AMPK Activates Cellulase Secretion in Penicillium funiculosum by Downregulating P-HOG1 MAPK Levels.

Cellulase production for hydrolyzing plant cell walls is energy-intensive in filamentous fungi during nutrient scarcity. AMP-activated protein kinase (AMPK), encoded by snf1, is known to be the nutrient and energy sensor in eukaryotes. Previous studies on AMPK identified its role in alternate carbon utilization in pathogenic fungi.

Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli.

The impact of recombinant protein production (RPP) on host cells and the metabolic burden associated with it undermine the efficiency of the production system. This study utilized proteomics to investigate the dynamics of parent and recombinant cells induced at different time points for RPP. The results revealed significant changes in both transcriptional and translational machinery that may have impacted the metabolic burden, growth rate of the culture and the RPP.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses.

Background: Marine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses.

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum.

Background: Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig1 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment.

Systematic identification of CAZymes and transcription factors in the hypercellulolytic fungus Penicillium funiculosum NCIM1228 involved in lignocellulosic biomass degradation.

Background: Penicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes.

Profiling of the β-glucosidases identified in the genome of : insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with β-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in the genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome.

Isobutanol production by combined and metabolic engineering.

The production of the biofuel, isobutanol, in faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both with metabolic engineering to produce isobutanol. The production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in and inhibition of competitive valine pathway using CRISPRi technology.

Characterization of a GH5 endoxylanase from Penicillium funiculosum and its synergism with GH16 endo-1,3(4)-glucanase in saccharification of sugarcane bagasse.

The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification.

Genome editing in using assembled CRISPR-Cas9 ribonucleoprotein complexes.

The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in , which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment.

Adaptation on xylose improves glucose-xylose co-utilization and ethanol production in a carbon catabolite repression (CCR) compromised ethanologenic strain.

Background: Sugar hydrolysates from lignocellulosic biomass are majorly composed of glucose and xylose that can be fermented to biofuels. Bacteria, despite having the natural ability to consume xylose are unable to consume it in presence of glucose due to a carbon catabolite repression (CCR) mechanism. This leads to overall reduced productivity as well as incomplete xylose utilization due to ethanol build-up from glucose utilization.

Engineering E. coli to synthesize butanol.

Biobutanol is gaining much attention as a potential biofuel due to its superior properties over ethanol. Butanol has been naturally produced via acetone-butanol-ethanol (ABE) fermentation by many Clostridium species, which are not very user-friendly bacteria. Therefore, to improve butanol titers and yield, various butanol synthesis pathways have been engineered in Escherichia coli, a much more robust and convenient host than Clostridium species.

A genetic toolkit for co-expression of multiple proteins of diverse physiological implication.

Construction of plasmids is crucial for expression of functional proteins of diverse physiological impact in . Here, we first designed and constructed a novel pair of bacterial expression vectors, i.e.

Insights into cyanobacterial alkane biosynthesis.

Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels. Though biological production of alkanes has been reported in various microorganisms, the reports citing photosynthetic cyanobacteria as natural producers have been the most consistent for the long-chain alkanes and alkenes (C15-C19). However, the production of alkane in cyanobacteria is low, leading to its extraction being uneconomical for commercial purposes.

Overexpression of Oxidoreductase YghA Confers Tolerance of Furfural in Ethanologenic Escherichia coli Strain SSK42.

Furfural is a common furan inhibitor formed due to dehydration of pentose sugars, like xylose, and acts as an inhibitor of microbial metabolism. Overexpression of NADH-specific FucO and deletion of NADPH-specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in Escherichia coli, which highlights the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified the gene as an overexpression target to confer tolerance against furfural.

Accessory enzymes of hypercellulolytic Penicillium funiculosum facilitate complete saccharification of sugarcane bagasse.

Background: Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig1, in response to SCB induction and examined their role in the saccharification of SCB.

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase.

Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding.

Microbial engineering to produce fatty alcohols and alkanes.

Owing to their high energy density and composition, fatty acid-derived chemicals possess a wide range of applications such as biofuels, biomaterials, and other biochemical, and as a consequence, the global annual demand for products has surpassed 2 million tons. With the exhausting petroleum reservoirs and emerging environmental concerns on using petroleum feedstock, it has become indispensable to shift to a renewable-based industry. With the advancement in the field of synthetic biology and metabolic engineering, the use of microbes as factories for the production of fatty acid-derived chemicals is becoming a promising alternative approach for the production of these derivatives.

Quantification of Alkanes in .

Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane.

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228.

Background: Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools.

Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from in Cellulose Saccharification under High-Level Substrate Loading.

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation.

Improved Butanol Production Using FASII Pathway in .

-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e.

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture.

Background: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF.

Building cell factories for the production of advanced fuels.

Synthetic biology-based engineering strategies are being extensively employed for microbial production of advanced fuels. Advanced fuels, being comparable in energy efficiency and properties to conventional fuels, have been increasingly explored as they can be directly incorporated into the current fuel infrastructure without the need for reconstructing the pre-existing set-up rendering them economically viable. Multiple metabolic engineering approaches have been used for rewiring microbes to improve existing or develop newly programmed cells capable of efficient fuel production.

Advances in the development and application of microbial consortia for metabolic engineering.

Recent advances in metabolic engineering enable the production of high-value chemicals via expressing complex biosynthetic pathways in a single microbial host. However, many engineered strains suffer from poor product yields due to redox imbalance and excess metabolic burden, and require compartmentalization of the pathway for optimal function. To address this problem, significant developments have been made towards co-cultivation of more than one engineered microbial strains to distribute metabolic burden between the co-cultivation partners and improve the product yield.

Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts.

Background: Efficient deconstruction of lignocellulosic biomass into simple sugars in an economically viable manner is a prerequisite for its global acceptance as a feedstock in bioethanol production. This is achieved in nature by suites of enzymes with the capability of efficiently depolymerizing all the components of lignocellulose. Here, we provide detailed insight into the repertoire of enzymes produced by microorganisms enriched from the gut of the crop pathogen rice yellow stem borer ().