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Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens.

Feng J, Gu Y, Quan Y, Zhang W, Cao M, Gao W, Song C, Yang C, Wang S - Sci Rep (2015)

Bottom Line: Microbial levan is an important biopolymer with considerable potential in food and medical applications.Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins.This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Nankai University, Tianjin 300071, China.

ABSTRACT
Microbial levan is an important biopolymer with considerable potential in food and medical applications. Bacillus amyloliquefaciens NK-ΔLP strain can produce high-purity, low-molecular-weight levan, but production is relatively low. To enhance the production of levan, six extracellular protease genes (bpr, epr, mpr, vpr, nprE and aprE), together with the tasA gene (encoding the major biofilm matrix protein TasA) and the pgsBCA cluster (responsible for poly-γ-glutamic acid (γ-PGA) synthesis), were intentionally knocked out in the Bacillus amyloliquefaciens NK-1 strain. The highest levan production (31.1 g/L) was obtained from the NK-Q-7 strain (ΔtasA, Δbpr, Δepr, Δmpr, Δvpr, ΔnprE, ΔaprE and ΔpgsBCA), which was 103% higher than that of the NK-ΔLP strain (ΔpgsBCA) (15.3 g/L). Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins. This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

No MeSH data available.


Related in: MedlinePlus

Metabolic pathways associated with levan biosynthesis in Bacillus amyloliquefaciens and engineering strategies for levan production.The red font indicates the genes deleted in this study and the corresponding deficient products. Metabolite symbols: Sucrose-6P, sucrose-6-phosphate; Bpr, bacillopeptidase F; Epr, extracellular serine protease; Mpr, extracellular metalloprotease; Vpr, extracellular serine protease; NprE, extracellular neutral metalloprotease; AprE, extracellular alkaline serine protease; TasA, major biofilm matrix protein; pgsBCA, poly-γ-glutamate synthase.
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f1: Metabolic pathways associated with levan biosynthesis in Bacillus amyloliquefaciens and engineering strategies for levan production.The red font indicates the genes deleted in this study and the corresponding deficient products. Metabolite symbols: Sucrose-6P, sucrose-6-phosphate; Bpr, bacillopeptidase F; Epr, extracellular serine protease; Mpr, extracellular metalloprotease; Vpr, extracellular serine protease; NprE, extracellular neutral metalloprotease; AprE, extracellular alkaline serine protease; TasA, major biofilm matrix protein; pgsBCA, poly-γ-glutamate synthase.

Mentions: In the current study, we proposed a new strategy for metabolic engineering of the B. amyloliquefaciens NK-1 strain to improve levan production. TasA is the main protein in biofilm matrix1718. The extracellular matrix surrounds the cells and might block the secretion of levansucrase. Previous works demonstrated that the tasA gene deletion strain could only form deficient biofilm17. The tasA gene was deleted in this work to determine its effects on levan production. Bacillus can produce high level of extracellular proteases to degrade the extracellular protein for cell use19. We speculated that deleting protease genes can expect to find other extracellular proteins more stable. Thus, we decided to delete the extracellular protease genes to improve levan production. In our previous work, we found that NK-1 strain could co-produce γ-PGA and levan simultaneously; moreover, their purification procedures are similar. Thus, pgsBCA genes deletion may increase levan purity as well as its production. A schematic of this proposed genetically engineered metabolic pathway of B. amyloliquefaciens NK-1 is shown in Fig. 1. We aimed to improve levan production by carrying out the above-mentioned three tasks: (1) delete the tasA gene to make the biofilm formation deficient; (2) delete six extracellular proteases genes bpr2021, epr22, mpr2324, vpr25, nprE26 and aprE2728, to decrease the degradation of the levansucrase; and (3) detete the pgsBCA cluster to block the γ-PGA synthesis pathway and obtain a higher purity and yield of the levan product. The final engineered NK-Q-7 strain could produce 31.1 g/L levan in flask, which was 103% higher compared with the production in the NK-ΔLP strain.


Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens.

Feng J, Gu Y, Quan Y, Zhang W, Cao M, Gao W, Song C, Yang C, Wang S - Sci Rep (2015)

Metabolic pathways associated with levan biosynthesis in Bacillus amyloliquefaciens and engineering strategies for levan production.The red font indicates the genes deleted in this study and the corresponding deficient products. Metabolite symbols: Sucrose-6P, sucrose-6-phosphate; Bpr, bacillopeptidase F; Epr, extracellular serine protease; Mpr, extracellular metalloprotease; Vpr, extracellular serine protease; NprE, extracellular neutral metalloprotease; AprE, extracellular alkaline serine protease; TasA, major biofilm matrix protein; pgsBCA, poly-γ-glutamate synthase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561895&req=5

f1: Metabolic pathways associated with levan biosynthesis in Bacillus amyloliquefaciens and engineering strategies for levan production.The red font indicates the genes deleted in this study and the corresponding deficient products. Metabolite symbols: Sucrose-6P, sucrose-6-phosphate; Bpr, bacillopeptidase F; Epr, extracellular serine protease; Mpr, extracellular metalloprotease; Vpr, extracellular serine protease; NprE, extracellular neutral metalloprotease; AprE, extracellular alkaline serine protease; TasA, major biofilm matrix protein; pgsBCA, poly-γ-glutamate synthase.
Mentions: In the current study, we proposed a new strategy for metabolic engineering of the B. amyloliquefaciens NK-1 strain to improve levan production. TasA is the main protein in biofilm matrix1718. The extracellular matrix surrounds the cells and might block the secretion of levansucrase. Previous works demonstrated that the tasA gene deletion strain could only form deficient biofilm17. The tasA gene was deleted in this work to determine its effects on levan production. Bacillus can produce high level of extracellular proteases to degrade the extracellular protein for cell use19. We speculated that deleting protease genes can expect to find other extracellular proteins more stable. Thus, we decided to delete the extracellular protease genes to improve levan production. In our previous work, we found that NK-1 strain could co-produce γ-PGA and levan simultaneously; moreover, their purification procedures are similar. Thus, pgsBCA genes deletion may increase levan purity as well as its production. A schematic of this proposed genetically engineered metabolic pathway of B. amyloliquefaciens NK-1 is shown in Fig. 1. We aimed to improve levan production by carrying out the above-mentioned three tasks: (1) delete the tasA gene to make the biofilm formation deficient; (2) delete six extracellular proteases genes bpr2021, epr22, mpr2324, vpr25, nprE26 and aprE2728, to decrease the degradation of the levansucrase; and (3) detete the pgsBCA cluster to block the γ-PGA synthesis pathway and obtain a higher purity and yield of the levan product. The final engineered NK-Q-7 strain could produce 31.1 g/L levan in flask, which was 103% higher compared with the production in the NK-ΔLP strain.

Bottom Line: Microbial levan is an important biopolymer with considerable potential in food and medical applications.Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins.This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Nankai University, Tianjin 300071, China.

ABSTRACT
Microbial levan is an important biopolymer with considerable potential in food and medical applications. Bacillus amyloliquefaciens NK-ΔLP strain can produce high-purity, low-molecular-weight levan, but production is relatively low. To enhance the production of levan, six extracellular protease genes (bpr, epr, mpr, vpr, nprE and aprE), together with the tasA gene (encoding the major biofilm matrix protein TasA) and the pgsBCA cluster (responsible for poly-γ-glutamic acid (γ-PGA) synthesis), were intentionally knocked out in the Bacillus amyloliquefaciens NK-1 strain. The highest levan production (31.1 g/L) was obtained from the NK-Q-7 strain (ΔtasA, Δbpr, Δepr, Δmpr, Δvpr, ΔnprE, ΔaprE and ΔpgsBCA), which was 103% higher than that of the NK-ΔLP strain (ΔpgsBCA) (15.3 g/L). Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins. This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

No MeSH data available.


Related in: MedlinePlus