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

Abstract

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.

Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

“…Bacillus amyloliquefaciens LL3 is a glutamic acid‐independent γ‐PGA‐producing strain, which suggests that the intracellular glutamate is the only substrate for γ‐PGA synthesis (Cao et al ., ). We previously reported that the deletion of RocR, a transcriptional regulator of glutamate metabolism, may have contributed to the increase of γ‐PGA production in B. amyloliquefaciens LL3 (Zhang et al ., ).…”

“…Metabolic evolution had been successfully used to enhance the production of many fermentation products, such as succinate (Jantama et al ., ; Zhu et al ., ), l ‐lactate (Grabar et al ., ), D‐lactate (Zhou et al ., ), ethanol (Yomano et al ., ) and l ‐alanine (Zhang et al ., ). Since γ‐PGA synthesis was relevant to cell growth of B. amyloliquefaciens (Cao et al ., ), metabolic evolution was performed in the strain NK‐A9 to improve cell growth and γ‐PGA production. After the 10th time transfer, growth of the recombinant strain had been much better than that of NK‐A9 and NK‐A7 (Figs S6A and B).…”

Development of bio-based fine chemical production through synthetic bioengineering

“…B. amyloliquefaciens was engineered to enhance γ-PGA production by heterologous expression of the Vitreoscilla gene (vgd) encoding hemoglobin to overcome the low concentration of dissolved oxygen [67,88]. In contrast, Chao et al developed a γ-PGA-producing E. coli strain by heterologous expression of γ-PGA synthetase and glutamate racemase from B. licheniformis or B. amyloliquefaciens [89]. The tri-peptide glutathione, which is the most abundant antioxidant, thiol-containing compound among organisms [100], is produced enzymatically and by microbial fermentation.…”

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

“…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(Feng et al, , 2015Ogunleye 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, 2013Cao et al, , 2011Jiang 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.…”