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Thermophilic endoglucanases have become of significant interest for effectively catalyzing the hydrolysis of cellulose. Myceliophthora thermophila is an ideal source of thermophilic enzymes. Interestingly, different hosts differently express the same enzymes. In this study, we successfully overexpressed endoglucanase (MtEG5-1) from M. thermophila in the methylotrophic yeast, Pichia pastoris GS115, via electroporation. We found that purified MtEG5-1 exhibited optimum activity levels at pH 5 and 70 °C, with 88% thermal stability after being incubated at 70 °C for 2 h. However, we observed that purified MtEG5-1 had a molecular weight of 55 kDa. The Km and Vmax values of purified MtEG5-1 were approximately 6.11 mg/mL and 91.74 μmol/min/mg at 70 °C (pH 5.0), respectively. Additionally, the optimum NaCl concentration of purified MtEG5-1 was found to be 6 g/L. Furthermore, we observed that the activity of purified MtEG5-1 was significantly enhanced by Mn2+ and was inhibited by K+. These results indicated that MtEG5-1 expressed by P. pastoris GS115 is more heat-tolerant than that expressed by A. niger and P. pastoris X33. These properties of MtEG5-1 make it highly suitable for future academic research and industrial applications.

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

Abstract Agricultural residues, such as lignocellulosic materials (LM), are the most attractive renewable bioenergy sources and are abundantly found in nature. Anaerobic digestion has been extensively studied for the effective utilization of LM for biogas production. Experimental investigation of physiochemical changes that occur during pretreatment is needed for developing mechanistic and effective models that can be employed for the rational design of pretreatment processes. Various-cutting edge pretreatment technologies (physical, chemical and biological) are being tested on the pilot scale. These different pretreatment methods are widely described in this paper, among them, microaerobic pretreatment (MP) has gained attention as a potential pretreatment method for the degradation of LM, which just requires a limited amount of oxygen (or air) supplied directly during the pretreatment step. MP involves microbial communities under mild conditions (temperature and pressure), uses fewer enzymes and less energy for methane production, and is probably the most promising and environmentally friendly technique in the long run. Moreover, it is technically and economically feasible to use microorganisms instead of expensive chemicals, biological enzymes or mechanical equipment. The information provided in this paper, will endow readers with the background knowledge necessary for finding a promising solution to methane production.