Analysis of nutritional composition in opaque2- and crtRB1-based single- and double-biofortified super sweet corn.

Sweet corn has emerged as a favorite food item worldwide owing to its kernel sweetness. However, traditional sweet corn cultivars are poor in provitamin-A (proA) and essential amino acids, viz., lysine and tryptophan. So far, no sweet corn hybrid with high nutritional qualities has been commercialized elsewhere. Here, we analyzed accumulation of provitamin-A (proA), lysine, and tryptophan in a set of mutant versions of (i) crtRB1-, (ii) o2-, and (iii) crtRB1 + o2-based sweet corn inbreds and hybrids with (iv) traditional sweet corn (wild-type: O2 + CrtRB1). The crtRB1- and crtRB1 + o2-based genotypes possessed significantly higher proA (17.31 ppm) over traditional sweet corn (2.83 ppm), while o2- and crtRB1 + o2-based genotypes possessed significantly higher lysine (0.345%) and tryptophan (0.080%) over traditional sweet corn (lysine 0.169%, tryptophan 0.036%). Late sowing favored high kernel lysine, proA, and green cob yield among hybrids. Sweetness (17.87%) among the improved inbreds and hybrids was comparable to the original sweetcorn genotypes (17.84%). Among the four genotypic classes, crtRB1 + o2-based improved genotypes showed stronger association among traits over genotypes with o2 and crtRB1 genes alone. Significant association was observed among (i) proA and BC (r = 0.99), (ii) proA and BCX (r = 0.93), (iii) lysine and tryptophan (r = 0.99), and (iv) green cob yield with fodder yield (r = 0.73) in sweet corn hybrids. The study demonstrated that combining crtRB1 and o2 genes did not pose any negative impact on nutritional, yield, and agronomic performance. Sweet corn with crtRB1 + o2 assumes significance for alleviating malnutrition through sustainable and cost-effective approach.

Provitamin A, lysine and tryptophan enrichment in shrunken2-based sweet corn genotypes through genomics-assisted breeding for crtRB1 and opaque2 genes.

BACKGROUND: Malnutrition affects large section of population worldwide. Vitamin A and protein deficiencies have emerged as the major global health-issue. Traditional shrunken2 (sh2)-based sweet corn is deficient in provitamin A (proA), lysine and tryptophan. Natural variant of ß-carotene hydroxylase1 (crtRB1) and opaque2 (o2) enhances proA, lysine and tryptophan in maize. So far, no sweet corn hybrid rich in these nutrients has been released elsewhere. Development of biofortified sweet corn hybrids would help in providing the balanced nutrition. METHODS AND RESULTS: We targeted three sh2-based sweet corn inbreds (SWT-19, SWT-20 and SWT-21) for introgression of mutant crtRB1 and o2 genes using molecular breeding. The gene-based 3'TE-InDel and simple sequence repeat (SSR) (umc1066) markers specific to crtRB1 and o2, respectively were utilized in foreground selection in BC1F1, BC2F1 and BC2F2. Segregation distortion was observed for crtRB1 and o2 genes in majority of populations. Background selection using 91-100 SSRs revealed recovery of recurrent parent genome (RPG) up to 96%. The introgressed progenies possessed significantly higher proA (13.56 µg/g) as compared to the original versions (proA: 2.70 µg/g). Further, the introgressed progenies had accumulated moderately higher level of lysine (0.336%) and tryptophan (0.082%) over original versions (lysine: 0.154% and tryptophan: 0.038%). Kernel sweetness among introgressed progenies (17.3%) was comparable to original sweet corn (17.4%). The introgressed inbreds exhibited higher resemblance with their recurrent parents for yield and morphological characters. CONCLUSION: These newly developed biofortified sweet corn genotypes hold immense promise to alleviate malnutrition.

Microbial xylitol production.

