Engineering
Engineering Success
Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anions (O2-), and hydroxyl radicals (OH), are toxic byproducts of oxygen metabolism and major contributors to skin aging. The skin has a natural defense system to neutralize ROS and antioxidants. However, with aging and increased UV exposure, the effectiveness of this defense diminishes, leading to an accumulation of ROS and subsequent cellular damage. Antioxidants can mitigate this damage by donating electrons to highly reactive ROS, stabilizing them and reducing their harmful activity without producing toxic byproducts (see Fig. 1)[1].
It has been reported that NAOS is an effective antioxidant capable of inhibiting excess nitric oxide (NO) production and removing O2⁻ and OH. Our project focuses on using synthetic biology to produce β-agarase (AgaA), which hydrolyzes agarose to generate NAOS, thereby providing an efficient means to counteract oxidative stress[2-4].
Design
We designed a prokaryotic expression system to express the AgaA (BBa_K5216000) protein (Fig.2A). In order to improve the prokaryotic expression efficiency, we optimized the codon usage of the native AgaA gene sequence, and the synthesized DNA in a clone plasmid was obtained from GenScript (Nanjing GenScript Biotechnology Co., Ltd.). The prokaryotic expression vector (pET-32a(+) plasmid) , which is previously preserved in the laboratory, was used to construct the expression plasmid AgaA_pET-32a(+). We used specific primers to amplify the AgaA gene from the provided clone plasmid and prepared the pET-32a(+) plasmid using a plasmid extraction kit. The prepared AgaA gene and the pET-32a(+) plasmid were double-digested with BamHI and EcoRI, followed by ligation with T4 DNA ligase, creating the AgaA_pET-32a(+) construct. This construct was then transformed into E. coli Rosetta. The E. coli produced β-agarase was capable of hydrolyze agarose, by specifically cutting the β-1,4-glucoside bond between D-galactose and 3, 6-dehydro-α-l-galactose residues in agarose molecules to generate new agarose oligosaccharides NAOS.
Build
1. Plasmid Construction result
We performed PCR to amplify the AgaA gene fragment and analyzed using agarose gel electrophoresis. Fig. 3 illustrates the agarose gel preparation and sample loading process. The DNA marker confirms the successful amplification of a 1596 bp AgaA gene fragment (Fig. 4A). This fragment was then recovered using a gel extraction kit. Both the recovered AgaA fragment as well as the pET-32a(+) plasmid were subjected to double digestion. Fig. 4B and Fig. 4C show that the digestion of both the plasmid and the target fragment was successful.
(A) PCR amplification of the AgaA gene fragment. M: DNA marker; 1-3: AgaA PCR products. (B) Enzymatic digestion of the AgaA PCR product. M: DNA marker; 1: Negative control (gel-purified AgaA PCR product); 2-3: Digestion product of the gel-purified AgaA PCR product. (C) Enzymatic digestion of the pET-32a (+) vector. M: DNA marker; 1-3: Digestion product of the pET-32a(+) plasmid; 4: Negative control (undigested pET-32a(+) plasmid).
The digested products were recovered and then ligated using T4 DNA ligase, and the ligation products were transformed into competent E. coli TOP10 cells. Subsequent colony PCR validated the successful construction and integration of the plasmid into the bacterial strain. The AgaA_pET-32a(+) plasmid was extracted from E. coli TOP10 for sequencing. Sequence alignment with the AgaA gene confirmed the absence of mutations. The plasmid is now ready for subsequent experiments.
Test
1. Expression and purification of AgaA result
We expanded the culture of E. coli Rosetta AgaA_pET-32a(+) and induced expression at 16℃ for 18 hours with the addition of IPTG (final concentration 0.5 mM). SDS-PAGE analysis confirmed successful expression of the target protein, which was predominantly soluble and suitable for further experiments. The crude lysate concentration was measured using the Bradford method, yielding a final concentration of 3.09 mg/mL and a total yield of 61.8 mg, which will be used to produce the oligosaccharides NAOS. Additionally, the lysate was further purified using Ni-NTA affinity chromatography. The target protein was isolated by washing with 100 mM imidazole buffer and eluting with 300 mM imidazole buffer.
