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Engineering of recombinant Escherichia coli cells co-expressing poly-γ-glutamic acid (γ-PGA) synthetase and glutamate racemase for differential yielding of γ-PGA.

Cao M, Geng W, Zhang W, Sun J, Wang S, Feng J, Zheng P, Jiang A, Song C - Microb Biotechnol (2013)

Bottom Line: The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing γ-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria-Bertani medium containing glucose or L-glutamate.However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon γ-PGA production.Furthermore, RacE integration could enhance γ-PGA yield through improving the preferred d-glutamate content.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.

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Electrophoresis analysis profile of recombinant plasmid pTrcLRP and pTrcNRP in E. coli JM109. Lane 1–10, test of pTrc99A harbouring LL3 pgsBCA and racE genes; Lane 11–18, test of pTrc99A harbouring NK-03 pgsBCA and racE genes.Note: Lane M: Tiangen Marker III (Tiangen); Lane 1: pTrc99A/BamHI (4.2 kb); Lane 2: LL3 pgsBCA (3.0 kb); Lane 3: LL3 racE (0.8 kb); Lane 4: pTrcLRP (Supercoil, 8.0 kb); Lane 5: pTrcLpgs/BamHI (7.2 kb); Lane 6: pTrcLRP/KpnI (8.0 kb); Lane 7: pTrcLRP/BamHI (8.0 kb); Lane 8: pTrcLRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 9: pTrcLRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 10: pTrcLRP/BamHI+HindIII (5.0 and 3.0 kb); Lane 11: NK-03 pgsBCA (3.0 kb); Lane 12: NK-03 racE (0.8 kb); Lane 13: pTrcNpgs/BamHI (7.2 kb); Lane 14: pTrcNRP/KpnI (8.0 kb); Lane 15: pTrcNRP/BamHI (8.0 kb); Lane 16: pTrcNRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 17: pTrcNRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 18: pTrcNRP/BamHI+HindIII (5.0 and 3.0 kb).
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fig02: Electrophoresis analysis profile of recombinant plasmid pTrcLRP and pTrcNRP in E. coli JM109. Lane 1–10, test of pTrc99A harbouring LL3 pgsBCA and racE genes; Lane 11–18, test of pTrc99A harbouring NK-03 pgsBCA and racE genes.Note: Lane M: Tiangen Marker III (Tiangen); Lane 1: pTrc99A/BamHI (4.2 kb); Lane 2: LL3 pgsBCA (3.0 kb); Lane 3: LL3 racE (0.8 kb); Lane 4: pTrcLRP (Supercoil, 8.0 kb); Lane 5: pTrcLpgs/BamHI (7.2 kb); Lane 6: pTrcLRP/KpnI (8.0 kb); Lane 7: pTrcLRP/BamHI (8.0 kb); Lane 8: pTrcLRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 9: pTrcLRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 10: pTrcLRP/BamHI+HindIII (5.0 and 3.0 kb); Lane 11: NK-03 pgsBCA (3.0 kb); Lane 12: NK-03 racE (0.8 kb); Lane 13: pTrcNpgs/BamHI (7.2 kb); Lane 14: pTrcNRP/KpnI (8.0 kb); Lane 15: pTrcNRP/BamHI (8.0 kb); Lane 16: pTrcNRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 17: pTrcNRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 18: pTrcNRP/BamHI+HindIII (5.0 and 3.0 kb).

Mentions: Judging from the bands migration distance compared with Marker III (Tiangen) in 0.8% agarose gel electrophoresis shown in Fig. 2, it was revealed that the fusion expression vectors pTrcLRP and pTrcNRP were successfully introduced into E. coli JM109. To our knowledge, the PgsBCA synthetase and glutamate racemase (RacE) genes in recombinant strains that could synthesize γ-PGA were only from B. subtilis strains (Ashiuchi et al., 1999). Therefore, the related work about pgsBCA and racE cloned from B. licheniformis and B. amyloliquefaciens strains and transformed into E. coli will enrich the co-expression systems and supply the clue for differential synthesis of γ-PGA as well.


