We often gain some wounds accidentally and aren’t aware of them, but it is not a big problem because wounds will heal soon. Nonetheless, diabetic wounds are often difficult to heal because of excessive blood sugar concentration in the blood. In the previous study, we found Pyrroloquinoline quinone(PQQ),a cofactor that can promote plant growth. Moreover, from other research it is found that PQQ has antioxidant effects on mammalian cells, which may have an effect to promote wound healing. However, Current methods to produce PQQ is using chemical synthesis, but the production methods are expensive and have low yields. So we decide to use biosynthesis methods to produce PQQ in a large quantity. We use Bacillus subtilis natto to produce PQQ.
There are three main reasons why we use B. subtilis natto. First, B. subtilis natto is generally recognized as safe (GRAS) probiotic, which means harmless to human beings, and it has been widely used in health supplements.
Second, it is normal industrial bacteria that are commonly used as a production platform. We can secrete the product from B. subtilis natto.
Third, natto extraction can promote wound healing, and gamma-PGA has the eminent moisturizing ability. The all-natural product even has the distinguishing feature of being non-toxic.
Upon above, we design a dressing containing PQQ and natto extraction,so that the wound repairing material can be passed to destination directly.

The First Goal:

produce PQQ in large quantities at a low cost

We transform PQQ plasmid into B. subtilis natto so that B. subtilis natto can produce PQQ.

We extract the plasmid in Bacillus subtilis from 2021 NCHU-Taichung, and we do electroporation to transform PQQ plasmid into natto.

The process of pqq biosynthesis contant five gene, pqqA, pqqB, pqqC, pqqD, and pqqE. pqqA is a precursor, a short peptide with 26 amino acids that contains two fully conserved residues, glutamate, and tyrosine. First, pqqA, pqqD, and pqqE will form a pqqA/D/E complex. In the presence of PqqD, PqqE helps the Glu-Tyr cross-linking within PqqA polypeptides to be generated. It has been expressed in E. coli and found that PQQ can be produced. 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 are still many questions worth discussing PQQ biogenesis.

Among the biosynthesis of PQQ, PqqA and PqqB dominate the forming of PQQ. In view of this, we separated those two genes from the origin reading frame and developed a new operon to overexpress them. To regulate the transcription level of pqqC, D, E, we chose a new plasmid with xylose operon to regulate the expression of the gene of pqqC, D, E.
We created another plasmid optimized from BBa_K3830012, BBa_K4358011, with Pxyl, xylR and xylR-binding sites. The pqqA and pqqB are crucial for PQQ synthesis.
On the flip side, we designed the Muiti-pqqA plasmid, which inserted a pqqA gene into the old plasmid(ref3). Based on the document that 2021 NCHU_Taichung provided, it has been known that PqqA is the precursor of PQQ. To understand the effect of PqqA, in 2022, we, NCHU_Taichung, plan to amplify PQQ production by doubling pqqA in our new plasmid BBa_4358013.
Different from BBa_K3830012, BBa_4358013 had more potential to synthesize PqqA. And BBa_4358013 may improve the efficiency of PQQ production, and make the PQQ produced by modified bacteria much more likely to be applied to commercial use.

We use supramolecular to extract and purify PQQ. It is a green chemical method because we can reuse the supras. We add alkane, alkanol, and surfactant together as the supras. We put the supras into a solution containing PQQ, purified broth. And through back extraction, we can get PQQ in the lower layer. Uppe layer is supras, and we can reuse it.


We use EP2(transformed B. subtilis natto) supernatant to be our extraction and add it to wound gel. Hence, we can easily apply it to the diabetic chronic wounds


dressing application on diabetic wound

Induce normal Hacat cells to diabetes-induced Hacat cells by glucose and insulin.

  • Making the gap of the same size in Normal Hacat cell and Diabetes induced Hacat cell to simulate wound.
  • treating them with natto, PQQ, and their mixture respectively, and compared their healing situation with untreated Hacat cells.

We can realize that PQQ would improve the ability of wound healing.


  1. Potter H. (2003). Transfection by electroporation.Current protocols in molecular biology, Chapter 9, Unit–9.3. https://doi.org/10.1002/0471142727.mb0903s62
  2. Zhu, W., & Klinman, J. P. (2020). Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone. Current opinion in chemical biology, 59, 93–103. "https://doi.org/10.1016/j.cbpa.2020.05.001
  3. Ma, K., Wu, Z. Z., Wang, G. L., & Yang, X. P. (2021). Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction. Food chemistry, 361, 130067. https://doi.org/10.1016/j.foodchem.2021.130067