Team:SSTi-SZGD/Results
Module 1 - HA Production
Construction of HA production pathway in B. subtilis 168
Our project is to use food-grade B. subtilis 168 as a host, which is prokaryotic expression system with unique advantages (i.e. high-density growth, endotoxin free supernatant, secretory expression for easy protein purification, etc.). By cloning hasA gene, derived from S. Zooepidemicus, into B. subtilis 168, containing two precursor synthesis pathways (different from zooviral streptococcus), HA synthase can be expressed heterogeneously to regulate the biosynthesis of HA polymers that express extracellularly. hasA coding sequence was commercially synthesized and subcloned into the integration vector pAX01 at the restriction sites SacII and BamHI (Figure.1). The integration of hasA gene into B.subtilis genome to generate a 168E strain was confirmed by colony PCR polymerization.
①pAX01-HasA plasmid
Figure 1: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of hasA gene in pAX01-hasA using primer pair HasA-F and HasA-R, and the expected product size is 479 bp. Right: illustration of construction of the expression vector pAX01-HasA. HasA was inserted at the restriction site HindIII and kpnI of pAX01 plasmid.
②pP43NMK-gtaB-tuaD and pP43NMK-glmU plasmids
To further increase HA production in recombinant B.subtilis 168E, we constructed two expression vectors containing operon tuaD-gtaB and a coding sequence of glmU, respectively (Figure.2 ,3). Products of these genes regulate the biosynthesis of two HA precursors in native B. subtilis 168. Cloning of operon tuaD-gtaB and glmU coding sequence into B.subtilis 168E were confirmed by colony PCR polymerization respectively.
Figure 2: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GtaB-TuaD-F and GtaB-TuaD-R and the expected product size is 438 bp. Right: illustration of construction of the expression vector pP43NMK-gtaB-tuaD. Operon gtaB-tuaD was inserted at the restriction sites HindIII and KpnI of pP43NMK plasmid.
Figure 3: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GlmU-F and GlmU-R and the expected product size is 304 bp. Right: illustration of construction of the expression vector pP43NMK-glmU. GlmU coding sequence was inserted at restriction sites HindIII and BamHI of pP43NMK plasmid.
Fed-batch fermentation of recombinant hasA and HA production
A fed-batch fermentation strategy was employed to ensure an optimal HA production over a time course. We performed a 48 h high-cell-density fermentation experiment using MM medium with xylose as an inducer. Sucrose was fed to the batch at designated time points. HA concentrations and cell growth densities were measured every 12 h.
Figure 4: analysis of HA production level of recombinant hasA and cell growth. A typical time course of recombinant hasA expression in B. subitlis 168E during xylose induction phase. HA production (red, mg/L), and cell growth density (black, OD600 value) were measured at regular intervals.
The results showed that HA production increased at a steady rate as time passed, reaching over 300mg/L (CTAB method) at the 50 h; while bacterial cells maintained normal growth pattern and experienced lag phase dropping in the final hours. These results confirmed the success of HA production by recombinant B.subtilis 168E strain.
Optimizing HA production Level by Regulating Precursor Genes
We would like to take HA production to the next level by co-overexpressing some key native genes that play a role in biosynthesis of the two HA precursors, UDP-GlcUA and UDP-GlcNAc. There are a handful genes available (Figure 5) however due to time limits, we selected three of them, tuaD, gtaB, and glmU to analyze (Figure 6). tuaD and gtaB gene products regulate the last two steps in the synthetic pathway of UDP-GlcUA, while gene product of glmU regulates the final two steps in the synthetic pathway of UDP-G lcNAc. By conducting CTAB experiments that form turbidity from a reaction between HA and CTAB solution, the results showed a remarkable increase in HA production when co-overexpressed tuaD-gtaB together (488mg/L, a 38% increase) (Figure7b), suggesting that UDP-glucose dehydrogenase (encoded by tuaD) and UDP-glucose pyrophosphorylase (encoded by gtaB), are two key enzymes in the biosynthesis of UDP-GlcUA and HA in B. subtilis. Similar results have been reported in recombinant L. lactis (1). On the other hand, the overexpression of glmU, which encodes the bifunctional enzyme that catalyzes glucosamine-1-phosphate to UDP-N-acetyl gluco-samine, also gave rise to a greater amount of HA (448mg/L, a 32% increase) (Figure 7b), which is consistent with previous reports (2). Together these results suggested that optimization of precursor genes has positive effects on HA production. Co-overexpression of tuaD and gtaB genes seemed to have a slightly better effect on HA production compared to that of glmU alone.
