State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, 368 Youyi Avenue, Wuchang District, Wuhan, 430062, Hubei, People's Republic of China.
J Ind Microbiol Biotechnol. 2019 Dec;46(12):1745-1755. doi: 10.1007/s10295-019-02229-8. Epub 2019 Aug 30.
Cell surface engineering was proven as the efficient strategy for enhanced production of target metabolites. In this study, we want to improve the yield of target protein by engineering cell surface in Bacillus licheniformis. First, our results confirmed that deletions of D-alanyl-lipoteichoic acid synthetase gene dltD, cardiolipin synthase gene clsA and CDP-diacylglycerol-serine O-phosphatidyltransferase gene pssA were not conducive to cell growth, and the biomass of gene deletion strains were, respectively, decreased by 10.54 ± 1.43%, 14.17 ± 1.51%, and 17.55 ± 1.28%, while the concentrations of total extracellular proteins were improved, due to the increases of cell surface net negative charge and cell membrane permeability. In addition, the activities of target proteins, nattokinase, and α-amylase were also improved significantly in gene deletion strains. Furthermore, the triplicate gene (dltD, clsA, and pssA) deletion strain was constructed, which further led to the 45.71 ± 2.43% increase of cell surface net negative charge and 26.45 ± 2.31% increase of cell membrane permeability, and the activities of nattokinase and α-amylase reached 37.15 ± 0.89 FU/mL and 305.3 ± 8.4 U/mL, increased by 46.09 ± 3.51% and 96.34 ± 7.24%, respectively. Taken together, our results confirmed that cell surface engineering via deleting dltD, clsA, and pssA is an efficient strategy for enhanced production of target proteins, and this research provided a promising host strain of B. licheniformis for efficient protein expression.
细胞表面工程被证明是提高目标代谢产物产量的有效策略。在本研究中，我们希望通过工程化枯草芽孢杆菌的细胞表面来提高目标蛋白的产量。首先，我们的结果证实，缺失 D-丙氨酰-脂磷壁酸合成酶基因 dltD、心磷脂合酶基因 clsA 和 CDP-二酰基甘油-丝氨酸 O-磷酸丝氨酸转移酶基因 pssA 不利于细胞生长，基因缺失菌株的生物量分别降低了 10.54±1.43%、14.17±1.51%和 17.55±1.28%，而由于细胞表面净负电荷和细胞膜通透性的增加，细胞外总蛋白的浓度得到提高。此外，目标蛋白纳豆激酶和α-淀粉酶的活性在基因缺失菌株中也显著提高。此外，构建了三重复基因（dltD、clsA 和 pssA）缺失菌株，进一步导致细胞表面净负电荷增加 45.71±2.43%，细胞膜通透性增加 26.45±2.31%，纳豆激酶和α-淀粉酶的活性分别达到 37.15±0.89 FU/mL 和 305.3±8.4 U/mL，分别提高了 46.09±3.51%和 96.34±7.24%。总之，我们的结果证实，通过缺失 dltD、clsA 和 pssA 进行细胞表面工程是提高目标蛋白产量的有效策略，本研究为高效表达目标蛋白提供了一种有前景的枯草芽孢杆菌宿主菌株。
