Part:BBa_K5303014
RChemA-G244A
Usage and Biology
Name: RChemA-G244A Base Pairs: 1230 bp Mutate G244 of RChemA to A244 by site-directed mutagenesis.
Exploration of the Enhancement of ALAS Activity
5-Aminolevulinic acid (5-ALA) is a widely used biopesticide that is effective in increasing crop resistance to biotic and abiotic stresses. Promoting a high yield of 5-ALA is a priority in agricultural research. The natural synthesis pathway of this compound is comprised primarily of the C4 and C5 pathways. The C4 pathway is predominantly observed in mammals, yeast, and purple sulfur-free photosynthetic bacteria, with succinyl coenzyme A and glycine serving as the primary precursors. In contrast, the C5 pathway is predominantly observed in higher plants, algae, and certain bacteria, with glutamate serving as the primary precursor [1]. Escherichia coli is a frequently utilized chassis strain for the biosynthesis of 5-ALA in industrial fermentation, primarily due to its high productivity and short production cycle. Given the significant influence of the endogenous C5 pathway of E. coli on its growth, the exogenous introduction of the C4 pathway to achieve high production of 5-ALA has become a prominent area of research. 5-ALA synthase, also known as aminolevulinic acid synthase (ALAS), plays a pivotal role in this metabolic pathway [2]. ALAS catalyzes the conversion of glutamate to 5-ALA, which represents the rate-limiting step in the overall metabolic pathway. Consequently, the catalytic activity of ALAS exerts a direct influence on the efficiency of 5-ALA production.
Currently, the main sources of ALAS include Rhodobacter capsulatus, Agrobacterium tumefaciens and Pseudomonas denitrificans. However, despite the abundance of enzyme resources provided by these microorganisms, the activity and stability of ALAS in E. coli remain suboptimal. Consequently, the question of how to enhance the enzymatic activity and stability of ALAS has become a pivotal topic in contemporary research.</p>
Researchers have mainly used genetic engineering and enzyme modification techniques to enhance the activity of ALAS. For example, screening ALAS from different sources for higher activity and introducing them into the host bacteria through heterologous expression technology have gained some progress in industrial microorganisms such as E. coli and Saccharomyces cerevisiae [11]. In addition, directed evolution techniques have also been widely used for optimizing the activity and stability of ALAS [1, 12]. These modifications allowed ALAS to exhibit higher catalytic efficiency under laboratory conditions. Despite initial improvements in enzyme activity, the activity and stability of ALAS remain insufficient for industrial applications, necessitating further enhancement of both activity and expression stability [13].
In the future, the optimal design of the synthetic enzyme itself remains a central way to improve ALAS activity. By combining molecular dynamics simulations and protein design tools, the structure-function relationship of ALAS can be further revealed, leading to precise regulation of enzyme activity through targeted mutagenesis. Furthermore, research in metabolic engineering should remain a priority, as optimizing metabolic flux can enhance 5-ALA production by increasing carbon flux in the target pathway and boosting the availability of key cofactors (e.g., coenzyme A, NADPH).
Overall, despite the progress made in enhancing 5-ALA synthase activity, further in-depth exploration is required in optimizing enzyme structure and metabolic regulation to achieve more efficient 5-ALA production and to establish a clearer model of the metabolic network.
Experimental Design and Result
BBa_K5303000, BBa_K5303001, and BBa_K5303002 were designed to enhance the activity of ALAS in E. coli by engineering its bacteriophage to enhance the production efficiency of 5-ALA. Through a comprehensive literature review, we screened AFhemA-ALAS, RchemA-ALAS, and RPhemA-ALAS for their potential to achieve higher ALAS activity. These enzymes have shown high catalytic activities in preliminary experiments, which promoted the synthesis of 5-ALA more significantly. Our experiments showed that BBa_K5303001 exhibited higher activity, and to further improve the yield, we modified BBa_K5303001 in the hope of enhancing its expression activity and catalytic efficiency by targeted mutagenesis.
Fig. 1 BBa_K5303000、BBa_K5303001、BBa_K5303002 fermentation results
Under the guidance and assistance of Prof. Gu Yang from Nanjing Normal University, we obtained 12 potential mutation sites. These 12 mutation sites were based on the rational prediction of the BBa_K5303001 active site and its surrounding amino acids, and the possible effects of the mutations on the conformational stability, substrate affinity and catalytic efficiency of the enzyme were considered.