Engineering of recombinant Escherichia coli cells co-expressing poly-γ-glutamic acid (γ-PGA) synthetase and glutamate racemase for differential yielding of γ-PGA.

Cao M, Geng W, Zhang W, Sun J, Wang S, Feng J, Zheng P, Jiang A, Song C - Microb Biotechnol (2013)

Electrophoresis analysis profile of recombinant plasmid pTrcLRP and pTrcNRP in E. coli JM109. Lane 1–10, test of pTrc99A harbouring LL3 pgsBCA and racE genes; Lane 11–18, test of pTrc99A harbouring NK-03 pgsBCA and racE genes.Note: Lane M: Tiangen Marker III (Tiangen); Lane 1: pTrc99A/BamHI (4.2 kb); Lane 2: LL3 pgsBCA (3.0 kb); Lane 3: LL3 racE (0.8 kb); Lane 4: pTrcLRP (Supercoil, 8.0 kb); Lane 5: pTrcLpgs/BamHI (7.2 kb); Lane 6: pTrcLRP/KpnI (8.0 kb); Lane 7: pTrcLRP/BamHI (8.0 kb); Lane 8: pTrcLRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 9: pTrcLRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 10: pTrcLRP/BamHI+HindIII (5.0 and 3.0 kb); Lane 11: NK-03 pgsBCA (3.0 kb); Lane 12: NK-03 racE (0.8 kb); Lane 13: pTrcNpgs/BamHI (7.2 kb); Lane 14: pTrcNRP/KpnI (8.0 kb); Lane 15: pTrcNRP/BamHI (8.0 kb); Lane 16: pTrcNRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 17: pTrcNRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 18: pTrcNRP/BamHI+HindIII (5.0 and 3.0 kb).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Electrophoresis analysis profile of recombinant plasmid pTrcLRP and pTrcNRP in E. coli JM109. Lane 1–10, test of pTrc99A harbouring LL3 pgsBCA and racE genes; Lane 11–18, test of pTrc99A harbouring NK-03 pgsBCA and racE genes.Note: Lane M: Tiangen Marker III (Tiangen); Lane 1: pTrc99A/BamHI (4.2 kb); Lane 2: LL3 pgsBCA (3.0 kb); Lane 3: LL3 racE (0.8 kb); Lane 4: pTrcLRP (Supercoil, 8.0 kb); Lane 5: pTrcLpgs/BamHI (7.2 kb); Lane 6: pTrcLRP/KpnI (8.0 kb); Lane 7: pTrcLRP/BamHI (8.0 kb); Lane 8: pTrcLRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 9: pTrcLRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 10: pTrcLRP/BamHI+HindIII (5.0 and 3.0 kb); Lane 11: NK-03 pgsBCA (3.0 kb); Lane 12: NK-03 racE (0.8 kb); Lane 13: pTrcNpgs/BamHI (7.2 kb); Lane 14: pTrcNRP/KpnI (8.0 kb); Lane 15: pTrcNRP/BamHI (8.0 kb); Lane 16: pTrcNRP/KpnI+BamHI (7.2 and 0.8 kb); Lane 17: pTrcNRP/KpnI+HindIII (4.2 and 3.8 kb); Lane 18: pTrcNRP/BamHI+HindIII (5.0 and 3.0 kb).
Mentions: Judging from the bands migration distance compared with Marker III (Tiangen) in 0.8% agarose gel electrophoresis shown in Fig. 2, it was revealed that the fusion expression vectors pTrcLRP and pTrcNRP were successfully introduced into E. coli JM109. To our knowledge, the PgsBCA synthetase and glutamate racemase (RacE) genes in recombinant strains that could synthesize γ-PGA were only from B. subtilis strains (Ashiuchi et al., 1999). Therefore, the related work about pgsBCA and racE cloned from B. licheniformis and B. amyloliquefaciens strains and transformed into E. coli will enrich the co-expression systems and supply the clue for differential synthesis of γ-PGA as well.

Bottom Line: The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing γ-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria-Bertani medium containing glucose or L-glutamate.However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon γ-PGA production.Furthermore, RacE integration could enhance γ-PGA yield through improving the preferred d-glutamate content.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.

Show MeSH
Related in: MedlinePlus