Design of 5′-UTR to Enhance Keratinase Activity in Bacillus subtilis

Abstract

Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 expression element to enhance its secretory activity in Bacillus subtilis WB600 through Spacer design, RBS screening, and sequence simplification. First, the A/U content in Spacer was increased by the site-directed saturation mutation of G/C bases, and the activity of keratinase secreted by mutant strain B. subtilis WB600-SP was 7.94 times higher than that of KerZ1. Subsequently, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R was further increased to 13.45 times that of KerZ1 based on the prediction of RBS translation efficiency and the multi-site saturation mutation screening. Finally, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R-D reached 204.44 KU mL−1 by reducing the length of the 5′ end of the 5′-UTR, which was 19.70 times that of KerZ1. In a 5 L fermenter, the keratinase activity secreted by B. subtilis WB600-SP-R-D after 25 h fermentation was 797.05 KU mL−1, which indicated its high production intensity. Overall, the strategy of this study and the obtained keratinase mutants will provide a good reference for the expression regulation of keratinase and other industrial enzymes.

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production

“…In previous studies, we have obtained recombinant keratinase by strategies such as promoter optimization, RBS sequence screening, and pro-peptide engineering. − Here, we used the engineered strain B. subtilis WB600- ker expressing keratinase as a starting point to explore the effect of autolysis gene deletion on the ability of B.…”

“…licheniformis BBE11-1 in B. subtilis WB600 and enhanced the expression of keratinase by promoter optimization, RBS sequence screening, and lead peptide engineering strategies. − It is worth noting that the low titer of recombinant keratinase in B. subtilis remains the main problem limiting its industrial application, and cellular autolysis may be one of the important reasons hindering the enhancement of enzyme protein titer.…”

Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste

“…The massive accumulation of feather waste causes environmental pollution and wastage of resources . At present, the recovery of feather waste is primarily achieved through the physicochemical combination method, which results in energy wastage and pollution owing to the use of strong acids and alkalies. , Therefore, the development of efficient green conversion technology is the key to enabling the transformation of keratin waste into resource for protein feed. The enzymatic recovery of feather proteins is an effective strategy that combines sustainable green development and environmental protection in keratin waste utilization.…”

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

“…B. subtilis is a Gram-positive bacterium, that has been widely used as a cell factory for the production of various industrial enzymes due to its generally regarded as safe (GRAS) nature, excellent secretory capability, low nutritional demands, and well-established fermentation processes. Although many heterologous proteins have achieved high expression in B. subtilis , such as pullulanase [ 3 ], protease [ 4 ], keratinase [ 5 ], etc., the recombinant expression level of certain target proteins is still low due to sequence specificity. To overcome the above problems, various expression strategies including expression regulatory element modification and fermentation optimization have been developed to improve the expression level of target proteins [ 6 ].…”

“…Optimization of RBS sequences is a common and effective strategy to improve the yield of recombinant proteins in B. subtilis . For example, Fang et al improved the keratinase activity by 69% through the prediction of RBS translation efficiency and multi-site saturation mutation screening [ 5 ]. To efficiently obtain optimal RBS sequences, Salis et al developed an RBS Calculator to assist the optimization design of RBS, which can improve the target translation initiation rate in Escherichia coli by 100,000 times [ 14 ].…”