Among these 12 mutants, BBa_K5303014 showed the most significant activity enhancement. The fermentation results showed that BBa_K5303014 expressed in E. coli BL21(DE3) significantly increased 5-ALA production compared with the control. After 24 h, the fermentation yields of each mutant and the control stabilized, and BBa_K5303014 yielded 164% higher than the control. This result indicated that BBa_K5303014 effectively improved the catalytic activity and substrate utilization of BBa_K5303001. It may be because BBa_K5303014 enhanced the binding stability of the enzyme and substrate and optimized the conformation of the catalytic center, which enhanced the overall reaction efficiency.
Fig. 2 BBa_K5303014 (G244A) 24 h fermentation pictures
Fig. 3 The fermentation results of BBa_K5303014 (G244A) with RC and other controls are presented and discussed in this figure
This finding provides a new direction for further modification of BBa_K5303001. We hope to continue optimizing the active site and surroundings of the enzyme through targeted mutagenesis, thus maximizing the production of 5-ALA.
The experimental results also indicate that through further mutation screening and metabolic engineering means, the biosynthesis of 5-ALA is expected to achieve greater breakthroughs, providing a more efficient production pathway for the application of 5-ALA in agriculture as well as in other fields, and contributing to the alleviation of the world food crisis.
Reference
1. Wang, W., et al., Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters. Journal of Agricultural and Food Chemistry, 2024. 72(14): p. 8006-8017.
2. Jahn, D. and D.W. Heinz, Biosynthesis of 5-Aminolevulinic Acid, in Tetrapyrroles: Birth, Life and Death, M.J. Warren and A.G. Smith, Editors. 2009, Springer New York: New York, NY. p. 29-42.
3. Zhu, C., et al., Enhancing 5-Aminolevulinic Acid Tolerance and Production by Engineering the Antioxidant Defense System of Escherichia coli. Biotechnology Bioengineering, 2019. 116(8): p. 2018-2028.
4. Lin, J., W. Fu, and P. Cen, Characterization of 5-aminolevulinate Synthase from Agrobacterium Radiobacter, Screening New Inhibitors for 5-Aminolevulinate Dehydratase from Escherichia coli and Their Potential Use for High 5-Aminolevulinate Production. Bioresource Technology, 2009. 100(7): p. 2293-2297.
5. Pu, W., et al., System Metabolic Engineering of Escherichia coli for Hyper-production of 5-Aminolevulinic Acid. Biotechnology for Biofuels and Bioproducts, 2023. 16(1): p. 31.
6. Luo, Z., et al., Synergistic Improvement of 5-Aminolevulinic Acid Production with Synthetic Scaffolds and System Pathway Engineering. ACS Synthetic Biology, 2022. 11(8): p. 2766-2778.
7. Ding, W., et al., 5-Aminolevulinic Acid Production from Inexpensive Glucose by Engineering the C4 Pathway in Escherichia coli. Journal of Industrial Microbiology and Biotechnology, 2017. 44(8): p. 1127-1135.
8. Chen, J., et al., Efficient Bioproduction of 5-Aminolevulinic Acid, a Promising Biostimulant and Nutrient, from Renewable Bioresources by Engineered Corynebacterium glutamicum. Biotechnology for Biofuels, 2020. 13(1): p. 41.
9. Yu, X., et al., Engineering Corynebacterium glutamicum to Produce 5-Aminolevulinic Acid from Glucose. Microbial Cell Factories, 2015. 14(1): p. 183.
10. Zhang, C., et al., Metabolic Engineering of an Auto-regulated Corynebacterium glutamicum Chassis for Biosynthesis of 5-Aminolevulinic Acid. Bioresource Technology, 2020. 318: p. 124064.
11. Lou, J.-w., et al., High-level Soluble Expression of the HemA Gene from Rhodobacter capsulatus and Comparative Study of its Enzymatic Properties. Journal of Zhejiang University SCIENCE B, 2014. 15(5): p. 491-499.
12. Ting, W.-W. and I.S. Ng, Adaptive Laboratory Evolution and Metabolic Regulation of Genetic Escherichia coli W3110 toward Low-carbon Footprint Production of 5-Aminolevulinic Acid. Journal of the Taiwan Institute of Chemical Engineers, 2022. 141.
13. Yi, Y.-C., et al., Challenges and Opportunities of Bioprocessing 5-Aminolevulinic Acid Using Genetic and Metabolic Engineering: a Critical Review. Bioresources and Bioprocessing, 2021. 8(1).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 616
Illegal XbaI site found at 18 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 616
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 616
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 616
Illegal XbaI site found at 18 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 616
Illegal XbaI site found at 18 - 1000COMPATIBLE WITH RFC[1000]
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