Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

Abstract

Background: Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. Result: Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. Conclusion: We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate

“…It is considered as an efficient expression system with several advantages, such as rapid growth rate to result in short fermentation cycles, distinguished ability to secrete significant amounts of proteins into the extracellular medium, easy cultivation and genetic manipulation [ 1 ]. A variety of proteases have been successfully expressed in B. subtilis , including an alkaline protease (AprE) from Bacillus licheniformis 2709 [ 11 , 12 ], a serine protease (AprB) from Bacillus sp. strain B001 [ 13 ], a neutral protease (NprT) from G. stearothermophilus [ 14 ], and alkaline serine proteases from Bacillus clausii (aprE) [ 15 , 16 ].…”

Anaerobic Solid-State Fermentation of Soybean Meal With Bacillus sp. to Improve Nutritional Quality

“…Based on genomics, bioengineering methods were applied to enhance the α-amylase in the aprA-deficient strain of Bacillus licheniformis for increasing SBM utilization rate ( 34 ). The key enzymes proteases involved in FSBM can be modified to enhance the expression according to genome association analysis ( 35 , 36 ). Genome research will further improve the quality of SBM fermented by N-11 strain, which is the further research direction.…”

Lignocellulolytic Enzymes in Biotechnological and Industrial Processes: A Review

“…Liu et al [112] found that introducing specific mutations from one hyper-cellulolytic strain into another is a feasible strategy to improve cellulase production. Zhou et al [113] were able to improve enzyme activity by 62.19% using modified hosts when compared with wild-type alkaline protease-producing strain B. licheniformis. Presently, the use of lignocellulosic materials requires more technological developments to make it more feasible, cost-effective, and compatible in the real industrial sense.…”