Xylitol is pentahydroxy sugar alcohol, existing in very trace amount in fruits and vegetables, and finds varied application in industries like food, pharmaceuticals, confectionaries, etc. and is of prime importance to health. Owing to its trace occurrence in nature and considerable increase in market demand that exceeds availability, alternate production through biotechnological and chemical approach is in process. Biochemical production involves substrates like lignocellulosic biomasses and industrial effluents and is an eco-friendly process with high dependency on physico-chemical parameters. Although the chemical processes are faster, high yielding and economical, they have a great limitation as usage of toxic chemicals and thus need to be regulated and replaced by an environment friendly approach. Microbes play a major role in xylitol production through a biotechnological process towards the development of a sustainable system. Major microbes working on assimilation of xylose for production of xylitol include Candida tropicalis, Candida maltose, Bacillus subtilis, Debaromyces hansenii, etc. The present review reports all probable microbial xylitol production biochemical pathways encompassing diverse bioprocesses involved in uptake and conversion of xylose sugars from agricultural residues and industrial effluents. A comprehensive report on xylitol occurrence and biotechnological production processes with varied substrates has been encompassed. KEY POINTS: • Xylitol from agro-industrial waste • Microbial xylose assimilation.

Bioprospecting of xylanase producing fungal strains: Multilocus phylogenetic analysis and enzyme activity profiling.

The study aims to explore potential xylanase-producing indigenous fungi isolated from soil and vegetable wastes containing plant degraded matter, reporting multilocus phylogenetic analysis and xylanase enzyme activity from selective strains. Four potential xylanolytic fungi were identified through distinct primary and secondary screening of 294 isolates obtained from the samples. Morphological characterization and multigene analysis (ITS rDNA, 18S rDNA, LSU rDNA, ß-tubulin, and actin gene) confirmed them as Aspergillus sp. AUMS56, Aspergillus tubingensis AUMS60 and AUMS64, and Aspergillus fumigatus AUKEMS24; achieving crude xylanase activities (through submerged fermentation using corn cobs) of 18.9, 32.29, 30.68, and 15.82 U ml-1 , respectively. AUMS60 and AUMS64 (forming lineage with A. tubingensis and Aspergillus niger in the same phylogroup with 100% Bayesian posterior probability support) secreted single xylanase (Xyn60; 36 kDa) and multiple xylanases (Xyn64A and Xyn64B; 33.4 and 19.8 kDa) respectively, having pH optima of 6.0 and exhibiting maximal activity at 60°C. These enzymes were highly stable at 40°C (120 h) and retained more than 70% activity at 50°C and at pH 5-6 (upon 72 h incubation). Our analysis suggested these enzymes to be endoxylanases demonstrating substrate hydrolysis within 15 min of reaction and maximum efficiency of xylanases from AUMS60 and AUMS64 achieving 51.1% (13 h) and 52.2% (24 h) saccharification, respectively. They also showed enhanced catalytic activity with various cations. Based on our investigation on xylan hydrolysis, we believe that these xylanases may find significant industrial applications as they have a real potential of working as a bio-catalytic cocktail (patent file number: IN E1/38213/2020-DEL) for the enhanced saccharification of lignocelluloses.

Characterization, cloning and functional expression of novel xylanase from Thermomyces lanuginosus SS-8 isolated from self-heating plant wreckage material.

Extracellular cellulase free xylanase from Thermomyces lanuginosus sp. SS-8, isolated from self heating plant wreckage material was identified as ß-1,4-endo-xylanase precursor, a monomer of 21.3 kDa with no carbohydrate residue. This xylanase retained 80 % activity at 60 °C for 96 h, was active at a wide pH range of 3-11 and uniquely hydrolyzed xylan to xylose without production of xylo-oligosaccharides. Gene xynSS8 encoding xylanase from T. lanuginosus SS-8 was cloned and functionally expressed in Escherichia coli XL1 Blue using pTZ57R/T plasmid and xynSS8/pQE-9 expression vector construct respectively. Gene xynSS8 was of 777 bp and deduced amino acid sequence was a mature xylanase of 258 amino acids. XynSS8 has extra 33 amino acids compared to its nearest homolog and was thermo-alkali tolerant as that of native protein. The xylanase could degrade pulp and release substantial chromophoric materials and lignin derived compounds indicating its effective utility in pulp bleaching. Novel characteristics of the enzyme may contribute to its wide industrial usage. This is first report of cloning and functional expression of the novel xylanase from T. lanuginosus SS-8.

