Efficient hydrolysis of lignocellulosic materials to sugars for conversion to biofuels and chemicals is a key step in biorefinery. Designing an active saccharifying enzyme system with synergy among their components is considered a promising approach. In this study, a lignocellulose-degrading enzyme system of Chaetomium globosum BCC5776 (CG-Cel) was characterized for its activity and proteomic profiles, and synergism with accessory enzymes. The highest cellulase productivity of 0.40 FPU/mL was found for CG-Cel under the optimized submerged fermentation conditions on 1% (w/v) EPFB (empty palm fruit bunch), 2% microcrystalline cellulose (Avicel®) and 1% soybean meal (SBM) at 30 °C, pH 5.8 for 6 d. CG-Cel worked optimally at 50–60 °C in an acidic pH range. Proteomics analysis by LC/MS/MS revealed a complex enzyme system composed of core cellulases and accessory hydrolytic/non-hydrolytic enzymes attacking plant biopolymers. A synergistic enzyme system comprising the CG-Cel, a β-glucosidase (Novozyme® 188) and a hemicellulase Accellerase® XY was optimized on saccharification of alkaline-pretreated rice straw by a mixture design approach. Applying a full cubic model, the optimal ratio of ternary enzyme mixture containing CG-Cel: Novozyme® 188: Accellerase® XY of 44.4:20.6:35.0 showed synergistic enhancement on reducing sugar yield with a glucose releasing efficiency of 256.4 mg/FPU, equivalent to a 2.9 times compared with that from CG-Cel alone. The work showed an approach for developing an active synergistic enzyme system based on the newly characterized C. globosum for lignocellulose saccharification and modification in bio-industries.

Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, Saccharomyces cerevisiae harboring the D-lactate dehydrogenase (DLDH) gene from Leuconostoc mesenteroides was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of gpd1, gpd2, and adh1 to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔgpd and CEN.PK2_DLDHΔgpdΔadh1, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔgpd was crossed with the weak acid-tolerant S. cerevisiae BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of S. cerevisiae for the production of industrially potent products.

Sandalwood and agarwood essential oils are rare natural oils comprising fragrant terpenoids that have been used in perfumes and incense for millennia. Increasing demand for these terpenoids, coupled with difficulties in isolating them from natural sources, have led to an interest in finding alternative production platforms. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fragrant terpenoids from sandalwood and agarwood. Specifically, we constructed strain FPPY005_39850, which overexpresses all eight genes in the mevalonate pathway. Using this engineered strain as the background strain, we screened seven distinct terpene synthases from agarwood, sandalwood, and related plant species for their activities in the context of yeast. Five terpene synthases led to the production of fragrant terpenoids, including α-santalene, α-humulene, δ-guaiene, α-guaiene, and β-eudesmol. To our knowledge, this is the first demonstration of β-eudesmol production in yeast. We further improved the production titers by downregulating ERG9, a key enzyme from a competing pathway, as well as employing enzyme fusions. Our final engineered strains produced fragrant terpenoids at up to 101.7 ± 6.9 mg/L. We envision our work will pave the way for a scalable route to these fragrant terpenoids and further establish S. cerevisiae as a versatile production platform for high-value chemicals.