Engineering Trichoderma reesei Rut-C30 with the overexpression of egl1 at the ace1 locus to relieve repression on cellulase production and to adjust the ratio of cellulolytic enzymes for more efficient hydrolysis of lignocellulosic biomass.

Variable industrial strains have been applied in the fermentation of bulk amino acids. Strain discovery and evolution, process optimization are traditional approaches to improve the yield and efficiency of the bio-production process, hence to compete with chemical or enzymatic process in amino acids production. With the fast development of bioengineering and synthetic biology, the strains can be rationally engineered to achieve better performance and gain the capacity in the fermentation of broader range of amino acids, especially for value-added amino acids. This proposed review aims to summarize traditional and recent strains in the microbial production of amino acids, characterize their metabolic pathways and present potential objectives for rational evolution. In addition, this proposed review prospect the recent opportunities and challenges in the microbial production of value-added amino acids (rare amino acids, non-canonical amino acids, and unnatural amino acids).

Improving the Microbial Production of Amino Acids: From Conventional Approaches to Recent Trends

From an anthropocentric point of view, human culture has been intricately involved in harnessing the potential of lignocellulosic feedstock to bring the bio-competitive alternative of fossil-based fuel resources. In today's scenario, the impact of hyperthermophiles and their enzymes has been intensely investigated for implementation in various high-temperature biotechnological processes. Already characterized archaeal and eubacterial cellulolytic glycoside hydrolase have shown highly impressive catalytic structures and mechanisms. Several sequence and structural factors have simultaneously been proposed to contribute towards the augmented stability of thermophilic proteins. However, state-of-the-art technologies like the rational designing approach and mechanism of directed evolution have emerged as critical toolkits for broadened industrial applications of recombinant proteins. This manuscript discusses the cellulase engineering techniques to enhance the biological production and stability of thermostable cellulolytic enzymes.

Thermostable cellulases: Structure, catalytic mechanisms, directed evolution and industrial implementations

Endoglucanase: revealing participation in open cellulosic chains

Wucai (Brassica campestris L.) is a leafy vegetable that originated in China, its soluble sugars accumulate significantly to improve taste quality during maturation, and it is widely accepted by consumers. In this study, we investigated the soluble sugar content at different developmental stages. Two periods including 34 days after planting (DAP) and 46 DAP, which represent the period prior to and after sugar accumulation, respectively, were selected for metabolomic and transcriptomic profiling. Differentially accumulated metabolites (DAMs) were mainly enriched in the pentose phosphate pathway, galactose metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and fructose and mannose metabolism. By orthogonal projection to latent structures-discriminant s-plot (OPLS-DA S-plot) and MetaboAnalyst analyses, D-galactose and β-D-glucose were identified as the major components of sugar accumulation in wucai. Combined with the transcriptome, the pathway of sugar accumulation and the interact network between 26 DEGs and the two sugars were mapped. CWINV4, CEL1, BGLU16, and BraA03g023380.3C had positive correlations with the accumulation of sugar accumulation in wucai. The lower expression of BraA06g003260.3C, BraA08g002960.3C, BraA05g019040.3C, and BraA05g027230.3C promoted sugar accumulation during the ripening of wucai. These findings provide insights into the mechanisms underlying sugar accumulation during commodity maturity, providing a basis for the breeding of sugar-rich wucai cultivars.

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Combined Metabolome and Transcriptome Analysis Elucidates Sugar Accumulation in Wucai (Brassica campestris L.)

To enhance the specific endo-β-1,4-glucanase activity (EGI) of three mixed substances: Trichoderma sp. (Trichoderma reesei, Trichoderma longibrachiatum, and Trichoderma pseudokoningii), we optimized the efficiency of the encoding gene, eg1, using DNA shuffling and Saccharomyces cerevisiae INVSc1 as a host. One variant, SEGI8 (the high activity protein encoded by eg1 gene using DNA shuffling) was found through screening approximately 1000 variants. The extracellular enzyme work of SEGI8 reached its highest level when cultured for 96 h, at 50 °C and a pH of 5.6, akin to the wild types. Clone harboring of the best mutant SEGI8 (selected via first-round mutagenesis) produced 169–257% more activity than transformants with three wild-type EGIs. Mutant SEGI8 produced by the clone showed broad pH stability (5.6–6.6) while performing comparable thermotolerance (60–70 °C) compared to wild EGIs; it also showed a high homology of 95%, 96%, and 97%, identical to the EGI of T. reesei, T. longibrachiatum, and T. pseudokoningii, respectively. Analysis of the sequences indicated that four point mutations causing amino acid substitution (V10A/G100S/F192L/S318G) were replaced. The major structure of the active site in SEGI8 was comprised of loop regions, with the same location as β-helix in NTEGI (the protein encoded by eg1 genes in T. reesei). It has been suggested that the mutation S318G might affect substrate binding and improved actions based on an analysis of the predicted 3D structural-modeling of SEGI8. Overall, this novel system could facilitate cellulose activity and create a foundation for constructing an efficient cellulose degradation system.

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Yong Zhao

Papers

Improving the activity of endoglucanase I (EGI) from Saccharomyces cerevisiae by DNA shuffling