Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses
Graphical abstract
Introduction
The growth of the second-generation biofuel industry across the globe is of prudent importance and appropriately coincides with the ample availability of agricultural waste that is currently being burned, causing pollution. The second-generation biofuel industry has a wide range of advantages, including, but not limited to, boosting agribusiness, meeting ever-growing energy needs, and minimizing environmental pollution caused by crop residual burning and fossil fuel usage. Since second-generation biofuels would compete with fossil fuels in the global market, there is a predetermined upper limit to its cost. This causes a significant challenge in this upcoming industry. Two factors contributing to the price of bio-fuels are (1) capital expenditure (CAPEX), which includes physical assets and equipment, and (2) operating expenses (OPEXs) that are required to run business operations. Since OPEXs make up the bulk of a company’s regular costs, a reduction in operating expenses would reduce the overall cost of biofuels. The saccharification of lignocellulosic biomass is the costliest step and requires optimization to reduce OPEXs [1]. Enzyme cost is generally measured with a “top-down’’ approach that accounts for the type of feedstock, enzyme load, and overall biofuel yield in addition to actual enzyme cost [2]. An ideal saccharifying cocktail is one that has broad substrate specificity, fast enzymatic action, minimal product inhibition and a low cost of production.
Currently, Trichoderma reesei dominates the industrial arena for the production of cellulases for use in the detergent, textile, feedstock and pulp and paper industries [3]. Additionally, tailor-made cellulolytic secretomes have also been introduced to the biofuel industry and are optimized and supplemented versions of the T. reesei secretome [4]. The cumulative action of many cellulases, including cellobiohydrolases, endoglucanases, β-glucosidases, xylanases, lytic polysaccharide monooxygenases and several other accessory enzymes, is critical for the conversion of biomass into fermentable sugars [5]. The most important cellulase found in the secretome of T. reesei is cellobiohydrolase, which hydrolyses strands of cellulose into cellobiose units that are further converted into glucose by the action of β-glucosidases. The major issue with T. reesei cellobiohydrolase I (TrCBH1) is feedback inhibition by cellobiose and other reducing sugars produced during cellulose hydrolysis [6]. Additionally, low levels of β-glucosidase and other accessory enzymes require supplementation of the T. reesei cocktail with β-glucosidase and other important enzymes from other sources, such as Aspergillus and Talaromyces species [[7], [8], [9]]. Recent bioprospecting in our lab identified Penicillium funiculosum NCIM1228 as having a secretome of superior saccharification competence when compared with that of T. reesei [10]. Approximately 58 % of the proteins found in the secretome of P. funiculosum were CAZymes (carbohydrate-active enzymes). Although there was a prevalence of cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) in the secretome of P. funiculosum, they constituted only 15 % of the total proteins, contrary to the secretome of T. reesei, where they represented 95 % of the total proteins [10,11]. Hence, the high performance of the secretome of P. funiculosum can be attributed to other auxiliary proteins working in tandem with CBHs to synergistically degrade biomass [12]. Structural and biochemical studies showed that P. funiculosum CBH I (PfCBH1) was five-fold more efficient than T. reesei CBH I (TrCBH1) [6]. Swapping of subdomains between PfCBH1 and TrCBH1 demonstrated that the enhancement in enzymatic activity is mainly caused by the catalytic domain of PfCBH1 [13]. PfCBH1 was also found to have higher thermostability and tolerance to competitive inhibitors, such as cellobiose, than that of TrCBH1. Furthermore, the P. funiculosum secretome outperformed the T. reesei-based commercial preparation in terms of biomass saccharification [10]. Quantitative multiplexing studies on secretomes produced in the presence of different cellulosic inducers confirmed the potential of the non-model fungus P. funiculosum to produce exhaustive enzymes needed for diverse biomass saccharification [14].
Once the superiority of the P. funiculosum secretome over that of T. reesei was confirmed, it was modified genetically to remove catabolite repression from cellulase expression [15]. The resultant derepressed PfMig188 strain showed better growth characteristics and higher expression and activity of cellobiohydrolases, endoglucanases, β-glucosidases and xylanases than those of the precursor strain. Total cellulase activity measured in terms of filter paper unit (FPU) per mL was found to be 4.7 FPU/mL, which was two-fold higher than that of the precursor strain, and more than 14 g/L secretome protein was produced at the shake flask level when cultured in the presence of 40 g/L cellulosic carbon source and 24 g/L protein source [15]. Therefore, in the present study, an attempt was made to produce an efficient yet inexpensive cellulolytic cocktail that has a broader saccharification performance on different cellulosic feedstocks than that of conventional secretomes. Optimization for less expensive substrates to produce quality secretomes was performed at the shake flask level and further tested in a 20-L bioreactor. The resultant secretome was tested for optimal working conditions, and its performance was tested on a range of chemically treated biomasses. The results obtained showed that the secretome was very effective for the production of fermentable sugar irrespective of the type of pretreatment or the biomass utilized. Similarly, when the secretome was compared with a commercial formulation specifically designed for the biofuel industry, it was found to outperform the commercial cocktail. The results thus highlight the secretome of PfMig188 strain as a well-balanced cocktail for second-generation bio-industries and demonstrate its potential as a possible alternative to T. reesei-based cellulolytic cocktails.
Section snippets
Materials
Two forms of microcrystalline cellulose (MCC), i.e., Microcel MCC(A) purchased from Blanver Farmoquimica (Brazil) and MCC(B) (pretreated rice straw produced at the 2G-Ethanol demonstration plant at India Glycols Ltd.), were used in this study. Soy flour and soy protein isolate were purchased from Kanoria Chemicals & Industries Ltd. (India), while wheat bran was purchased from a local flour mill in New Delhi, India. Avicel PH-101, carboxymethyl cellulose, p-nitrophenol, p-nitrophenyl-β-d
Chemical composition of pretreated rice straw and sugarcane bagasse
Cellulosic feedstocks usually contain cellulose, which is the dominant structural polysaccharide in plant cell walls, followed by hemicellulose and lignin. Since the amount of carbohydrate polymers and lignin varies across different feedstocks existing in nature, it becomes imperative to evaluate the composition of the different constituents of cellulosic biomass available to be used for biofuel production [20,21]. For this, chemical characterization of the treated and untreated feedstocks to
Conclusions
In this study, a cost-effective approach was used to optimize the production of cellulase by a recombinant strain of P. funiculosum, PfMig188, without compromising its ability to produce a high amount of enzymes. The results obtained in the study showed that the PfMig188 strain produced a well-balanced cellulolytic and hemicellulolytic concoction of enzymes exhibiting sufficient amounts of cellobiohydrolase, endoglucanase, β-glucosidase and xylanase activity. Furthermore, the secretome was
Authorship contribution statement
OAO, AR and SSY conceptualized the idea, designed and coordinated the study, OAO and AR performed molecular biology and shake flask enzyme production study, MJ and KKJ performed bioreactor studies, PW, AAO and AML performed biomass analysis, pretreatment and comparative saccharification with commercial enzyme, SSY and AAO supervised and generated fund for the study. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study was funded by Department of Biotechnology, Government of India via Bioenergy Centre grant nos. BT/PR/Centre/03/2011 and BT/EB/ICT-extension/2012.
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Authors contributed equally to this work.