As described in project description, our goal is to develop a multifunctional live bacterial skincare product. Using the principle of synthetic biology, Gluconacetobacter hansenii ATCC53582 is engineered to produce glutathione (GSH) efficiently. In addition, an optogenetic system is designed to control the engineered bacteria. Within the system, NIR light is used to regulate the production of bacterial cellulose for moisturizing, while blue light is used to regulate the release of glutathione and bacterial lysate to provide antioxidative effect and maintain skin microbiome.
To these ends, we designed three modules in the chassis strain, namely the GshF expression and GSH production module, the c-di-GMP signaling and BC film production module, the lysis and safety module. The resultant engineered bacteria could be used to develop a skincare product with the cosmetic effects of moisturizing, brightening, anti-spot, repairing, anti-aging, and skin microbiome maintenance.
The biosynthetic pathway of GSH usually contains two ATP-dependent consecutive reactions catalyzed by γ-glutamylcysteine (γ-GC) synthetase (γ-GCS or GSH I) and GSH synthetase (GS or GSH II). However, it is difficult to accumulate high levels of intracellular GSH since the enzymatic activity of the native GSH synthetases (the above mentioned GSH I and GSH II) would be strongly inhibited by the GSH. To overcome this inhibition effect of end-product, the gene encoding a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus, which is less sensitive to GSH, is codon optimized and expressed in G. hansenii for enhanced GSH production.
This module is kept the same as that of last year's project. Bacterial cellulose (BC) production in G. hansenii is positively regulated by the second messenger cyclic diguanylate (c-di-GMP). Therefore, we can control BC film production by regulating the concentration of c-di-GMP. For effective and accurate regulation of c-di-GMP, we designed two sub-modules. One is the diguanylate cyclase (DGC) sub-module, which is responsible for the synthesis of c-di-GMP.
While the other one is c-di-GMP phosphodiesterase (PDE) sub-module, which directs the hydrolysis of c-di-GMP. When the rate of c-di-GMP synthesis surpasses its hydrolysis rate, the concentration of c-di-GMP will increase, and the bacteria will produce BC film accordingly. In this module, we use bphS that encodes a photo-activated DGC named BphS and fcsR that encodes a c-di-GMP PDE named FcsR. Upon illumination with near-infrared light, BphS will be activated with changed protein conformation and start to synthesize c-di-GMP.
The lysis and safety module is further improved on the basis of last year's project in which a blue light responsive system pDawn was opted to express the lysis genes from bacteriophages to induce bacterial cell death. However, we found that the inducible suicide system (BBa_K3740033) became disabled in rapidly growing cells owing to the leaky, low-level expression of the lysis gene in the dark. To create evolutionarily stable kill switches, pDawn and lysis genes as well as RBSs are optimized in our project.
We removed the degradation tag LVA in the original cI of pDawn to enhance the repression of the lysis gene in the dark for reduction of the deleterious evolutionary pressure. A small library of potential constructs by altering RBS and lysis gene have been created for the screening of the system with optimal lethality and stability. Efficient lysis will result in the release of GSH as well as bacterial lysate. This system functions as a safety module since the self-lysis of engineered bacteria could be induced under natural light in case of environmental release.
[1] https://2021.igem.org/Team:SZPT-CHINA/Design
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