Team:H14Z1 Hangzhou/Applied Design
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Product Design
Abstract
Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied to prevent and treat a variety of liver diseases. Due to low stability and short half-life, oral table supply of GSH or SAM might be replaced by supplying these molecules in-vivo using our smart yogurt strategy. In the experiment, two-functional GSH synthetase gene (gshF) and SAM synthetase gene (metK) were in tandem inserted into the expression vector (pNZ8148), and the resulted plasmid (pNZ-GM) was further employed to construct the target vector pNZ-GMcA by introducing adhesion factor gene (cwaA). This combinatory vector was transformed into food-grade Lactococcus lactis NZ9000 for their functional expressions. The results showed that our smart yogurts not only have much higher contents of SAM and GSH with regard to commercial yogurts in the daily life, but also hold high density of colonization-prone cells which will produce these two liver-protective factors continuously in our gastrointestinal tracts.
Background
Glutathione (GSH) is biosynthesized from glutamic acid, cysteine and glycine, and distributes in many organs in human body, especially in the liver where the highest concentration of GSH exists. GSH plays an important role in liver biochemical metabolism. GSH can inhibit the formation of fatty liver, directly antagonize the toxicity of oxygen free radicals, prevent hepatocyte injury and treat of fulminant hepatic failure caused by drug poisoning.
S-adenosyl methionine (SAM) is an important intermediate product in liver metabolism. As a methyl donor and a precursor of physiological sulfhydryl compounds, SAM participates in important metabolic reactions in vivo. SAM can delay the need for liver transplantation and reduce mortality in patients with alcoholic cirrhosis in a 24-month trial and significantly increase hepatic glutathione levels in nonalcoholic liver disease with oral administration of 1200mg SAM daily (J Hepatol 1999, 30(6) 1081-1089)
CwaA protein encoded by gene cwaA is a cell wall-anchored protein. The C terminus of CwaA contains an LPQTDE (LPxTG-like cell wall anchoring) motif belonging to the gram-positive LPxTG anchor superfamily. Aside from the hexapeptide motif at the C terminus, CwaA possesses five cell wall surface anchor repeat domains. The specific hit domains of CwaA included epiglycanin (tandem-repeating region of mucin), OmpC (outer membrane protein), PT (the tetrapeptide XPTX repeat). Taken together, CwaA is a multidomain-containing cell wall-anchored protein that is very likely involved in cell adhesion. Thus, by expressing CwaA protein might be useful for improving the adhesive ability of strains, which would prolong the deliver time of healthy factors produced by the strains.
Lactococcus lactis is a food-grade bacterium that is widely used in the dairy industry, such as yogurt production, and is commonly used in bacterial drug delivery system. Since the oral table administration of GSH and SAM have some disadvantages, such as low stability and short life span, here we tried to develop a novel in-vivo strategy of producing and delivering them simultaneously in L. lactis. And then integrate cwaA gene to enhance the adhesion ability if L. lactis for better delivery of GSH and SAM in human body. In the experiment, two-functional GSH synthetase gene (gshF) and SAM synthetase gene (metK) were in tandem inserted into the expression vector (pNZ8148), and the resulted plasmid (pNZ8148-MG) was employed to construct the target vector pNZ8148-GMcA by introducing adhesion factor gene (cwaA). This target vector was transformed to get recombinant Lactococcus lacti, which was employed to produce our “smart yogurt”.
The design of our food-grade L. lactis containing three functional modules
In the experiment, two-functional GSH synthetase gene (gshF) and SAM synthetase gene (metK) were in tandem inserted into the expression vector (pNZ8148), and the resulted plasmid (pNZ-GM) was further employed to construct the target vector pNZ-GMcA by introducing adhesion factor gene (cwaA). The construction process was described in Fig.1
Figure. 1 Schematic diagram of constructing food-grade L. lactis containing three functional modules
Production of “smart yogurt” with our constructed L. lactis NZ9000/ pNZ-GMcA
After validation of the function of the final engineered strain L. lactis/pNZ-GMcA, we applied it to produce smart yogurts. As depicted below, we produced three kinds of smart yogurts. One was produced by using wild-type strain L.lactis NZ9000 adding GSH and SAM in the process. Another one was produced by using engineered L. lactis/pNZ-GMcA and the last one using wild-type strain L.bulgaricus and engineered L. lactis/pNZ-GMcA without adding GSH and SAM.
Figure. 2 Schematic diagram of producing smart yogurts.
Advantages of our “Smart yogurt” compared with daily-life yogurt
1) GSH and SAM content in the smart yogurts
The GSH and SAM content of the smart yogurts in the fermentation at 6 and 12 hours were detected. As shown in Figure. 3, the smart yogurt made by using engineered L. lactis contained obvious more GSH and SAM. The content increased with the increase of cell numbers.
Figure. 3 GSH and SAM content in the smart yogurts at 6 and 12 hours. Asterisk represented not detected.
2) High density of our constructed cells (L. lactis/pNZ-GMcA)
As shown in Figure. 4, after 12 h, about 1.2×109 cells existed in 1 mL yogurt, which was a rather high density of strains. The higher the density is, the more helpful factors such as GSH and SAM could be produced and delivered. Especially, high density cells will be deliver into gastrointestinal tract and make colonization here to synthesize GSH and SAM in-vivo continuously.
Figure. 4 The cell numbers in smart yogurts at 12 h.
3) Low-cost production
The price of health products and medicine containing GSH and SAM are usually expensive. In our work, we innovatively integrate GSH and SAM module in L. lactis endowing the ability of producing GSH and SAM. When the engineered L. lactis was used for producing yogurts, it would be a low-cost product for replenishing GSH and SAM for human body.
4) Easy to deliver factors in-vivo
Our final engineered L.lactis/pNZ-GMcA contained adhesion module, and the assay of self-aggregation value showed that the adhesion ability of the engineered strain was improved by integrating the adhesion module. The enhanced adhesion ability can prolong the time of L.lactis in our body and thus deliver more GSH, SAM and other helpful factors produced by L. lactis.
Figure. 5 Self-aggregation value of L. lactis NZ9000 and L. lactis/pNZ-cwaA
5) Easy to be accepted by most of people after safety evaluation
Compared with other health products and medicine containing GSH and SAM, people would be favor of drinking yogurts containing GSH and SAM. Because yogurts are known as safe and healthy for long time.
6) Protection of the technologies for producing smart yogurts
As the engineered L. lactis was innovative, we have applied for a Chinese patent for producing smart yogurts using this engineered strain containing three modules. And the application was received.
Figure. 6 The patent for producing smart yogurts.