Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

Ogunyewo, Olusola A.; Randhawa, Anmoldeep; Joshi, Manish D.; Jain, Kavish Kumar; Wadekar, Prathamesh; Odaneth, Annamma A.; Lali, Arvind; Yazdani, Syed Shams

doi:10.1016/j.procbio.2020.02.029

Cited by 26 publications

(39 citation statements)

References 45 publications

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“…11B). The result showed similar saccharification efficiency to what was earlier reported when the secretome of Pf Mig1 88 was used for saccharification of acid treated sugarcane bagasse and rice straw (26). The remarkable increase in biomass hydrolyzing capacity of Pf Mig1 88 secretome could majorly be attributed to an increased proportion of CBH, EG and BGL being secreted due to the absence of functional CCR in Pf Mig1 88 as recently reported (26).…”

Section: Resultssupporting

confidence: 86%

“…The result showed similar saccharification efficiency to what was earlier reported when the secretome of Pf Mig1 88 was used for saccharification of acid treated sugarcane bagasse and rice straw (26). The remarkable increase in biomass hydrolyzing capacity of Pf Mig1 88 secretome could majorly be attributed to an increased proportion of CBH, EG and BGL being secreted due to the absence of functional CCR in Pf Mig1 88 as recently reported (26). The 64% hydrolysis achieved with the secretome of Pf Mig1 88 at 20% solid loading was highly significant as it has previously been reported that most saccharification experiments carried out at high substrate loading may not proceed beyond 60% even after physical optimization of the saccharification process (48–50).…”

Section: Resultssupporting

confidence: 86%

“…This deregulation was done by deleting a homolog of a carbon catabolite repressor (CCR) Mig1 in Penicillium funiculosum NCIM1228 thereby yielding a catabolite derepressed strain, Penicillium funiculosum Mig1 88 ( Pf Mig1 88 ), which showed more than two-fold improvement in enzyme activity and protein titer in the secretome (25). When the saccharification potential of Pf Mig1 88 secretome was evaluated towards different chemically pretreated cellulosic biomass, it was found that Pf Mig1 88 secretome could hydrolyze ∼75% of total cellulose into glucose from nitric acid + ammonium hydroxide pretreated biomass in 72 h at 30 mg/g DBW (dry biomass weight) and 20% biomass loading (26). Under this pretreatment condition, even without recombinant expression or supplementation, Pf Mig1 88 secretome could also achieve the benchmark of ∼70% hydrolysis of sugars using 20 mg/g DBW at 20% solid loading recommended for cellulolytic enzymes to be utilized for second-generation biofuels (27).…”

Section: Introductionmentioning

confidence: 99%

“…Under this pretreatment condition, even without recombinant expression or supplementation, Pf Mig1 88 secretome could also achieve the benchmark of ∼70% hydrolysis of sugars using 20 mg/g DBW at 20% solid loading recommended for cellulolytic enzymes to be utilized for second-generation biofuels (27). However, pretreatment with either nitric acid or ammonium hydroxide alone resulted in the relatively lower release of 60% and 63% sugars, respectively, which suggested that accessibility to the cellulosic fibres was hindered by lignin and hemicellulose (26). This drawback thus necessitated the need to prospect into better ways of increasing the cellulase content of the secretome to increase the probability of substrate-enzyme proximity even in the presence of lignin and hemicellulose.…”

Section: Introductionmentioning

confidence: 99%

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Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Ogunyewo

Randhawa

Gupta

et al. 2020

Preprint

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1 synergistic saccharification performance with CBH1 on high substrate 2 loading upon simultaneous overexpression 3 4 5 Abstract 20 Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in 21 the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess 22 auxiliary activity are produced by numerous bacterial and fungal species to assist in the 23 degradation of cellulosic biomass. In this study, we annotated and performed structural 24 analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum 25 (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. 26 PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus 27 (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular 28 interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding 29 affinity with a ∆G free energy of -46 k kcal/mol when compared with that of TaLPMO9A (-30 31 kcal/mol). The enzyme was further found to be highly thermostable at elevated 31 temperature with a half-life of ~88 h at 50 o C. Furthermore, multiple fungal genetic 32 manipulation tools were employed to simultaneously overexpress this LPMO and 33 Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, 34PfMig1 88 , in order to improve its saccharification performance towards acid pretreated wheat 35 straw (PWS) at 20% substrate loading. The resulting transformants showed ~200% and ~66% 36 increase in LPMO and Avicelase activities, respectively. While the secretomes of 37 individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 38 6% and 13%, respectively, over PfMig1 88 at same enzyme concentration, the simultaneous 39 overexpression of these two genes led to 20% increase in saccharification efficiency over 40 PfMig1 88 , which accounted for 82% saccharification of PWS at 20% substrate loading. 41 42 43 44 3 Importance 45Enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant 46 bottleneck in the development of second-generation bio-based industries. While efforts are 47 being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high 48 production cost of this enzyme remains a crucial challenge confronting its wide availability 49 for efficient utilization of cellulosic materials. This is because it is challenging to get an 50 enzymatic cocktail with balanced activity from a single host. This report provides for the first 51 time the annotation and structural analysis of an uncharacterized thermostable lytic 52 polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in 53 biomass deconstruction upon overexpression in catabolite derepressed strain of P. 54 funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by 55 many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed 56 simultaneous...

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Section: Resultssupporting

confidence: 86%

Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Ogunyewo

Randhawa

Gupta

et al. 2020

Preprint

Self Cite

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“…For comparing growth on agar plates, P. funiculosum NCIM1228 and ∆ cbh1 strains were grown at 30 °C for 2, 7 and 14 days. For determining individual enzyme activities, NCIM1228 and Δ cbh1 strains were inoculated at 10 7 conidiospores/ml in 50 ml of PD broth, grown for 24 h and 10% of these cultures were used to inoculate secondary modified RCM medium for further 5 days of growth [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

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

Randhawa

Pasari

Sinha

et al. 2021

Biotechnol Biofuels

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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. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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Utilizing fungal biodegradation for valorisation of lignocellulosic waste biomass and its diverse applications

Mehta,

Chelike

2024

Applied Research

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Myco degradation is an effective technique for breaking down waste plant substances made of lignin, cellulose, and hemicellulose, which are collectively known as lignocellulose. This abundant organic material is found throughout the world. Due to its recalcitrant nature, lignocellulose poses a challenge for efficient conversion into biofuels, biochemicals, and other valuable products. Myco degradation, which involves the use of fungi to degrade lignocellulosic materials, offers a sustainable and cost‐efficient resolution to this challenge. This review provides an overview of the mechanisms and applications of myco degradation for lignocellulosic biomass degradation. The review discusses the various types of fungi involved in lignocellulose degradation, their enzymatic systems, and the factors that influences their performance. Furthermore, the potential applications of myco degradation products, such as biofuels, enzymes, and bioplastics, are reviewed. It also highlights the implications of myco degradation for waste management and sustainable development. Overall, myco degradation represents a promising technology for the efficient deprivation of lignocellulosic waste biomass, and further research in this field holds great potential for the sustainable creation of bio‐based products.This article is protected by copyright. All rights reserved.

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Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

Cited by 26 publications

References 45 publications

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

Insights into structure ofPenicillium funiculosumLPMO and its synergistic saccharification performance with CBH1 on high substrate loading upon simultaneous overexpression

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

Utilizing fungal biodegradation for valorisation of lignocellulosic waste biomass and its diverse applications

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