Initiate PLL production by cloning pls to Bacillus subtilis
To get a steady production of PLL in Bacillus subtilis, we tried to clone the ε-PLL production gene pls from Streptomyces albulus. Since Streptomyces albulus is not generally regarded as safe and Bacillus subtilis' genetic information varies hugely from Streptomyces albulus, we synthesized the pls gene with codon optimization(Figure1.a). The synthesized pls gene is ligated to the pMA5 vector backbone with T4 ligase(Figure1.b). After being introduced into Bacillus subtilis with chemical transformation and verified successful transformation(Figure1.c), recombinant pMA5-pls vectors showed a significant increase in production of ε-PLL(Figure1.d).
Increase production by introducing ppk2 gene
The synthesis of ε-PLL requires ATP to provide energy. However, the ATP regeneration system in Bacilli subtilis is not efficient enough to provide adequate energy for this process. Thus, in order to increase the production of ε-PLL, we selected the ppk2 form Corynebacterium glutamicum to accelerate the ε-PLL synthesis. The ppk2 gene is cloned from Corynebacterium glutamicum (Figure2.a) and ligated to the pMA5-pls vector after the pls gene, generating a new recombinant pMA5-pls-ppk2 vector(Figure2.b). The vector is then transformed into Bacillus subtilis via chemical transformation(Figure 2.c). Our results showed that with the help of the ppk2 gene, ε-PLL concentration increased from 0.3g/L to 0.8g/L after 24 hours of fermentation of Bacillus subtilis(Figure2.d).
Boost lysine production by replacing thrD gene with lysC311
Another key point to limit ε-PLL synthesis is the abundance of ingredient L-lysine. Bacillus subtilis has an intact lysine synthesis pathway and can produce lysine for its own use. However, this pathway has a natural feedback loop, which leads to only adequate lysine for bacterial survival and no accumulation of extra lysine for ε-PLL production(Figure 3.a). In order to boost the production of lysine in Bacillus subtilis. We looked into literature and decided to replace the thrD gene in the lysine production pathway with the mutated lysC311 gene from Corynebacterium glutamicum. The lysC gene was cloned with a mutation at amino acid 311 from Corynebacterium glutamicum, together with the upstream and downstream of the thrD gene(Figure 3.b), and we recombined a vector for homologous recombination use. This replacing vector is then tranformed into Bacillus subtilis together with Cas9 vector and sg-thrD vector, resulting a new Bacillus subtilis strain that can accumulate lysine(Figure 3.c). The pMA5-pls-ppk2 vector is then transformed into the new strain, and an extra production of ε-PLL is observed(Figure 3.d). We then measured the production of ε-PLL in this recombinant Bacillus subtilis over time, a steady and robust production of ε-PLL is shown without harming bacteria growth(Figure 3.e,f).
The biocontainment system in Bacillus subtilis
To ensure the safety of our engineered bacteria, we designed a lac operon-coordinated biocontainment system for Bacillus subtilis(Figure 4a). First, the alrA gene is depleted using CRISPR, resulting in a strain that requires extra D-Alanine to grow. Then, we incorporated the alrA gene with the lac operon(Figure 4.b), and the vector was then transformed into Bacillus subtilis. We then confirmed that without adding IPTG, the bacteria showed no growth(Figure 4.c), proving the biocontainment system works well. Finally, we tried to combine the biocontainment system with the production system. The lac-alrA is ligated to the production vector pMA5-pls-ppk2(Figure 4.d). Therefore, after depletion of alrA and replacing the thrD gene with lysC311, we have a single vector, pMA5-pls-ppk2-lac-alrA, to ensure both robust production and safety(Figure 4.e).
Membrane development
After ensuring the safe and robust production of ε-PLL, we made three different types of membranes with ε-PLL and chitosan. We ultimately chose sodium alginate-chitosan bilayer membrane to be the carrier of ε-PLL since it presented relatively good overall performance among the three types of membrane we had tested. We conducted tensile strength test using tensile testing machine. The result showed that compared to commercial cling film, the tension that our membrane can hold is even better(Figure 5.a). On the other hand, our membrane lacks elasticity as it breaks easily compared to commercial plastic ones, indicating that we still need to improve our receipe for later use. Furthermore, we studied the effect of drying time during the casting of the membrane, and proved that it works best at 4 hours(Figure 5.b).
Antibacterial test of the membrane
Finally, we tested the anti-effect of our cling film using the Kirby-Bauer test. We showed that the membrane showed certain anti-bacterial effects in both the gram-positive and gram-negative bacteria (Figure 5.ab). Thereafter, we tried to wrap food to check the antibacterial effect of our cling film. We chose three commonly seen foods: beef, salmon, and blueberries as our study objects. After 5 days with commercial cling film and our antibacterial film, we can see that the food appears to be fresher compared to the control ones. We then collect microbiome on the food using a wet cotton swab and check the bacteria and fungi colony numbers. Our product shows a significant decrease in both bacteria and fungi numbers compared to common cling film in all three subjects we studied (Figure 6.c-r).