In addition, Ubblelohde viscometer method indicated that HA products secreted from these recombinant expression systems were with comparable molecular weights, suggesting that they are all high-molecular-weight HA products (~4.233*106Da) (Figure 8).
Figure 5: Biosynthetic pathway of HA in B.subtilis 168
Figure 6: organization of the constructed HA artificial operons in pP43NMK vector
Figure 7: CTAB analysis of HA concentraton, a. Illustration of the turbidity by mixing different source of HA with CTAB solution. b: effects of overexpressing the precursor genes on HA production in recombinant B.
Figure 8: Molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.
Conclusion: we successfully constructed a recombinant B. subtilis 168E strain secreting high molecular weight HA. By overexpressing three of the precursor genes in B. subtilis, HA production level increased by 38%, indicating augmentation in the biosynthesis of the two precursors, UDP-GlcUA and UDP-GlcNAc, is vital to achieve high level of HA production. In the future we will investigate the effects of co-overexpression of other precursor genes on HA production in B. subtilis168E.
Module 2 - LHAase Secretion
Construction of secretory expression of LHAase in B. subtilis 168
In order to construct a recombinant B. subtilis 168 strain that is able to secrete low-molecular-weight HA, a leech HA hydrolyzing gene (LHAyal) is cloned into the wildtype B. subtilis 168 to express hyaluronidase (HAase). An a-amylose signal peptide (AmyX) was added to the N-terminus of the LHAyal coding sequence to construct an extracellular secretory system. Furthermore, 6xHis tag has been extensively used in recombinant protein expression to facilitate protein purification process. Compare with other lengths of his tags, 6xHis tag provides sufficient space that ensures the proper folding of mature protein. We added a 6xHis tag between AmyX and LHAyal coding sequence. Two plasmid vectors, pDG1730 and pMA0911, were used to compare protein expression efficiency. Colony PCR polymerization confirmed the construction of the recombinant B. subtilis strains.
①pDG1730-AmyX-H6LHyal plasmid
Figure 9: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of LHAyal gene in pDG1730-AmyX-H6LHAyal using primer pair LHAase-F and LHAase-R and the expected product size is 420 bp. Right: illustration of construction of the expression vector pDG1730-AmyX-H6LHAyal. LHAyal coding sequence was inserted at the restriction site HindIII and BamHI of pDG1730 plasmid.
②pMA0911-AmyX-H6LHyal plasmid
Figure 10: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of LHAyal gene in pMA0911-AmyX-H6LHAyal using primer pair pMA0911-LHyal-F and pMA0911-LHyal-R, and the expected product size is 512 bp. Right: illustration of construction of the expression vector pMA0911-AmyX-H6LHAyal. LHAyal coding sequence was inserted at the restriction site NdeI and BamHI of pMA0911 plasmid.
LHAase functional and expression level analysis
①Growth rate vs. LHAase expression level
Similar to HA production, a fed-batch fermentation strategy was also employed to ensure maximal expression of LHAase over a time course. We performed a 60 h high-cell-density fermentation experiment with feeding of fresh culture media every 12 h. LHAase production and cell growth density were measured at regular interval. As expected, fusion of the signal peptide and 6xHis tag resulted to a distinct accumulation of extracellular LHAase in the flask culture. (Figure 11) showed that LHAase expression increased exponentially as time passed, enzymatic activity up to 1.149*105 U/ml at the final 60 h (in pDG1730 vector); on the other hand, overexpression of LHAyal did not seem to affect normal bacterial growth (Figure 11). Therefore N-terminal engineering enables an extracellular secretion of hyaluronidase. A visual HA plate assay was also conducted to verify and confirm the activity of LHAase. HA hydrolysis by the crude culture supernatant resulted to the appearance of a circle zone around the hole, indicating that leech LHAase has distinct hyaluronidase function and can be successfully overexpressed in B. Subtilis (Figure 12).
Figure 11: analysis of enzyme activity of recombinant LHAase and cell growth. A typical time course of recombinant LHAase expression in B. subitlis 168. LHAase enzyme activity (red, U/ml), and cell growth density (black, OD600 value) were measured at regular intervals.
Figure12: Determination of LHAase activity using a HA plate assay, where cylindrical holes were injected with fermenting supernatant of LHAase-expression (pDG1730, pMA0911) or control (empty vector) B. subtilis 168. A positive control denoted to commercial BTH, while a negative control denoted to water. The white circles in pDG1730 and pMA0911 showing the color residues from fermenting culture.
