Heterogenous expression of poly-γ-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3

Wang, N.; Yang, Ge; Che, Chuanyan; Liu, Y.

doi:10.1134/s0003683811040193

Cited by 7 publications

(6 citation statements)

References 14 publications

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“…amyloliquefaciens LL3 were capable of synthetizing γ-PGA with different molecular weight in the presence or absence L-glutamate, respectively. It is known that the γ-PGA synthetase complex consists of three functional subunits (PgsB, PgsC and PgsA) and is responsible for catalysing glutamate to synthesize γ-PGA with the type of membranous adenosine triphosphate (ATP)-dependent amide-ligase ( Ashiuchi et al ., 2004 ; Wang et al ., 2011 ). Therefore, the cloning and sequence alignment of pgsBCA genes will be fundamental to understand the molecular mechanism of γ-PGA synthesis.…”

Section: Resultsmentioning

confidence: 99%

“…Although the co-expression of racE and pgsBCA had successfully presented an appreciable productivity in glucose medium (without L-glutamate), compared with that of other reported engineered E. coli strains harbouring only pgsBCA genes ( Ashiuchi et al ., 1999 ; Jiang et al ., 2011 ; Wang et al ., 2011 ), the optimized production of γ-PGA by the candidate 109LRP in flask culture was just 1.26 g l −1 , which was still not as high as the wild type of Bacillus strains ( Cao et al ., 2010 ; 2011 ). Therefore, lots of works will be done to regulate the composition and the biosynthesis of γ-PGA, including the synergism and combinational performances of the concentration of substrate, the stability and permeability of cell membrane, and the activities of glutamate synthase, glutamate dehydrogenase, glutamate racemase, γ-PGA synthetase and depolymerase.…”

Section: Resultsmentioning

confidence: 99%

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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

Geng

Zhang

et al. 2013

Microbial Biotechnology

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Poly-γ-glutamic acid (γ-PGA) is a promising environmental-friendly material with outstanding water solubility, biocompatibility and degradability. However, it is tough to determine the relationship between functional synthetic enzyme and the strains' yield or substrate dependency. We cloned γ-PGA synthetase genes pgsBCA and glutamate racemase gene racE from both L-glutamate-dependent γ-PGA-producing Bacillus licheniformis NK-03 and L-glutamate-independent B. amyloliquefaciens LL3 strains. The deduced RacE and PgsA from the two strains shared the identity of 84.5% and 78.53%, while PgsB and PgsC possessed greater similarity with 93.13% and 93.96%. 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. This is the first report about co-expression of pgsBCA and racE from the two Bacillus strains, which will be of great value for the determination of the biosynthetic mechanism of γ-PGA.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Geng

Zhang

et al. 2013

Microbial Biotechnology

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“…PGA is mainly found as a capsular polymer; therefore, its accumulation as an exo ‐polymer, as observed for B. subtilis and B. licheniformis , the PGA synthetic system of which is virtually the same as that of B. subtilis (Wang et al ., ; Yangtse et al ., ), is currently considered a peculiar phenomenon. Comparative genetic analysis of the pgs and cap operons (Fig.…”

Section: Localizationmentioning

confidence: 99%

Microbial production and chemical transformation of poly‐γ‐glutamate

Ashiuchi

2013

Microbial Biotechnology

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Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.

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“…For industrial applications, most studies have relied on biosynthesizing PGA in B. subtilis ( Ashiuchi, 2013 ; Halmschlag et al., 2019 ; Park et al., 2005 ; Wang et al., 2017 ) and other Bacillus species ( Feng et al., 2017 , 2015 ; Ogunleye et al., 2015 ; Tian et al., 2014 ; Xavier et al., 2019 ; Yoon et al., 2000 ), although there have been significant efforts in moving the complex to recombinant hosts such as E. coli ( Ashiuchi et al., 1999 ; Cao et al., 2013 , 2011 ; Jiang et al., 2006 ; Liu et al., 2019 ; Wang et al., 2011 ), Corynebacterium glutamicum ( Xu et al., 2019 ), and even tobacco plants ( Tarui et al., 2005 ). A major advantage of recombinant hosts is that PGA synthesis can be decoupled from native cellular regulatory processes and engineered for higher productivity, yield, stereochemical composition, and molecular weight.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli

Nascimento

Nair

2020

Metabolic Engineering Communications

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Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli , we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.

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Heterogenous expression of poly-γ-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3

Cited by 7 publications

References 14 publications

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

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

Microbial production and chemical transformation of poly‐γ‐glutamate

Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli

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