analysis of expressed sequence tags (ESTs) of Camelina sativa to mine drought stress-responsive genes.

Camelina sativa is an oil-producing crop belonging to the family of Brassicaceae. Due to exceptionally high content of omega fatty acid, it is commercially grown around the world as edible oil, biofuel, and animal feed. A commonly referred 'false flax' or gold-of-pleasure Camelina sativa has been interested as one of biofuel feedstocks. The species can grow on marginal land due to its superior drought tolerance with low requirement of agricultural inputs. This crop has been unexploited due to very limited transcriptomic and genomic data. Use of gene-specific molecular markers is an important strategy for new cultivar development in breeding program. In this study, Illumina paired-end sequencing technology and bioinformatics tools were used to obtain expression profiling of genes responding to drought stress in Camelina sativa BN14. A total of more than 60,000 loci were assembled, corresponding to approximately 275 K transcripts. When the species was exposed to 10 kPa drought stress, 100 kPa drought stress, and rehydrated conditions, a total of 107, 2,989, and 982 genes, respectively, were up-regulated, while 146, 3,659, and 1189 genes, respectively, were down-regulated compared to control condition. Some unknown genes were found to be highly expressed under drought conditions, together with some already reported gene families such as senescence-associated genes, CAP160, and LEA under 100 kPa soil water condition, cysteine protease, 2OG, Fe(II)-dependent oxygenase, and RAD-like 1 under rehydrated condition. These genes will be further validated and mapped to determine their function and loci. This EST library will be favorably applied to develop gene-specific molecular markers and discover genes responsible for drought tolerance in Camelina species.

Expression, reconstruction and characterization of codon-optimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application.

The high production of functional carbonic anhydrase (CA) is required for practical CO2 sequestration application mediated by CA. Here, the synthetic gene based on Escherichia coli codon usage of new α-type CA (HC-aCA) of Hahella chejuensis, a Korea marine microorganism, was highly expressed in E. coli. We obtained a high yield of functional HC-aCA by denaturing/refolding process and incorporating zinc ion into its active site. The refolded HC-aCA displayed a half-deactivation temperature of 60 °C with maximal activity at 50 °C, and had high pH stability in alkali condition with maximal activity at pH 10.0. The esterase activity of HC-aCA almost doubled at high salt concentration ranging from 0.67 to 2.0 M NaCl. HC-aCA catalyzed the conversion of CO2 to CaCO3 as calcites form in the presence of Ca(2+). The refolded HC-aCA could be a promising candidate for the development of efficient CA-based CO2 sequestration processes.

Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease.

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes-LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12-were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

Expression and characterization of codon-optimized carbonic anhydrase from Dunaliella species for CO(2) sequestration application.

Carbonic anhydrases (CAs) have been given much attention as biocatalysts for CO(2) sequestration process because of their ability to convert CO(2) to bicarbonate. Here, we expressed codon-optimized sequence of α-type CA cloned from Dunaliella species (Dsp-aCAopt) and characterized its catalyzing properties to apply for CO(2) to calcite formation. The expressed amount of Dsp-aCAopt in Escherichia coli is about 50 mg/L via induction of 1.0 mM isopropyl-ß-D-thiogalactopyranoside at 20 °C (for the case of intact Dsp-aCA, negligible). Dsp-aCAopt enzyme shows 47 °C of half-denaturation temperature and show wide pH stability (optimum pH 7.6/10.0). Apparent values of K (m) and V (max) for p-nitrophenylacetate substrate are 0.91 mM and 3.303 × 10(-5) µM min(-1). The effects of metal ions and anions were investigated to find out which factors enhance or inhibit Dsp-aCAopt activity. Finally, we demonstrated that Dsp-aCAopt enzyme can catalyze well the conversion of CO(2) to CaCO(3), as the calcite form, in the Ca(2+) solution [8.9 mg/100 µg (172 U/mg enzyme) with 10 mM of Ca(2+)]. The obtained expression and characterization results of Dsp-aCAopt would be usefully employed for the development of efficient CA-based system for CO(2)-converting/capturing processes.

Superoxide dismutase (SOD) genes in Streptomyces peucetius: effects of SODs on secondary metabolites production.

Two superoxide dismutase (SOD) genes; sod1 and sod2, from Streptomyces peucetius ATCC 27952 show high similarity to other known SODs from Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680. These sod1 and sod2 were cloned into pIBR25 expression vector under a strong ermE* promoter to enhance secondary metabolites from Streptomyces strains. The recombinant expression plasmids; pIBR25SD1 and pIBR25SD2, were constructed to overexpress sod1 and sod2 respectively to enhance production of doxorubicin (DXR) in S. peucetius, clavulanic acid (CA) in Streptomyces clavuligerus NRRL 3585 and actinorhodin (ACT) and undecylprodigiosin (Red) in Streptomyces lividans TK24. Biomass variation, antibiotics production and transcriptional analysis of regulatory genes in recombinant strains have been studied to understand the effect of sod1 and sod2. The cell growth analysis shows that life span of all recombinant strains was found to be elevated as compared to wild type cells. In S. peucetius, overexpression of sod1 and sod2 was not effective in DXR production but in case of S. clavuligerus, CA production was increased by 2.5 and 1.5 times in sod1 and sod2 overexpression, respectively while in case of S. lividans, ACT production was increased by 1.4 and 1.6 times and Red production by 1.5 and 1.2 times upon sod1 and sod2 overexpressions, respectively as compared to the corresponding wild type strains.

Hydrogen peroxide-mediated dealkylation of 7-ethoxycoumarin by cytochrome P450 (CYP107AJ1) from Streptomyces peucetius ATCC27952.

Cytochrome P450 CYP107AJ1, which was isolated from Streptomyces peucetius and showed high homology with peroxygenases, catalyzed a dealkylation reaction with hydrogen peroxide to provide electrons, protons and oxygen, evading the requirement for a supporting redox protein. Preliminary investigation of its transcriptional level in S. peucetius showed significant expression. Homology modeling and subsequent docking with 7-ethoxycoumarin yielded a reasonable docked structure. cyp107AJ1 cloned into pET28a(+) was expressed in Escherichia coli, and soluble protein was subjected to column-chromatographic purification in order to carry out enzyme assays with 7-ethoxycoumarin. HPLC analysis of the extracted product, corresponding to its LC/MS analysis, showed the dealkylated 7-ethoxycoumarin, which was further established by subsequent GC/MS spectral analysis. We suggest that CYP107AJ1 bypassed the requirement for NAD(P)H and redox partners for generating novel analogues.

Homology modeling, binding site identification and docking in flavone hydroxylase CYP105P2 in Streptomyces peucetius ATCC 27952.

Homology models of cytochrome P450 105P2 (CYP105P2) were constructed using four P450 structures, CYP105A1, CYP105, CYP165B3 and CYP107L1, as templates for the model building. Using Accelrys Discovery Studio 2.1 software, the lowest energy CYP105P2 model was then assessed for stereochemical quality and side-chain environment. Further active site optimization of the CYP105P2 model built using these templates was performed by molecular dynamics to generate the final CYP105P2 model. The substrates, flavone, flavanone, quercetin and naringenin, were docked into the model. The model-flavone complex was used to validate the active site architecture, and structurally and functionally important residues were identified by subsequent characterization of the secondary structure.