1. Incorporating the green eco component - recycling straw
Every year the world produces a lot of straw, and what to do with it is a problem. Straw is rich in cellulose, which is a polysaccharide. With proper treatment, we can extract glucose from straw, and we can use this glucose to cultivate our modified Corynebacterium glutamicum, which can provide a better idea for straw treatment in the future.
After treatment of the straw, we obtained the relevant solution, the main component of which is glucose. The composition of the sugar solution of the treated straw is as follows. 6 g of dried solid, after treatment in 60 mL system, the concentration of the sugar solution obtained is 27.6 g/L and 23.25 g/L.
| Absolutely dry solid | Straw sugar concentration | |
| 60ml system | 6 g | 27.6 g/L |
| 60ml system | 6 g | 23.25 g/L |
We configured the same concentration of glucose solution and cultured continuously at the same concentration for 120h and measured the growth curves every 24h. The strains we used were unmodified Corynebacterium glutamicum ATCC13032, ΔcrtEb Corynebacterium glutamicum ATCC13032 and ΔcrtEb:crtR Corynebacterium glutamicum ATCC13032.We obtained the following growth curves. We get the following growth curve.
We can find that the sugar solution produced after straw treatment is good for cultivating Corynebacterium glutamicum. For group h, after knocking out the crtEb gene, the culture with straw sugar solution grows more rapidly in a short period of time compared to using glucose solution. The genes we ultimately wanted to knock out were crtEb and crtR, and in the growth curve below, group h was able to reach an average level, a result that is very promising. In future experiments, we need to explore the mechanisms that allow the straw sugar solution to better cultivate Corynebacterium glutamicum.
During the experiment we chose to use cellulase to enzymatically degrade the straw and use the glucose produced for the culture of Corynebacterium glutamicum. In order to test the effect of using the glucose produced by the enzymatic degradation of straw to cultivate Corynebacterium glutamicum, and compared with the use of a standard glucose solution, the results are shown in the figure below. By observing the glucose content in the solution, we can measure whether the strain is able to utilize the glucose and whether the glucose produced from straw has any effect on the growth of the strain.
We performed glucose backfilling after measuring the OD600 value once. The glucose produced by the enzymatic digestion of straw was not highly utilized compared to the blank group. The glucose from the enzymatic digestion of straw without any treatment was not well utilized by Corynebacterium glutamicum, compared to the standard glucose solution. In this regard, we need to treat the glucose obtained from enzymatic digestion of straw.
In our experiments, we pay special attention to combining environmental protection and sustainable development, and are committed to solving the current problem of straw that cannot be reasonably utilized. The use of straw sugar solution for bacterial culture is a good idea, which not only can effectively utilize straw resources, but also can further promote environmentally friendly research.
In the future, we will continue to explore more ways to treat straw and integrate them more closely into our programs. In this way, we hope to make a greater contribution to solving energy problems and environmental governance, and to play our part in realizing the Sustainable Development Goals. We believe that through continuous efforts and innovation, we can make significant contributions to building a better future.
2. Promoter Optimization
After identifying the PAM site with the highest editing efficiency of MAD7, in order to further improve the editing efficiency of MAD7 nuclease, we constructed different promoters using pEC plasmid as a vector in order to improve the transcriptional-translational efficiency of MAD7. We constructed four promoters, namely, PlacM, Ptuf, Ptac, and Ptrc, respectively, to enhance the transcriptional efficiency of MAD7. Eventually, the average editing efficiency of the PlacM promoter reached 10.97%, the average editing efficiency of the Ptuf promoter was 2.98%, and the editing efficiency of the Ptac promoter was the lowest, 0. In this regard, we can continue to optimize the promoters on the basis of PlacM to finally achieve the effect of a strong promoter.
3. Selection of the optimal plasmid
The editing efficiency of MAD7 nuclease can be maximized by using appropriate plasmids to carry MAD7 nuclease in different situations. In our project, we selected different plasmids to carry fragments of MAD7 nuclease into Corynebacterium glutamicum ATCC13032. We used four plasmids, pJYS1, pBluescript, pEC-XK99E, and pXMJ19. as can be seen from the graphs, the average editing efficiency and the number of transformations did not differ much when using the pXMJ19 and pBluescript plasmids as vectors. pEC-XK99E plasmid vector, although it transformed the largest number of colonies, the editing efficiency was the The pEC-XK99E plasmid vector had the highest number of transformed colonies but the lowest editing efficiency. The highest editing efficiency was for pJYS1, which reached 80% and had a high number of transformations relative to pXMJ19 and pBluescript plasmids. We transferred the plasmids into Corynebacterium glutamicum ATCC 13032, and continuously constructed and optimized the plasmids based on our screened plasmids.
