Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans Abstract Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of d-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s−1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M−1 s−1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides d-(+)-galactose, the purified enzyme also acted against d-(+)-raffinose, α-d-(+)-melibiose, and methyl-α-d-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

Degradation of Triclosan from Domestic Wastewater by Biosurfactant Produced from Bacillus licheniformis Abstract The use of triclosan (TCS), an antimicrobial agent in consumer product, results in adverse effects on the environment due to its wide usage all over the world. The present study focused on TCS detection and attempted for degradation by biosurfactant produced by Bacillus licheniformis from domestic wastewater in Surathkal region, Karnataka, India. The experimental investigation includes biosurfactant production using crude sunflower oil and detection and degradation of TCS from wastewater by High-Performance Liquid Chromatography (HPLC). Results exhibited that maximum biosurfactant yield (7.8 g/L) was achieved using 1 g/L of glycerol as carbon and 5.5 g/L of ammonium bicarbonate as a nitrogen source. Detection of TCS from domestic wastewater (0.36 mg/L) and degradation was carried out by HPLC. The result discloses that 47.2% and 100% removal of TCS was achieved in 2 h and 16 h for 1:1(v/v) ratio of wastewater and biosurfactant.

Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology Abstract Molecular characteristics including information of insertion site, flanking sequence, and copy numbers are the base for the safety assessment and subsequent monitoring of genetically modified organisms (GMOs), which has to be revealed thoroughly in a case-by-case manner. Although both polymerase chain reaction (PCR)-based and next-generation sequencing (NGS)-based approaches are proven to be effective in the molecular characterization of most of GM events, they often fail to work with GM maize events, mainly due to the genome complexity. In this study, by using NGS, we successfully identified the 3′ end T-DNA insertion site and flanking sequence of a GM maize event IE09S034, which were confirmed by PCR amplification and Sanger sequencing. Notably, insertions of unintended exogenous elements were revealed in this event although the single copy of target exogenous genes was also confirmed by digital PCR. The output of this study provides novel and important genetic evidence for the safety assessment and monitoring of GM maize event IE09S034.

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label Abstract 1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

Biochemical and Molecular Characterizations of a Novel pH- and Temperature-Stable Pectate Lyase from Bacillus amyloliquefaciens S6 for Industrial Application Abstract In this paper, we report cloning of a pectate lyase gene from Bacillus amyloliquefaciens S6 (pelS6), and biochemical characterization of the recombinant pectate lyase. PelS6 was found to be identical with B. subtilis 168 pel enzyme with 100% amino acid sequence homology. Although these two are genetically very close, they are distinctly different in physiology. pelS6 gene encodes a 421-aa protein with a molecular mass of 65,75 kDa. Enzyme activity increased from 12.8 ± 0.3 to 49.6 ± 0.4 units/mg after cloning. The relative enzyme activity of the recPel S6 ranged from 80% to 100% at pH between 4 and 14. It was quite stable at different temperature values ranging from 15 to 90 °C. The recPEL S6 showed a maximal activity at pH 10 and at 60 °C. 0.5 mM of CaCl2 is the most effective metal ion on the recPEL S6 as demonstrated by its increased relative activity with 473%. recPEL S6 remained stable at − 20 °C for 18 months. In addition recPEL S6 increased juice clarity. This study introduces a novel bacterial pectate lyase enzyme with its characteristic capability of being highly thermostable, thermotolerant, and active over a wide range of pH, meaning that it can work at both acidic and alkaline environments, which are the most preferred properties in the industry.

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton Abstract The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

Functional Analysis of the Marigold ( Tagetes erecta ) Lycopene ε - cyclase ( TeLCYe ) Promoter in Transgenic Tobacco Abstract Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5′-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of − 910 to − 429 bp 5′ to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its’ deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.

The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase Abstract Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5β,6α,8β-trihydroxycariolan, 5β,8β-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.

RETRACTED ARTICLE: Characterization and Evaluation of Mango Germplasm Through Morphological, Biochemical, and Molecular Markers Focusing on Fruit Production: An Overview

Recent Trends in Enhancing the Resistance of Cultivated Plants to Heavy Metal Stress by Transgenesis and Transcriptional Programming Abstract Normal growth and development of high plants strongly depends on the concentration of microelements, including essential heavy metals, in the substrate. However, an excess of those elements may become harmful. Therefore, micronutrient concentrations in plant tissue should be well-balanced and controlled by homeostatic mechanisms. The advancement of knowledge on the regulation of metal homeostasis in plants is important for phytoremediation of metal-contaminated soil and for micronutrient malnutrition control. Experimental data from loss-of-function and gain-of-function studies, including functional descriptions and classifications have presented new opportunities for multiplex CRISPR/dCas9-driven control of gene expression and have opened up new prospects for the goal-seeking regulation of metal homeostasis in plants. The aim of this review is to help for multiplex transcriptional programming targets search by summarizing and analyzing data on possible ways to handle a plant’s ability to maintain metal homeostasis.