Team:SSTi-SZGD/Demonstrate

From the integrated human practices , we understand that there is a need for safer and more effective methods for anti-aging and wrinkle removal purpose. Therefore, the way to meet these requirements is the goal of our project. We started our project by biosynthesis of a substance, hyaluronic acid (HA), which is commonly used in preparing dermal fillers in plastic surgery via synthetic biology engineering, then we developed a safe and effective anti-wrinkle product--HA microneedle. Our methods for HA biosynthesis is via using a food grade safe strain, B.subtilis , which has indigenous synthetic pathway for the biosynthesis of HA precursors. By cloning a HA synthase gene from Streptococcus zooepidermicus , we obtained an engineered B. subtilis strain that is able to biosynthesize HA. We managed to increase HA production level by regulating some of the precursor genes in the upstream synthetic pathway. Furthermore, we cloned a leech HA hydrolytic gene into the engineered B.subtilis strain so that low molecular weight HA can be produced for the purpose of microneedle fabrication. By covalently cross-linking HA molecules, a stabilized hydrogel is obtained that serves the starting material for microneedle production.

What we have achieved?

In summary, we demonstrate that in this project we achieved the following results

  • We successfully constructed a recombinant B.subtilis 168E strain that secretes high-molecular-weight HA. In addition, by overexpressing some of the precursors gene in the HA synthesis pathway, HA production level was elevated.
  • We further obtained a recombinant B.subtilis 168 strain that directly secretes low-molecular-weight HA in large quantity by cloning leech HA hydrolase LHAyal gene.
  • Using our purified and freeze-dried HA powder, we successfully fabricated HA microneedles that is an innovative design in dermal filler development.

① High-molecular weight HA
② Leech hyaluronidase
③ Low-molecular-weight HA
④ HA microneedles

We successfully constructed a recombinant B.subtilis 168E strain, containing a HA synthase ( hasA ) gene, to produce HMW-HA, and overexpressed some of the precursor genes ( tuaD, gtaB, glmU ) obtained from the HA synthesis pathway. CTAB methods verified that overexpression of the precursor genes further helped increasing HA production. Molecular weight analysis showed that the increase of HA yield did not affect the molecular weight. We also purified and freeze-dried our HA product!

Effects of hasA and precursors genes on HA production

Molecular weight analysis of HA products

Our purified and freeze-dried HA powder

We successfully expressed recombinant leech hyaluronidase ( LHAyal ) in B. subtilis 168. Enzyme activity of LHAase was visualized using a HA agarose plate assay and verified by DNS reducing sugar assay. With its confirmed hydrolase activity, we were one step closer to in vivo hydrolysis of high molecular weight HA.

LHAase enzyme activity analysis (DNS reducing sugar)

LHAase enzyme activity vs. Bacterial growth

LHAase enzyme activity visualized by HA agarose plate

We cloned leech LHAyal gene into the recombinant B.subtilis 168E strain and successfully made it hydrolyzing HMW-HA to become LMW-HA. Reduction in the molecular weight of HA was confirmed by gel chromatography (GPC-RI-MALS). CTAB results showed an increasing LMW-HA concentrations, reflecting a decrease of the viscosity of the fermentation liquid. Therefore, we successfully produced LMW-HA in B.subtilis !

Molecular weight analysis of hydrolyzing HA

Analysis of HMW-HA and LMW-HA concentrations

We covalently cross-linked HA molecules (cHA) to make hydrogel powder, and designed a specially crafted micromold. By decoding the optimal weight ratio of HA-cHA (5:1) to make a hydrogel mixture, we successfully fabricated a HA microneedle patch with good mechanical strength for skin epidermal penetration.

The cross-linked HA hydrogel powder

Demoulding of HA microneedle patch after overnight curing

Display of our HA microneedles!

Summary

To conclude, we successfully obtained three HA products with commercial values via synthetic engineering approach, and managed to obtain a miniature of the innovated cosmetic product--HA microneedles--that has a great potential in future beauty industry!

Copyright © 2018 SSTi-SZGD All Rights Reserved.
Copyright © 2018 SSTi-SZGD All Rights Reserved.