②LHAase expression at transcriptional level
In addition, we extracted total RNA at each indicated time point to understand the expression pattern at transcriptional level. By reverse transcribing mRNA to cDNA and performed RT-PCR using LHAyal primers, the results showed that mRNA levels accumulated up to 48 h and seemed to drop at 60 h (Figure 13). Given Figure 11 showing that LHAase production continued to rise up to 60 h, it is possible to assume that LHAase production may cease to increase after 60 h, due to a drop in mRNA transcripts after 48 h of bacterial growth. In the future we will explore this further.
Figure13: Agarose gel electrophoresis of mRNA transcript obtained at various time points. (1-7): 6h, 12h, 24h, 36h, 48h, 60h, NTC. Top strand showing RT-PCR products amplifying a section of LHAyal coding sequence, bottom strand showing RT-PCR products amplifying a housekeeping gene sequence (16s rRNA).
③LHAase purification and analysis
LHAase was then purified from crude supernatant sample using a Ni-NTA affinity column and analyzed by SDS-PAGE. A clear band was shown with a molecular weight approximately 58 kDa (Figure 14), which is consistent with the value published in previous research (3).
Figure 14: SDS-PAGE analysis of LHAase in total extracellular crude protein fraction from recombinant B.subtilis harboring pMA0911-AmyX-H6LHAyal. A 12% SDS-PAGE gel was used. Lane 1: purified LHAase by Ni-NAT affinity column; Lane 2: the crude extracellular protein fraction
Enzymatic characterization
①DNS reducing sugar assay and ELISA
Compare protein expression levels in vectors pDG1730 and pMA0911, results from DNA reducing sugar assay showed a comparable enzymatic activity for both vectors (1.126x*105 U/ml for pDG1730 vs. 1.149*105 U/ml for pMA0911) (Figure 15).
Figure 15: enzymatic activity of recombinant LHAase expressed from B.subtilis strains harboring pDG1730-AmyX-H6LHAyal or pMA0911-AmyX-H6LHAyal, analyzing by DNS reducing sugar assay.
②ELISA analysis
We further analyzed LHAase enzyme activity using ELISA assay. By interacting with a HAase antibody, the ELISA results confirmed that LHAase has hyaluronidase property (Figure 16). It is interesting to note that LHAyal subcloned in pMA0911 vector seemed to have a better LHAase activity than that of in pDG1730 vector, which is an integration vector. This may suggest that integration of LHAyal expression cassette into the genome of B. subtilis 168 somehow inhibits LHAyal overexpression.
Figure 16: enzymatic activity of recombinant LHAase expressed from B.subtilis strains harboring pDG1730-AmyX-H6LHAyal or pMA0911-AmyX-H6LHAyal, analyzing by ELISA.
LMW-HA biosynthesis in recombinant B. subtilis 168E
By transforming pDG1730-LHAyal vector into recombinant B. subtilis 168E, we devoted to achieve a cell factory that is able to directly produce low molecular weight HA. Although recombinant LHAyal in pMA0911 vector seemed to have more prominent enzymatic activity, we still decided to use pDG1730-LHAyal vector to construct a recombinant strain based on two reasons: 1) recombinant LHAyal in pDG1730 had a comparable enzymatic activity compare to that in pMA0911, 2) pDG1730 is an integration vector that help inserting LHAyal gene into the genome of B.subtilis to avoid plasmid loss. It also provides convenience for further introduction of other plasmid into the recombinant strain.
HA secreted in culture supernatant was separated and purified, gel chromatography (GPC-RI-MALS) was performed to evaluate the molecular weight of HA molecules(4). The results showed that the average molecular weight of HA decreased significantly from HA produced in 168E strain to that of 168E+LHAyal strain (Mw from ~6 x 106 to ~5x 104) (Figure 17), suggesting that LHAase expressed extracellularly was able to hydrolyze HA polymers in culture environment. We also analyzed HA concentration by CTAB methods. Surprisingly, the resulted showed a significant increase in HA production in 168E+LHAyal strain (780mg/L), compared to that of 168E strain (303mg/L) (Figure 18). This 260% increase indicated a new approach to overcome the bottleneck in HA production via microbial fermentation. It is likely that reduced molecular weight results to a reduction in cultural viscosity, which in turn increases dissolved oxygen level that provides an improved condition for bacterial growth.
Figure 17: GPC-RI-MALS analysis of average molecular weight of HA secreted from 168E and 168E+LHAyal strains.
Figure 18: CTAB concentration analysis of HA secreted from 168E or 168E+LHAyal strains.
