Engineering Expression Cassette of pgdS for Efficient Production of Poly-γ-Glutamic Acids With Specific Molecular Weights in Bacillus licheniformis

Abstract

Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer with various applications and γ-PGAs with different molecular weights exhibit distinctive properties. However, studies on the controllable molecular weights of biopolymers are limited. The purpose of this study is to achieve production of γ-PGAs with a wide range of molecular weights through manipulating the expression of γ-PGA depolymerase (PgdS) in Bacillus licheniformis WX-02. Firstly, the expression and secretion of PgdS were regulated through engineering its expression elements (four promoters and eight signal peptides), which generated γ-PGAs with molecular weights ranging from 6.82 × 10 4 to 1.78 × 10 6 Da. Subsequently, through combination of promoters with signal peptides, the production of γ-PGAs with a specific molecular weight could be efficiently obtained. Interestingly, the γ-PGA yield increased with the reduced molecular weight in flask cultures (Pearson correlation coefficient of −0.968, P < 0.01). Finally, in batch fermentation, the highest yield of γ-PGA with a weight-average molecular weight of 7.80 × 10 4 Da reached 39.13 g/L under glutamate-free medium. Collectively, we developed an efficient strategy for one-step production of γ-PGAs with specific molecular weights, which have potential application for industrial production of desirable γ-PGAs.

Efficient molasses utilization for low-molecular-weight poly-γ-glutamic acid production using a novel Bacillus subtilis strain

“…PgdS (the γ-DL-glutamyl hydrolase) is an endo-type hydrolase that cleaves the γ-glutamyl bond and depolymerizes high-Mw-γ-PGA to low-Mw-γ-PGA [ 3 ]. It is the key enzyme responsible for the Mw control in γ-PGA producers [ 21 , 22 ]. To investigate the reason for low-Mw-γ-PGA production by B. subtilis 242, the transcripts of pgdS at different fermentation periods (6, 12, 24, 24 and 36 h) were detected using quantitative real-time (RT)-PCR.…”

“…High-Mw-γ-PGAs (Mw > 1 × 10 3 kDa) have strong viscosity and could be used as thickeners or flocculant [ 20 ]. Low-Mw-γ-PGAs (Mw < 500 kDa) could be used as drug delivery (45–60 kDa), tissue engineering nanocomposites (20–275 kDa), water-retaining agents (< 20 kDa), and so on [ 21 , 22 ]. At present, the Mws of commercial γ-PGA produced Bacillus species are approximately 1 × 10 3 kDa, which limits the wide uses of γ-PGA [ 21 ].…”

Bacterial Biopolymer: Its Role in Pathogenesis to Effective Biomaterials

“…Another method for gellan production is by using a newer strain called S. azotofigens strain GL-1 [ 72 , 73 ]. This bacterial strain was cultivated inside shake flasks at 30 °C and pH 6.5 by utilization of CW medium or molasses.…”

“…Another type of biopolymer Poly-γ-glutamic acid (γ-PGA) showed varied applications depending upon its varied molecular weights. The enhancement in the production of Poly-γ-glutamic acid (γ-PGA) can be achieved by regulation of γ-PGA depolymerase (PgdS) within Bacillus licheniformis.This can be achieved by engineering its promoter and signal peptide, thereby producing poly-γ-glutamic acid of varied molecular weight [ 73 ].…”

Development of a Conjugation-Based Genome Editing System in an Undomesticated Bacillus subtilis Strain for Poly-γ-glutamic Acid Production with Diverse Molecular Masses

“…licheniformis by manipulating the promoter and signal peptide. γ-PGA with an average M w of 78 kDa was obtained . As in previous studies, different sources of γ-PGA hydrolases were heterologously expressed to investigate their hydrolytic capacities.…”