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A fungal nitrilase gene from Fusarium proliferatum AUF-2 was cloned through reverse transcription-PCR. The open reading frame consisted of 903 bp and potentially encoded a protein of 301 amino acid residues with a theoretical molecular mass of 33.0 kDa. The encoding gene was expressed in Escherichia coli strain BL21 and the recombinant protein with His-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity in the range of 35-40 °C and pH 8.0. EDTA, Mg, Zn, Ca, Fe, Fe and Mn stimulated hydrolytic activity, whereas Cu, Co and Ni had inhibitory effect on nitrilase activity. Ag ions showed a strong inhibitory effect on the recombinant nitrilase activity. This nitrilase was specific towards aliphatic, heterocyclic and aromatic nitriles. The kinetic parameters V and K for benzonitrile substrate were determined to be 14.6 μmol/min/mg protein and 1.55 mM, respectively. Homology modelling and molecular docking studies provided an insight into the substrate specificity and the proposed catalytic triad for recombinant nitrilase consisted of Glu-54, Lys-133 and Cys-175. This is the first report on the cloning and heterologous expression of nitrilase from Fusarium proliferatum. [ABSTRACT FROM AUTHOR]

• Engineering a natural diploid S. cerevisiae for increased FAEE production. • Rational allocation of metabolic fluxes towards expanding FAEE precursor pools. • Process optimization in shake flask increased the titer to ∼40-folds. • Scale-up production in fed-batch bioreactor yielded a final 5.0 g/L FAEE titer. Developing microbial chassis for synthesizing value-added compounds is the most eco-friendly approach to minimize the impacts of overusing fossil fuels and other harsh chemicals. Recent research has focussed on harnessing microbial fatty acid metabolism to generate oleochemicals for use in industries as diverse as fuel production, cosmetics, lubricants and more. Considering the economic viability of generating "green biocatalysts", we reconfigured the fatty acid biosynthesis pathway in an environmentally robust strain of Saccharomyces cerevisiae MTCC4796 to produce fatty acid ethyl ester (FAEE) with high yield and titer. We applied the push–pull-block strategy wherein a heterologous wax ester synthase (WS2) was initially integrated to establish a FAEE biosynthetic pathway (pull) with additional rewiring including overexpression of PDH-bypass pathway genes for increasing the supply of precursor fatty acyl-CoA (push) and single allele disruption of non-essential genes from competing pathways (block) to maximize the metabolic flux towards FAEE overproduction. Our final engineered strain NGYT carrying 11 genetic modifications produced 26 mg/L and 1 g/L of FAEE before and after glucose optimization respectively. This titer was further scaled-up to reach 5 g/L in fed-batch bioreactors, which, to our knowledge, is the highest reported FAEE titer achieved from Saccharomyces cerevisiae with glucose as carbon source. Additionally, the FAEE's produced by NGYT strain showed a higher percentage of monounsaturated fatty acids, which are ideally suitable for biodiesel applications. Given the global emphasis on the development of cleaner fuels, the findings from this study expedite our progress towards industrial scale-FAEE production as a substitute to conventional diesel. [ABSTRACT FROM AUTHOR]

Trichoderma is an anamorphic filamentous fungal genus with immense potential for production of small valuable secondary metabolites with indispensable biological activities. Microbial dynamics of a psychrotrophic strain Trichoderma velutinum ACR-P1, isolated from unexplored niches of the Shiwalik region, bestowed with rich biodiversity of microflora, was investigated for production of nonribosomal peptides (NRPs) by metabolite profiling by intact-cell mass spectrometry (ICMS) employing matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. Being the first report on NRPs production by T. velutinum, studies on optimization of growth conditions by Response Surface Methodology (RSM) for production of NRPs by ACR-P1 was carried out strategically. Multifold enhancement in the yield of NRPs belonging to subfamily SF4 with medium chain of amino acid residues having m/ z 1437.9, 1453.9, and 1452.0 at pH 5.9 at 20 °C and of subfamily SF1 with long-chain amino acid residues having m/ z 1770.2, 1784.2, 1800.1, 1802.1, and 1815.1 was achieved at pH 7.0 at 25 °C. Complexities of natural mixtures were thus considerably reduced under respective optimized culture conditions accelerating the production of novel microbial natural products by saving time and resources. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic hydrolysates contain a mixture of hexose (C6)/pentose (C5) sugars and pretreatment-generated inhibitors (furans, weak acids and phenolics). Therefore, robust yeast isolates with characteristics of C6/C5 fermentation and tolerance to pretreatment-derived inhibitors are pre-requisite for efficient lignocellulosic material based biorefineries. Moreover, use of thermotolerant yeast isolates will further reduce cooling cost, contamination during fermentation, and required for developing simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SScF), and consolidated bio-processing (CBP) strategies. Results: In this study, we evaluated thirty-five yeast isolates (belonging to six genera including Saccharomyces, Kluyveromyces, Candida, Scheffersomyces, Ogatea and Wickerhamomyces) for pretreatment-generated inhibitors {furfural, 5-hydroxymethyl furfural (5-HMF) and acetic acid} and thermotolerant phenotypes along with the fermentation performances at 40 °C. Among them, a sugarcane distillery waste isolate, Saccharomyces cerevisiae NGY10 produced maximum 49.77 ± 0.34 g/l and 46.81 ± 21.98 g/l ethanol with the efficiency of 97.39% and 93.54% at 30 °C and 40 °C, respectively, in 24 h using glucose as a carbon source. Furthermore, isolate NGY10 produced 12.25 ± 0.09 g/l and 7.18 ± 0.14 g/l of ethanol with 92.81% and 91.58% efficiency via SHF, and 30.22 g/l and 25.77 g/l ethanol with 86.43% and 73.29% efficiency via SSF using acid- and alkali-pretreated rice straw as carbon sources, respectively, at 40 °C. In addition, isolate NGY10 also produced 92.31 ± 3.39 g/l (11.7% v/v) and 33.66 ± 1.04 g/l (4.26% v/v) ethanol at 40 °C with the yields of 81.49% and 73.87% in the presence of 30% w/v glucose or 4× concentrated acid-pretreated rice straw hydrolysate, respectively. Moreover, isolate NGY10 displayed furfural- (1.5 g/l), 5-HMF (3.0 g/l), acetic acid- (0.2% v/v) and ethanol-(10.0% v/v) tolerant phenotypes. Conclusion: A sugarcane distillery waste isolate NGY10 demonstrated high potential for ethanol production, C5 metabolic engineering and developing strategies for SSF, SScF and CBP. [ABSTRACT FROM AUTHOR]