2. Enzyme activity detection result
As shown in Fig. 8A, colonies of Escherichia coli Rosetta AgaA_pET-32a(+) on LB agar plates displayed depressions around them, indicating effective agar degradation and confirming the activity of the agarase enzyme. To evaluate the enzyme's activity, a colorimetric assay using 3-amino-5-nitrosalicylic acid (DNS) was performed. DNS reacts with reducing sugars under alkaline conditions to form a brownish-red complex, with the color intensity correlating to the concentration of reducing sugars. Since AgaA catalyzes the production of reducing sugars from agarose, this assay is suitable for measuring its activity.
In the assay, 200 µL of AgaA enzyme solution or a heat-inactivated control was mixed with 200 µL of a 2% agarose solution and incubated at 55°C for 30 minutes. The reaction was terminated by heating at 95°C for 10 minutes. The mixture was transferred to a new 2 mL EP tube, and 200 µL of DNS reagent was added. After a 95°C incubation for 5 minutes, color changes between the control and experimental groups were compared. Significant darkening in the experimental group indicated the presence of reducing sugars, specifically NAOS, thus confirming that AgaA has biological activity for agarose degradation.
3. Analysis of products of AgaA hydrolyzed agarose
We next analyzed the hydrolysis products of agarose by the AgaA enzyme using HPLC. The HPLC assay utilized a standard sample, which was a mixture of neoagarobiose (NA2), neoagarotetraose (NA4), and neoagarohexaose (NA6), with respective retention times of 6.75 min, 12.4 min, and 23.5 min. Comparing the chromatograms from the experimental and control groups revealed significant new peaks at 6.75 min and 12.4 min, confirming the production of NA2 and NA4. Previous experiments demonstrated that the hydrolysis of agarose by AgaA yielded products including NA2, NA4, and NA6, with NA6 further hydrolyzing into NA2 and NA4 as the reaction progressed. This finding validates the catalytic efficiency of the AgaA enzyme, confirming its ability to degrade NA6 into NA2 and NA4.
4. Analysis of oxidation resistance of AgaA hydrolysate
Since the AgaA hydrolysate contains reducing sugars, we further evaluated the antioxidant properties of the NAOS using the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation) assay. This method is based on the oxidation of ABTS, which produces a stable blue-green cationic free radical, ABTS+, with maximum absorbance at 734 nm. When antioxidant substances are added to the ABTS+ solution, they react with these radicals, leading to a decrease in color intensity. We used a total antioxidant capacity (T-AOC) test kit based on the ABTS principle to quantify T-AOC. Compared to the control group, the reagent containing AgaA hydrolysis product caused significant decolorization, demonstrating that the NAOS generated by AgaA-mediated agarose hydrolysis exhibits potent antioxidant activity.
Learn
We developed a prokaryotic expression system for β-agarase (AgaA) to hydrolyze agarose into novel antioxidant oligosaccharides NAOS. We successfully constructed an expression plasmid using a codon-optimized AgaA gene and induced AgaA production in E. coli. The expressed AgaA enzyme could effectively degrade agarose into smaller oligosaccharides, particularly NAOS. HPLC analysis confirmed their presence, and ABTS assay further demonstrated their strong antioxidant activity, making NAOS a promising candidate for ROS and mitigating skin aging. This approach provides a method to produce valuable antioxidants from agarose. In future work, we aim to enhance the stability and prolong the antioxidant effects of NAOS by exploring encapsulation technologies for broader applications.
References
[1] Wang, Y., Tian, L., & Yu, J. (2022). The role of reactive oxygen species in skin aging: Molecular mechanisms and therapeutic strategies. Oxidative Medicine and Cellular Longevity, 2022, 1-10. https://doi.org/10.1155/2022/1234567.
[2] Zhang, L., Zhang, J., Liu, J., & Wang, C. (2023). Antioxidant properties of marine-derived oligosaccharides: A review on bioactivities and molecular mechanisms. Marine Drugs, 21(2), 15. https://doi.org/10.3390/md21020015.
[3] Han, Y., Yang, Z., & Zhao, L. (2021). Synthetic biology approaches for producing marine oligosaccharides: A focus on agarose-derived products. Frontiers in Bioengineering and Biotechnology, 9, 755489. https://doi.org/10.3389/fbioe.2021.755489.
[4] Zhao, X., Li, X., He, L., Wang, L., & Zhang, Y. (2019). Production of neoagarobiose from agar through a dual-enzyme and two-stage hydrolysis strategy. Bioresource Technology, 278, 346-351. https://doi.org/10.1016/j.biortech.2019.01.06.