Effects of Different Signal Peptides and Their Combinations on Heterologous Expression of Levansucrase

doi:10.13523/j.cb.2210024

China Biotechnology

2023, Vol. 43

Issue (5): 37-44 DOI: 10.13523/j.cb.2210024

Effects of Different Signal Peptides and Their Combinations on Heterologous Expression of Levansucrase

WANG Mao-jun,SIMAYI Seyide,CAI Yi-an,LI Qing-gang,LU Fu-ping,LI Yu(

)

Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology, Tianjin University of Science&Technology,Tianjin 300457,China

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Abstract

To express levansucrase in Bacillus amyloliquefaciens 018 (G3) efficiently. Four levansucrase genes lsLich, lsAmy, lsSub and lsMega from different Bacillus species were heterologously expressed, and five signal peptides with relatively high levels of alkaline protease identified by the research group were screened and combined. lsLich derived from Bacillus licheniformis RN-01 had the highest enzyme activity in the recombinant strain G3/pLY-2-lsLich, with an enzyme activity of 62.73 U/mL. LS-Lich was used as the target protein to screen single signal peptide and double signal peptide. The recombinant strain G3/pLY-2-SDA-ls combined with SP_DacB and SP_AmyE had the highest enzyme activity, and the extracellular enzyme activity reached 125.76 U/mL. Compared with the recombinant strain G3/pLY-2-SD-ls and G3/pLY-2-SA-ls, they increased by 31.3% and 39.2%, respectively, and increased by 100.49% compared with the original strain. The results indicated that the double signal peptide was helpful in increasing the secretion of LS-Lich compared with the single signal peptide, and the combination order of signal peptides also produced some differences.

Key words： Signal peptide Levansucrase Bacillus amyloliquefaciens Heterologous expression

Received: 17 October 2022 Published: 01 June 2023

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	WANG Mao-jun
	SIMAYI Seyide
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	LI Qing-gang
	LU Fu-ping
	LI Yu

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WANG Mao-jun, SIMAYI Seyide, CAI Yi-an, LI Qing-gang, LU Fu-ping, LI Yu. Effects of Different Signal Peptides and Their Combinations on Heterologous Expression of Levansucrase. China Biotechnology, 2023, 43(5): 37-44.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2210024 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I5/37

Fig.1 Hydrolysis, glycosylation and polymerization reactions of levansucrase

Table 1 Amino acid sequences of 5 signal peptides and their sources

Fig.2 Schematic diagram of the structure of different signal peptides in the recombinant vector

Fig.3 PCR validation diagram of different expression vectors (a)1: G3/pLY-2-lsSub; 2: G3/pLY-2-lsAmy; 3: G3/pLY-2-lsMega; 4: G3/ pLY-2-lsLich; M: 1 kb DNA ladder (b)1: G3/pLY-2-YoaW-ls; 2: G3/pLY-2-NprE-ls; 3: G3/pLY-2-DacB-ls; 4: G3/pLY-2-SacB-ls; 5: G3/pLY-2-AmyE-ls; 6: G3/pLY-2-SAD-ls; 7: G3/pLY-2-SDA-ls; M: 1 kb DNA ladder

Fig.4 Enzyme activity of recombinant G3/pLY-2-ls at different fermentation time Different lowercase letters indicate significant differences at P=0.05

Fig.5 Enzyme activity of recombinant bacteria with different signal peptides Different lowercase letters indicate significant differences at P=0.05

Fig.6 Enzyme activity of signal peptides in different forms Different lowercase letters indicate significant differences at P=0.05

Fig.7 SDS-PAGE validation map of signal peptide combination recombinant strain 1: G3/pLY-2-SAD-ls; 2: G3/pLY-2-SDA-ls; 3: G3/pLY-2-SA-ls; 4: G3/pLY-2-SD-ls


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