Difference between revisions of "Part:BBa K2593004"

Latest revision as of 07:28, 29 September 2024

LHyal gene

Biology

LHyal gene encodes LHAase (Mw=58kD), which is a hyaluronidases from the hyaluronate 3-glycanohydrolases sub-family of leech origin. Hyaluronidase (HAase) is a large family of glycoside hydrolases that predominantly degrades hyaluronic acid (HA), which is a polysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization. Based on substrate specificity and hydrolysis products, HAases are commonly grouped into three families: first group is hyaluronate lyases (EC 4.2.2.1, Streptococcus hyaluronate lyase), it’s commonly use to produce LHAase but has some potential risk on product. Second group is hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH) Commercial BTH has been widely used in clinical medicine, and its hydrolysis mechanism has been studied extensively. The disadvantages of the enzymatic production of specific or narrow-spectrum HA oligosaccharides by BTH include the limited source material (bovine testes), its considerably high price and the broad range of degradation products. Third group is hyaluronate 3-glycanohydrolases(EC 3.2.1.36, Leech HAase).Compared with BTH and Streptococcus hyaluronate lyase, leech HAase has higher substrate specificity and a narrow-spectrum of enzymatic products16,17. In particular, leech HAase is unable to degrade chondroitin or chondroitin sulfate compared to other HAase sources due to its strong substrate specificity. Although mammalian HAase has been widely used as a drug diffusion agent, such HAase activity is susceptible to heparin inhibition. Leech HAase activity, in theory, is not affected by heparin, and has more medical value in clinic and other medical aspects. In addition, the use of recombinant leech HAase does not pose any risk of animal cross-infection. Therefore, high-level production of recombinant leech HAase would be of great significance for both clinical medical treatment (such as surgery, ophthalmology and internal medicine) and producing narrow-spectrum HA oligosaccharides at the industrial scale.

Usage

In our project, this device worked to produce hyaluronic acid hydrolase (hyaluronidase) which is derived from leech genome(Fig 1). The SDS-PAGE method was used to prove the expression of the hydrolases, A clear band was shown with a molecular weight approximately 58 kDa (Figure 2), which is consistent with the value published in previous research.

Fig 1 . the LHyal gene was validated by using PCR primer pMA-LHyal-F and pMA-LHyal-R,the expected size is 520 bp

Figure 2: 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 .

Sequence and Features

Contribution By Team WFL-HangzhouBay

Group: WFL-HangzhouBay iGEM 2024

Summary

New Improved Part: BBa_K5070001 (pPICZαA-LHyal-WT)

Old Existing Part by Team iGEM18_SSTi_SZGD: BBa_K2593004 (LHyal-WT)

Based on BBa_K2593004 (LHyal-WT), we constructed a new plasmid, pPICZαA-LHyal-WT [BBa_K5070001], utilizing the pPICZαA vector and incorporating codon optimization techniques. The primary aim of codon optimization is to enhance the expression efficiency of HAase.

To optimize the expression of hyaluronidase derived from leeches in Pichia pastoris, we improved the codon usage based on the preferences of Pichia pastoris. This involved constructing an optimized expression sequence, denoted as OP, derived from BBa_K2593004 to enhance hyaluronidase production efficiency. The optimisation process included analyzing the codon usage frequency of the original component BBa_K2593004 and comparing it with the codon preferences of Pichia pastoris and using the codon optimization tool (https://www.genscript.com/tools/rare-codon-analysis). We replaced non-optimal codons in the original component with those preferred by Pichia pastoris. Subsequently, we designed and synthesized the optimized gene sequence.

Characterizing our engineered yeast includes verifying the presence and activity of the recombinant hyaluronidase. We utilized PCR to confirm the integration of the hyaluronidase gene into the yeast genome. Enzyme expression was validated through SDS-PAGE analysis, and its activity was measured using the DNS (Dimethylmethylene Blue) assay, a colorimetric method that quantifies the degradation of hyaluronic acid. We used the DNS assay to measure hyaluronidase production, directly indicating enzyme activity by reducing a blue-colored substrate. The absorbance at 530 nm is proportional to the concentration of hyaluronidase, enabling us to quantify the enzyme's activity in our yeast cell factory.

Figure 1: Technology Map

Construction Design and Engineering Principle

Usage and Biology

Hyaluronidase is an enzyme that degrades hyaluronic acid, a vital component of the extracellular matrix in connective tissues. It has therapeutic uses in reducing inflammation and enhancing the absorption of topically applied drugs. Pichia pastoris, a methylotrophic yeast, serves as an excellent host for heterologous protein production due to its robust metabolism and ease of genetic manipulation. Hyaluronidases that break down hyaluronan are widely used to prepare low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties.

