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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.

SUBMITTER: Zhou C

PROVIDER: S-EPMC7041084 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Zhou Cuixia C Zhou Huiying H Li Dengke D Zhang Huitu H Wang Hongbin H Lu Fuping F

Microbial cell factories 20200224 1

<h4>Background</h4>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 pro ...[more]

PMID: 32093734

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Project description:Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

Dataset Information

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

Publications

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

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