The “slash and burn” method of processing agricultural waste, which produces an excess of useless ash, is still prominent in Taiwanese agriculture. This project aims to present a solution which uses organic agricultural waste to extract enzymes that can increase the efficiency of plant growth, helping to curtail the need for slash and burn. Rice straws are decomposed to produce sugar solution that is used to make PQQ and promote plant growth to increase agricultural yields and reduce waste to meet the needs of circular agriculture.
To accomplish this program, three main goals must be achieved:
The First Goal: Decompose the straws into sugar solution via saccharification.
Rice straw consists of cellulose, hemicellulose, and lignin. In the picture, the cellulose is enclosed circularly by hemicellulose and lignin. (Figure 1)
Decompose rice straw by Cellulosome
Cellulosome is aimed to decompose rice straw. Our cellulosome is composed of three kinds of cellulases and one scaffolding protein. Our cellulosomal enzyme system includes exo-glucosidase, endo-glucosidase, xylanase and Scaffolding protein. The scaffolding protein assembles cellulase and increases its decomposition efficiency.  
In our project, every cellulase has its own plasmid for enzyme production. The cellulosome gene is from Clostridium thermocellum. (Figure 3)
XynC is xylanase, which can degrade hemicellulose. CelA and celK are glucosidases. The former is endoglycosidase, and the latter is exoglycosidase. CipA is the scaffolding protein.
Make rice straw into powder, add cellulase water and NaOAc which acts as a buffer, incubate in 60°C, add Okara solution for nitrogen source then filter the solution.
The Second Goal: Apply PQQ-producing endosymbiotic bacteria to promote plant growth.
What are endosymbionts?
Endosymbionts are microorganisms, fungi or bacteria, that can co-exist in plants and do not cause plant diseases. Some of them help plants absorb nutrients, such as nitrogen-fixing bacteria, and metabolites secreted by endophytes which act as an important role in plant growth, such as Bacillus subtilis to produce glutamate. Others protect plants from disease, such as Burkholderia seminalis strain 869T2 against soil borne Fusarium wilt of banana.
Endophyte in our project.
At first, we used E. coli as the host to produce PQQ and inoculated it into the soil. However, the result did not achieve the way we hoped, in the complex soil system, the competition between E. coli and other organisms would reduce the productivity of PQQ. Compared to E. coli, a rhizosphere microorganism, endophytes can live in plant tissue, a more stable and nutritious environment. Bacillus subtilis, which would be the best host for the PQQ gene, not only because it is an endophyte, but also because it is a widely used model organism, and has been publicly verified that it has no virulence gene and is capable of entering the higher plants.
What is PQQ?
Pyrroloquinoline quinone (PQQ) is a coenzyme that combines with lactate dehydrogenase to convert lactic acid that is not available to organisms, into pyruvate, which they need for survival. PQQ is also a good antioxidant, which can reduce cell oxidation potential and protect cells. According to many essays, PQQ is beneficial to bacteria, plants and animals. Human can ingest it to resist cancer and Alzheimer's disease.PQQ is rare in nature. It can only be synthesized by a few bacteria and the yield is insufficient, which makes PQQ precious.
In addition, many studies have confirmed that PQQ can promote plant growth. Therefore, we plan to synthesize PQQ plastids and transfer them into Bacillus, and inoculate plants with endophytes that can produce PQQ to foster growth in plants.
Biosynthesis of PQQ
Five genes, including pqqA, pqqB, pqqC, pqqD, pqqE, are necessary in the biosynthesis of PQQ. pqqA is the precursor, a short peptide with 26 amino acids that contains two fully conserved residues, glutamate, and tyrosine. First, PqqA, PqqD and PqqE will form PqqA/D/E complex. In the presence of PqqD, PqqE helps the Glu-Tyr cross-linking within PqqA polypeptides to be generated. Second,PqqF/G complex trims the cross-linked product from the PqqA/D/E reaction. However the existence of these two gene is not found in all species. Our PQQ biosynthesis genes are from Gluconobacter oxydans, without pqqF and pqqG in it.It has been expressed in E. coli and found that PQQ can be produced. Third, PqqB, a hydroxylating enzyme, catalyzes the generation of AHQQ, which acts as the substrate in the final oxidative steps of the pathway catalyzed by PqqC. There is still many intriguing questions remain in PQQ biogenesis. 
Construct PQQ Plasmid
We use pET28a for plasmid backbone, add the f1 ori and sequences of PQQ, replace T7 promoter by PR promoter, make our gene can be copied in Bacillus subtilis, which can expect to act as a plant endophyte.
Culture the strain overnight, pellet down the strain, replace the supernatant with sterilized water and finally inoculate plants.
Collect the samples, after sterilizing the sample's surface, we use mortar and pestle, grind the samples, place samples into spread plates, and then select a single colony for plasmid extraction and sequencing.
The Final Goal: Circular Agriculture
The genetically modified Bacillus subtilis is cultivated in the saccharified rice straws to produce PQQ, which promotes the rice growth and completes the entire agricultural cycle. The larger and healthier plants grow via this method ultimately fix more CO2, and also it can reduce the usage of farm chemicals and fertilizer. Moreover, information technology such as big data, ICT , can be combined with this circular system and monitor plant growth, so farmers can know what time and how much our product should be used during planting.
- Chang, JJ., Anandharaj, M., Ho, CY. et al.(2018). Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis. Biotechnol Biofuels, 11(157).
- Ti Fang (2017). Improvement strategy of artificial cellulosomes’ efficiency.(Master’s thesis). NCHU, Taichung, Taiwan.
- Gupta G., Panwar J., Akhtar M.S., Jha P.N. (2012). Endophytic Nitrogen-Fixing Bacteria as Biofertilizer. Sustainable Agriculture Reviews,11.
- Qiu, X. M., Sun, Y. Y., Ye, X. Y., & Li, Z. G. (2020). Signaling Role of Glutamate in Plants. Plant science, 10, 01743.
- Ho, Y. N. et al. (2015). In planta biocontrol of soil borne Fusarium wilt of banana through a plant endophytic bacterium, Burkholderia cenocepacia 869T2. Plant and soil, 387(1-2), 295-306.
- Akagawa, M., Minematsu, K., Shibata, T., Kondo, T., Ishii, T., & Uchida, K. (2016). Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein. Scientific reports, 6, 26723.
- Naveed M, Tariq K, Sadia H, et al. (2016). The life history of pyrroloquinoline quinone (PQQ): a versatile molecule with novel impacts on living systems. Int J Mol Biol Open Access, 1(1), 29-46.
- Choi O, Kim J, Kim JG, Jeong Y, Moon JS, Park CS, Hwang I. (2008). Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16. Plant Physiol, 146(2), 657-68.
- Zhu W, Klinman JP. (2020). Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone. Curr Opin Chem Biol. (59), 93-103.
- Yao-Qing Shen et al. (2012). Distribution and Properties of the Genes Encoding the Biosynthesis of the Bacterial Cofactor, Pyrroloquinoline Quinone. 51(11), 2265–2275.