4. Selection of the best PAM site
In the course of this study, in order to explore the preference of PAM sites of CRISPR-MAD7(Cas12a) system in Corynebacterium glutamicum, to find the most suitable PAM sites for the work of MAD7 nuclease complex, and to improve the efficiency and accuracy of gene editing technology of CRISPR-MAD7(Cas12a) system in Corynebacterium glutamicum. We tried to design a total of 11 different PAM sites,which are TTTA(+), TTTC(-), TTTT(-), TTTG(-), TTTC(+), TTTA(-), TTTT(-), CTTC(+), CTTA(+), CTTG(-), CTTT(+). The plasmids were constructed on this basis to explore their editing efficiency and to find the best PAM sites.
Through the results, we can find that TTTA(+), TTTC(-), TTTT(-), TTTG(-), TTTC(+), TTTA(-), TTTT(-), CTTG(-), CTTT(+), which are PAM sites, are helpful for the CRISPR-MAD7(Cas12a) system to perform better gene editing in Corynebacterium glutamicum. However, in terms of colony number and transformation efficiency, TTTC(+) is superior, which means that TTTC(+) can give the best performance compared with other PAM sites. Of course, this also means that gene editing at this PAM site is easier to achieve and performs well in terms of success rate and efficiency. Based on this finding, we determined that this PAM site would be an important basis for our subsequent experiments.
5. Large Segment Knockout Function Test for MAD7 System
Based on the efficiency and effectiveness of the CRISPR-MAD7(Cas12a) system, we verified the knockdown efficiency of the CRISPR-MAD7(Cas12a) system on Corynebacterium glutamicum gene fragments by randomly knocking down genes with sizes of 5 kb, 10 kb, 15 kb, 20 kb, and 25 kb, and obtained the following results, with specific editing efficiencies as follows.
From the experimental results, it can be observed that the CRISPR-MAD7 (Cas12a) system has the highest efficiency of up to 83% for knocking out 10kb genes in Corynebacterium glutamicum, and more than 50% for knocking out 5kb and 20kb genes, meanwhile, the CRISPR-MAD7 (Cas12a) system can knock out genes of 25kb size, which is something no other system can achieve. Of course, for the knockout of 25kb genome, we still need to further explore and optimise the experiment. Meanwhile, these results also indicate that the CRISPR-MAD7(Cas12a) system we found is more suitable for gene editing in Corynebacterium glutamicum .
6. Construction of strains
After completing the selection of promoters, PAM sites and plasmids, we started to achieve our ultimate goal of efficient lycopene production by knocking out the relevant genes. We used pJYS1 plasmid as a vector, PlacM as a promoter, and TTTC(+) as the final PAM site on the basis of which we carried out the construction of plasmids for knockdown genes. The construction of the final strain with high lycopene production was completed. We mainly performed pJYS1-MAD7-ΔcrtEb (Knock out the crtEb gene), pJYS1-MAD7-ΔcrtR (Knock out the crtR gene), pJYS1-MAD7-ΔcrtEb:ΔcrtR (Knock out the crtEb/crtR dual gene) strains were constructed. The results were shown as follows.
Construction of ΔcrtEb strains
From the results, we can see that the editing efficiency of the constructed pJYS1-MED7-ΔcrtEb strain is as high as more than 90%. This is a good verification that the system we constructed is very effective.
Construction of ΔcrtR strains
From the results, we can see that the constructed pJYS1-MED7-ΔcrtR strain changed its colour significantly.
Construction of ΔcrtEb:ΔcrtR strains
From the results, we can see that the colour of the constructed pJYS1-MAD7-ΔcrtEb:ΔcrtR strain changed significantly. It can be clearly seen that the colour of the strain has turned pink indicating a substantial increase in lycopene production.
By selecting plasmid vectors, choosing PAM sites, optimizing promoters and performing large fragment genome knockdown experiments, we successfully constructed a new editing system for targeting Corynebacterium glutamicum for transformation. Our experiments provide valuable references for future research teams and directions on where to start to modify a particular bacterium.
Besides, compared with the existing system, our constructed system is more convenient and suitable for industrialized production. And lycopene, as an excellent antioxidant, has an important role in biomedicine and food health. Through the modified Corynebacterium glutamicum, we realized the accumulation of lycopene, which opened a new way for the production of lycopene and promoted the development and progress of related fields.
We can find that the sugar solution produced after straw treatment is good for cultivating Corynebacterium glutamicum. For group h, after knocking out the crtEb gene, the culture with straw sugar solution grows more rapidly in a short period of time compared to using glucose solution. The genes we ultimately wanted to knock out were crtEb and crtR, and in the growth curve below, group h was able to reach an average level, a result that is very promising. In future experiments, we need to explore the mechanisms that allow the straw sugar solution to better cultivate Corynebacterium glutamicum.
7. Successful production of lycopene
After previous strain construction, we ended up with expanded cultures in shake flasks and ended up with the strains shown in the figure. On the left is the ΔcrtEb strain and on the right is the ΔcrtR/crtEb strain. Where we can see that the ΔcrtR:ΔcrtEb strain produced significantly higher lycopene yields.