Figure 2: Gene map

pPICZαA is a plasmid that is only expressed in Pichia pastoris. Although it can be inherited in Escherichia coli (E. coli), it cannot be expressed there. Moreover, pPICZαA is a high-copied plasmid that can form many copies per yeast. When LHyal is inserted into this plasmid, the highly copied plasmid leads to a high HAase yield. pPICZαA-LHyal-WT optimizes the sequence based on LHyal-WT, making it more efficient and high-yield for the production of HAase.

Figure 3: Plasmid map of pPICZαA-LHyal-WT

Characterization/Measurement

a. Real-time fluorescence quantitative PCR to detect gene copy number

A fluorescently labelled probe binds specifically to the target DNA sequence in qPCR. As the PCR reaction proceeds, the target DNA sequence is amplified, and the number of fluorescent molecules bound to the probe increases. This results in the generation of fluorescent signals proportional to the amount of DNA, and the amplification curve is shown in Fig. 4A.

By monitoring the changes in these fluorescent signals in real-time, the starting copy number of the target DNA sequence was calculated. In addition, the expression level of the target gene was normalized by comparing the Ct values of the internal reference gene ARG4 with that of the target gene LHyal. The experimental data were subjected to a t-test to compare the expression changes of the LHyal gene between the wild-type strain and the modified strain. As shown in Fig 4B, the results indicated a significant difference in gene expression between LHyal-WT and LHyal-WT(op), with P=0.0016<0.05. Further analysis of the gene copy number between the two strains indicated that the codon-optimized strain had elevated expression of the LHyal gene, providing an experimental basis for the subsequent study of high hyaluronidase expression.

Figure 4: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments, P=0.0016<0.05.

b. Determination of recombinant yeast hyaluronidase activity by DNS method

The DNS (dinitrosalicylic acid) method is a colorimetric method for determining the reducing sugar content. Under alkaline conditions, 3,5-Dinitrosalicylic acid (DNS) undergoes a redox reaction with reducing sugar to produce 3-amino-5-nitrosalicylic acid, which is brownish-red in color under boiling conditions. The depth of color is proportional to the amount of reducing sugar within a specific concentration range. Since hyaluronidase can produce glucose by decomposing hyaluronan, we characterized hyaluronidase activity by the amount of glucose produced per unit of time.

Before testing the samples' glucose content, a glucose standard solution of 2 mg/mL was prepared, and the glucose standard curve was prepared. The results are shown in Figure 5.

Figure 5: Glucose standard curve preparation

Experimentally, taking a milliard of Pichia pastoris yeast fermentation broth, the DNS method was used to detect glucose content and calculate hyaluronidase activity based on glucose production over time. As shown in Figure 6A, glucose content increased with the growth of the culture, and enzyme activity increased as well. The glucose content was measured at 540 nm, and the absorbance value (A540) for each sample was compared against the glucose standard curve to calculate enzyme activity. As shown in Figure 6B, the optimized strain, pPICZɑA-LHyal-WT(op), had higher glucose content and enzyme activity compared to the wild-type strain, pPICZɑA-LHyal-WT.

Figure 6: Determination of recombinant yeast hyaluronidase activity by DNS method; A: Glucose content over time; B: Enzyme activity.

Conclusion

Our team, WFL-HangzhouBay, successfully constructed and optimized the hyaluronidase expression plasmid, pPICZαA-LHyal-WT [BBa_K5070001], based on the existing part BBa_K2593004 (LHyal-WT) from iGEM18_SSTi_SZGD. By codon optimization tailored to the expression preferences of Pichia pastoris, we significantly enhanced the expression of hyaluronidase. The improved gene expression was confirmed through real-time fluorescence quantitative PCR, and the increased enzyme activity was demonstrated using the DNS method. These results indicate that codon optimization was highly effective, leading to higher hyaluronidase yields and activity. The optimized strain can serve as a valuable tool for future applications in pharmaceutical and cosmetic industries, where hyaluronidase is widely used.

References

[1] Cui, Y., et al. (2015). High-level expression of human hyaluronidase in Pichia pastoris and its potential application in tumour therapy. Applied Microbiology and Biotechnology, 99(21), 8817-8827.

[2] Liu, C., et al. (2017). Enhanced production of recombinant human hyaluronidase in Pichia pastoris by optimizing codon usage and fermentation conditions. Biotechnology Letters, 39(8), 1233-1239.

[3] Wang, J., et al. (2018). A novel method for quantitative detection of hyaluronidase activity using a DNS assay. Biotechnology Progress, 34(2), 434-440.

[4] Zhang, J., et al. (2019). Metabolic engineering of Pichia pastoris for the production of biopharmaceuticals. Microbial Cell Factories, 18(1), 1-12.

[5] Chen, X., et al. (2020). Synthetic biology approaches to enhance the production of heterologous proteins in yeast. Biotechnology Journal, 15(5), 1-15.

Reference

Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.25-27.