Conclusion
At present, BTH dominates commercial HAase market as the expression level of other recombinant HAases are too low to meet practical needs. By employing N-terminal engineering and fed-batch fermentation, considerable amount of LHAase was obtained, with a crude titre of 1.149*105 U/ml, a value that is higher than that of commercial BTH (1.05*103 U/mg, 20-25% pure (5) obtained from bovine testicle extraction. The hydrolysis property of LHAase was evaluated and confirmed in a HA-secreting recombinant B.subtilis 168E strain, where low molecular weight HA (around 5 x 104 Da) was produced at a relatively higher concentration compared to that of the HMW-HA.
Module 3 - Micro-needles production
Crosslinked HA hydrogel particle production
Figure 19: The crosslinked HA hydrogel was freeze-dried, grounded, squeezed out and screened using a 170 mesh sieve to obtain particles with diameters less than 90 μm.
Fabrication of HA-cHA Micro-needles
In order to successfully fabricate a HA microneedle, we tested different weight ratio of the HA molecules and cHA. The experimental results showed that the ratio of HA-cHA (0:1) forms a lump after overnight curing and is easily broken into powder. The ratio of HA-cHA (1:1) is difficult to be demolded after overnight curing, and could not form a complete needle shape, while being poor in mechanical properties and spreadability. The ratio of HA-cHA (5:1) forms complete microneedles after overnight curing, and the patch is easily to be demolded, with high mechanical strength and spreadability (Figure 20). Therefore, the ratio of HA-cHA (5:1) is the most suitable ratio for preparing microneedles that have appear to be strong and sharp enough to penetrate into the skin. It took less than 48 h to complete a microneedle patch and the required materials are minimal. Microneedle made from micromold-based fabrication technology is suitable for mass production due to high production efficiency, and repeat-using of the mold.
Figure 20: HA microneedle fabrication. (a) micromold is filled with HA-cHA homogenous suspension; (b) demolded after curing overnight; (c-e) display of needles made of different weight ratios of HA and cHA.
Water Solubility
We performed water solubility tests using microneedles made from HA-cHA (5:1). The results showed that HA-microneedles had high water solubility and can be completely dissolved in aqueous solution within 5 min (Figure 21).
Figure 21: Illustration of water solubility tests on HA microneedles made from HA-cHA (5:1) ratio. Microneedles dissolve quickly once in water at room temperature (left), and completely disappeared after 5 min (right).
Discussion
Microneedle experiments demonstrated that we successfully fabricated HA microneedles using a mixture of cHA and HA molecules. This particle microneedle product has high mechanical strength and spreadability, and a stabilized needle structure did not impair its water soluble property, as it can rapidly dissolve in aqueous solutions. We came a long way searching for the most suitalbe ratio for fabricating HA microneedles(6). At this stage, ratio of 5:1 (HA-cHA) seems to give the best outcome. However, after curing overnight, the surface of microneedle patch gets dry so quickly, possibly due to a quick evaporation, that sometimes causing the surface to crack. Our next step is to test other ratios (1:3:, 3:1, 1:4, 4:1, etc) in order to search for more appropriate weight ration for microneedle fabrication.
Due to time limits, we are unable to conduct all experiments that validate the function of HA microneedles. In the future we plan to conduct the following experiments, 1) test the in vitro degradation rate of microneedles by using hyluronidase, 2) use skin models (i.e. pig skin) to test the mechanical strength and penetration efficiency of the microneedles, and 3) test the penetration/insertion ability and in vivo
Reference
[1] Prasad et al., 2010 S.B. Prasad, G. Jayaraman, K.B. RamachandranHyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactisAppl. Microbiol. Biotechnol., 86 (2010), pp. 273-283
[2]Chen,W.Y.,Marcellin,E.,Hung,J.,Nielsen,L.K.,2009.Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus. J.Biol.Chem.284,18007–18014.
[3] Jin,P.,Kang,Z.,Zhang,N.,Du,G.C.,Chen,J.,2014.High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers.Sci.Rep. 4,4471.
[4] Tranchepain, F. Deschrevel, B.et al. (2006). A complete set of hyaluronan fragments obtainedfrom hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices. Analytical Biochemistry, [J]348,232–242.
[5] El-Safory, N. S., Fazary, A. E. & Lee, C. K. Hyaluronidases, a group of glycosidases:Current and future perspectives. Carbohyd. Polym. 81, 165–181 (2010).
[6] Jeong YI, Kim ST, Jin SG,et al., Cisplatin incorporated hyaluronic acid nanoparticles based on ion complex formation, Journal of pharmaceutical sciences, 2008;97(3):1268-76.
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