Overview

Superoxide dismutase (SOD) is a metalloenzyme that catalyzes the dismutation of superoxide anions into hydrogen peroxide and molecular oxygen, serving as a core component of the biological defense system against oxidative stress. Manganese superoxide dismutase (Mn-SOD), a key isozyme within the SOD family, utilizes manganese ions as its cofactor and is primarily localized in mitochondria and other subcellular compartments with high oxidative metabolic activity. It plays an indispensable role in scavenging mitochondrial reactive oxygen species (ROS). This project aims to employ synthetic biology strategies to produce highly active and stable Mn-SOD for use as a core ingredient in eye drops, targeting the early prevention of cataracts.

To achieve this goal, several genetic constructs were engineered. First, the native Mn-SOD gene sequence (BBa_K4907023) was obtained from the iGEM part database and codon-optimized for enhanced expression, yielding the construct designated pET28a-Mn-SOD. Subsequently, to improve the specific activity of the wild-type enzyme, a Transformer-based deep learning model was employed to computationally predict critical mutation sites. Site-directed mutagenesis was performed on pET28a-Mn-SOD (G93H, D107L, and K46S).

Enzymatic assays revealed that the G93H mutant exhibited the most significant enhancement in specific activity, with an increase of approximately 15.4% compared to the wild-type Mn-SOD. The D107L and K46S mutants, showed negligible or only minor fluctuations in activity. Thermal stability testing demonstrated that the D107L mutant possessed the highest stability among all variants, with a 22.26% improvement relative to the wild-type.

Parts List

NO.	Parts Name	Type	Description	Length
1	BBa_259XEISM	Basic	Mn-SOD gene	606bp
2	BBa_25GCTRP3	Basic	Mn-SOD-mutation1 gene	606bp
3	BBa_25EJ6QS0	Basic	Mn-SOD-mutation2 gene	606bp
4	BBa_25VC49J1	Basic	Mn-SOD-mutation3 gene	606bp
5	BBa_25DCMLTT	Composite	T7-Mn-SOD	5929bp
6	BBa_25D4LO9D	Composite	T7-Mn-SOD-mutation1	5929bp
7	BBa_25KIJ21V	Composite	T7-Mn-SOD-mutation2	5929bp
8	BBa_25H46944	Composite	T7-Mn-SOD-mutation3	5929bp