Demonstrate
After several months of effort, our system is proven to be functional. The core of our project is to eliminate the environmental problems caused by crop stalks - transforming the agricultural wastes into the high added-value products.
We use the AHP model to find a better pathway among several candidates. After the consideration of 5 factors, we found that astaxanthin synthesis got the highest score. Then we named our project: The E.coli cell factory that degrades Stalks and Produces Astaxanthin.
To achieve the purpose The Stalk Wastes to the High-value Products, we divided the project into the two subsections – Cellulose degradation and astaxanthin synthesis.
For the Cellulose degradation part, our team successfully added the INP-N(BBa_K3279006)sequence to the N terminus of β-1, 4-endoglucanase (BBa_K118023)and β-1,4-exoglucanase (BBa_K118022), both of which are from 2018 team UESTC-China, expressed fusion proteins were examined by immunofluorescence staining and enzyme activity assay. Observed with fluorescence confocal microscopy, we confirmed that the fusion proteins were anchored in the outer membrane surface of E.coli. And from the data of activity assay, we summarized that the cellulases' activities were not affected remarkably with the presence of INP-N . These fusion proteins allowed the cellulases to anchor in the cell outer membrane surface, which makes it possible for the cellulases to contact and degrade the substrate without cell disruption. The future plan for this subsection is to join the INP-N fused endoglucanase, exoglucanase, and glucosidase in a vector and be expressed, in this way, the bacteria would gain the intact power of breaking the cellulose into glucose.
Figure 1 Cellulose degradation system in the cell outer membrane surface
For the astaxanthin synthesis subsection, We borrowed endogenous DXP pathway and its product FPP(Farnesyl pyrophosphate), which is also the start point of many other secondary metabolic pathways. We constructed strains that can produce lycopene and β-carotene. After that, the essential genes in the astaxanthin synthesis pathway (CrtE CrtB CrtI CrtY CrtZ) were transferred into the E.coli strain BL21 and the color of cells turned yellow, which may be caused by zeaxanthin. In our subsequent work, we would examine these bacterial products and focus on the expression of CrtBKT, which converts the zeaxanthin to the astaxanthin in the final step of the astaxanthin synthesis pathway.
Figure 2 Comparison of liquid culture's color. Each group carries the different plasmid: pACYC184-M (control), pACYC184-M-EBI and pACYC184-M-EBIY. All groups are induced under the same conditions by 0.1mM IPTG.
Figure 3 Yield changes influenced by induction conditions (a) The changes of lycopene production with induction time. (b) The changes of β-carotene production with induction time.
We conducted the simulated bacteria fermentation using our engineered lycopene- and β-carotene-producing strains. We set several groups induced by different concentrations of IPTG (0.05mM, 0.1mM and 0.3mM) and the cultural volumes are 50mL and 100mL (Figure 4). The cell culture is collected every 30 minutes as samples for the yield analysis and cell quantities determination. We aim to find useful information in the small-scale trails that can guide the follow-up mass production.
Figure 4 Our simulated fermentation for bacteria producing lycopene and β-carotene(The culture's volume in each flask is 100mL in this case)
Our human practice also corroborated the positive effects our project can bring to the environmental issues made by crop stalks wastes. Our team members visited several rural villages and found that although the stalks incineration has been banned long ago, some stalks are still being burned secretly by villagers to save time and money, which has caused great damage to the environment. The locals usually use stalks as substitutes for coal and forage, or simply grind and spread to the fields, however, these methods all have problems such as high cost, low added value of output, and impeding marketization. Yet in our project, engineered bacteria would be able to convert the agricultural wastes into a variety of high value products. It is capable of synthesizing products such as lycopene, β-carotene and astaxanthin, which are now highly priced in the market. It is also possible to drive the formation of new industrial chains and solve employment problems.
Above all, our project provides not only an idea for recycling stalks and eliminating environmental pollution, but also a prospect of producing high-value products at low cost.