Alumina-supported oxime for the degradation of sarin and diethylchlorophosphate.

1-(4-Chlorophenyl))-N-hydroxymethanimine and cyclohexyl-N-hydroxymethanimine were synthesized and a well-established oxime, i.e., 2-[(hydroxyimino)methyl]-1-methylpyridinium chloride was purchased. Thereafter; all were loaded over Al(2)O(3) using incipient wetness technique. The prepared systems were characterized using surface area analyzer, scanning electron microscope, energy dispersive X-ray spectrophotometer, Fourier transform infrared spectrophotometer and thermogravimetric analyzer. Kinetics of the degradation of sarin (GB) and simulant, i.e. diethylchlorophosphate (DEClP) was studied over synthesized oxime impregnated Al(2)O(3) and results were compared with well reported oxime impregnated Al(2)O(3). Kinetics of reaction was found to be following the pseudo first order reaction kinetics. The order of reactivity of the prepared systems was found to be cyclohexyl-N-hydroxymethanimine/Al(2)O(3)>1-(4-chlorophenyl)-N-hydroxymethanimine/Al(2)O(3)>2-[(hydroxyimino)methyl]-1-methylpyridinium chloride/Al(2)O(3)>Al(2)O(3). From the reaction kinetics it was observed that the reaction with DEClP was faster than with GB. Cyclohexyl-N-hydroxymethanimine/Al(2)O(3) was found to be the most reactive system with half-life of 0.94 and 15 h for DEClP and GB respectively.

Purification and preliminary characterization of a xylanase from Thermomyces lanuginosus strain SS-8.

Thermomyces lanuginosus SS-8 was isolated from soil samples that had been collected from near self-heating plant material and its extracellular cellulase-free xylanase purified approximately 160-fold using ion exchange chromatography and continuous elution electrophoresis. This xylanase was thermoactive (optimum temperature 60 °C) at pH 6.0 and had a molecular weight of 23.79 kDa as indicated by SDS-PAGE electrophoresis. The xylanase rapidly hydrolyzed xylan directly to xylose without the production of intermediary xylo-oligosaccharides within 15 min of incubation under optimum conditions. This trait of rapidly degrading xylan to xylose as a sole end-product could have biotechnological potential in degradation of agro-wastes for bioethanol manufacturing industry.

Study of codon bias perspective of fungal xylanase gene by multivariate analysis.

Fungal xylanases has important applications in food, baking, pulp and paper industries in addition to various other industries. Xylanases are produced extensively by both bacterial and fungal sources and has tremendous potential of being active at extremes of temperature and pH. In the present study an effort has been made to explore the codon bias perspective of this potential enzyme using bioinformatics tools. Multivariate analysis has been used as a tool to study codon bias perspectives of xylanases. It was further observed that the codon usage of xylanases genes from different fungal sources is not similar and to reveal this phenomenon the relative synonymous codon usage (RSCU) and base composition variation in fungal xylanase genes were also studied. The codon biasing data like GC content at third position (GC(3S)), effective codon number (N(C)), codon adaptive index (CAI) were further analyzed with statistical softwares like Sigma1plot 9.0 and Systat 11.0. Furthermore, study of translation selection was also performed to verify the influences of codon usage variation among the 94 xylanase genes. In the present study xylanase gene from 12 organisms were analyzed and codon usages of all xylanases from each organism were compared separately. Analysis indicates biased codon among all 12 fungi taken for study with Aspergillus nidulans, Chaetomium globosum, Aspergillus terreus and Aspergillus clavatus showing maximum biasing. N(C) plot and correspondence analysis on relative synonymous codon usage indicate that mutation bias and translation selection influences codon usage variation in fungal xylanase gene. To reveal the relative synonymous codon usage and base composition variation in xylanase, 94 genes from 12 fungi were used as model system.

An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance.

Deinococcus radiodurans R1 recovering from acute dose of gamma radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or non-homologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double-strand break repair during post-irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in gamma radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from gamma radiation. The results strongly suggest that 3' (rho) 5' single-stranded DNA ends constitute an important component in recombination pathway involved in DNA double-strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.