Team:Nankai/project background
1. Pudding health kit — Wound healing hydrogel based on γ-PGA
Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer that is water soluble, non-toxic, edible, and biodegradable, which has shown to promote cell migration and enhance cell adhesion. Besides, γ-PGA has been reported to prevent postsurgical tissue adhesion and the γ-PGA drug-loaded hydrogel to promote wound healing.
Figure 1. Appearance and structural formula of γ-PGA.
We prepared two types of SOD-PGA hydrogels for wound healing. γ-PGA hydrogel had high water absorption properties delivering the important moist environment. SOD released from the hydrogel maintained high enzyme activity and SOD-PGA hydrogels could scavenge the superoxide anion effectively. In vivo results showed that SOD-PGAS/PGA-H could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore, SOD-PGA hydrogels would be a good candidate for wound healing applications.Learn about more here.
Figure 2. MCS/γ-PGA mixture in solution (A). MCS/γ-PGA hydrogel (B). Microstructure of γ-PGAS/PGA hydrogel (C, D)
2. γ-PGA producing strain — Bacillus amyloliquefaciens LL3
Strains capable for producing γ-PGA are divided into two categories based on their requirement for glutamate acid: glutamate-dependent strains and glutamate-independent strains. Glutamate-independent strains are preferable for industrial production because of their low cost and simplified fermentation process. However, compared with glutamate-dependent strains, their lower γ-PGA productivity limits their industrial application. Therefore, the construction of a glutamate-independent strain with high γ-PGA yield is important for industrial applications.
Figure 3. Stereoscan photograph of B.amyloliquefaciens LL3 strain
B. amyloliquefaciens strains are ubiquitous in the soil and are great reservoirs of important natural products, such as α-amylase, levansucrase, and fibrinolytic enzymes. Besides important cell factories, B. amyloliquefaciens strains are also used as plant growth-promoting and bio-control bacteria partly due to their ability to produce substances with antifungal, antibacterial and nematocidal activities. B. amyloliquefaciens LL3, isolated from fermented food, is a glutamate-independent strain, which can produce γ-PGA with sucrose as its carbon source and ammonium sulfate as its nitrogen source.
3. Methods to increase poly-γ-glutamic acid production
In this study, we aimed to improve the γ-PGA production based on the B. amyloliquefaciens NK-1 strain (a derivative of LL3 strain with its endogenous plasmid and upp gene deleted). In order to improve γ-PGA production, we employed two strategies to fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain.
3.1 Metabolic toggle switch
Figure 4. Biosynthetic pathway of poly-γ-glutamic acid. Red arrows shows the metabolic flux towards γ-PGA synthesis. The blue arrow is the leakage of metabolic flux.
Firstly, we aimed to increase the intracellular concentration of γ-PGA precursor-- glutamate. In B. amyloliquefaciens,2-oxoglutarate is very important for the synthesis of glutamate, yet large amount of 2-oxoglutarate is consume by TCA cycle to turn into succinl-CoA in the action of enzyme ODHC (2-oxoglutarate dehydrogenase complex). Scientists had found that the activity of ODHC was rather low when glutamate was highly produced in a Corynebacterium glutamicum strain. This made us wonder, could we be able to increase the amount of intracellular glutamate by inhibiting the expression of ODHC at the stationary phase in B. amyloliquefaciens? Therefore, we constructed a metabolic toggle switch in the NK-1 strain to inhibit the expression of ODHC by adding IPTG in the stationary stage, trying to distribute the metabolic flux more frequently to be used for γ-PGA precursor-glutamate synthesis.
Figure 5. Metabolic toggle switch to regulate the expression of ODHC.
Without IPTG, the promoter Pgrac is inhibited by suppressor LacI and the supreessor XylR will not synthesized, thus the promoter Pxyl is active and odhAB genes are expressed. When IPTG is added, the xylR gene is expressed and the suppressor XylR is synthesized thereafter inhibited the expression of odhAB genes.
3.2 Replacement of promoters
Secondly, to balance the increase of endogenous glutamate production, we optimized the expression level of pgsBCA genes (responsible for γ-PGA synthesis) by replacing its native promoter with six different strength promoters.
Figure 6.Genes responsible for γ-PGA synthesis
Through these two strategies, we aimed to obtain a γ-PGA production improved mutant strain.
These are all bioreactors used for fermentation.
Electroosmosis.
Chromosome of bacillus amyloliquefaciens LL3.
Ready for doing PCR.