We designed 65 parts for the project, including 39 basic parts and 26 composite parts, covering ALAS genes derived from different species, regulatory elements used in experiments, and RChemA sequences mutated through prediction. Our favorite parts are BBa_K5303001 and BBa_K5303014. The former initially provided us with higher-activity of the ALAS, the latter was built on the former to encode the highest-activity ALAS currently available. Additionally, CASTs-associated pQcasTns(Ptr)-array8 and ptDonor plasmids (BBa_K5303113 and BBa_K5303114) were constructed to address the problem of unstable ALAS free expression during the experiment. Furthermore, enzyme constrained model was employed to predict a series of potential targets and plasmids, such as BBa_K5303103, were constructed for their regulation. All of the parts we use have been submitted to the iGEM Registry following the BioBricks assembly standards. Please find below a brief form for registering our parts. For more information on our parts, please visit our Parts page.

Name	Type	Description	Designer
BBa_K5303000	Coding	AFhemA	Mengying Zhao
BBa_K5303001	Coding	RChemA	Mengying Zhao
BBa_K5303002	Coding	RPhemA	Mengying Zhao
BBa_K5303007	Coding	RChemA-S139E	Mengying Zhao
BBa_K5303008	Coding	RChemA-M393L	Mengying Zhao
BBa_K5303014	Coding	RChemA-G244A	Mengying Zhao
BBa_K5303017	Coding	RChemA-A218I	Mengying Zhao
BBa_K5303019	Coding	RChemA-G60A	Mengying Zhao
BBa_K5303024	Coding	RChemA-S378M	Mengying Zhao
BBa_K5303025	Coding	RChemA-T245Q	Mengying Zhao
BBa_K5303028	Coding	RChemA-V252W	Mengying Zhao
BBa_K5303031	Coding	RChemA-I292A	Mengying Zhao
BBa_K5303032	Coding	RChemA-K13R	Mengying Zhao
BBa_K5303039	Coding	RChemA-I203L	Dongxun Li
BBa_K5303055	Coding	RChemA-G58A	Dongxun Li
BBa_K5303056	T7	T7 promotor	Dongxun Li
BBa_K5303057	RBS	Ribosome Binding Site(RBS)	Dongxun Li
BBa_K5303058	Promotor	J23119 promotor	Dongxun Li
BBa_K5303059	Other	TetR	Dongxun Li
BBa_K5303060	Other	Tns	Dongxun Li
BBa_K5303061	DNA	Adenylate kinase(adk)	Dongxun Li
BBa_K5303062	DNA	Aspartate-semialdehyde dehydrogenase(asd)	Dongxun Li
BBa_K5303063	DNA	Phosphoenolpyruvate carboxylase(ppc)	Dongxun Li
BBa_K5303064	DNA	Succinate--CoA ligase [ADP-forming] subunit beta(SucC)	Dongxun Li
BBa_K5303065	DNA	Succinate--CoA ligase [ADP-forming] subunit alpha(SucD)	Dongxun Li
BBa_K5303066	DNA	Homoserine kinase(thrB)	Dongxun Li
BBa_K5303067	DNA	Adenine deaminase(adE)	Dongxun Li
BBa_K5303068	DNA	Aspartokinase(apE)	Dongxun Li
BBa_K5303069	DNA	Bifunctional aspartokinase/homoserine dehydrogenase 1(baE)	Dongxun Li
BBa_K5303070	DNA	Threonine synthase(tsE)	Dongxun Li
BBa_K5303071	Terminator	T7 Terminator	Dongxun Li
BBa_K5303078	Other	Kanamycin	Dongxun Li
BBa_K5303079	Other	Lac operator(LacO)	Dongxun Li
BBa_K5303080	Other	LacI	Dongxun Li
BBa_K5303081	Other	F1 origin	Dongxun Li
BBa_K5303101	RBS	SRBS	Dongxun Li
BBa_K5303102	Plasmid_Backbone	pTarget	Dongxun Li
BBa_K5303113	Plasmid	pQcasTns(Ptr)-array8	Dongxun Li
BBa_K5303114	Plasmid	ptDonor	Dongxun Li

Name	Type	Description	Designer
BBa_K5303082	Composite	pET24a	Dongxun Li
BBa_K5303083	Composite	pET24a-AFhemA	Dongxun Li
BBa_K5303084	Composite	pET24a-RChemA	Dongxun Li
BBa_K5303085	Composite	pET24a-RPhemA	Dongxun Li
BBa_K5303088	Composite	pET24a-RChemA-S139E	Dongxun Li
BBa_K5303089	Composite	pET24a-RChemA-A218I	Dongxun Li
BBa_K5303090	Composite	pET24a-RChemA-G244A	Dongxun Li
BBa_K5303091	Composite	pET24a-RChemA-T245Q	Dongxun Li
BBa_K5303092	Composite	pET24a-RChemA-G58A	Dongxun Li
BBa_K5303093	Composite	pET24a-RChemA-G60A	Dongxun Li
BBa_K5303094	Composite	pET24a-RChemA-V252W	Dongxun Li
BBa_K5303095	Composite	pET24a-RChemA-I292A	Dongxun Li
BBa_K5303096	Composite	pET24a-RChemA-S378M	Dongxun Li
BBa_K5303097	Composite	pET24a-RChemA-M393L	Dongxun Li
BBa_K5303098	Composite	pET24a-RChemA-K13R	Dongxun Li
BBa_K5303099	Composite	pET24a-RChemA-I203L	Dongxun Li
BBa_K5303103	Composite	pTarget-adk	Dongxun Li
BBa_K5303104	Composite	pTarget-asd	Dongxun Li
BBa_K5303105	Composite	pTarget-ppc	Dongxun Li
BBa_K5303106	Composite	pTarget-SucC	Dongxun Li
BBa_K5303107	Composite	pTarget-SucD	Dongxun Li
BBa_K5303108	Composite	pTarget-thrB	Dongxun Li
BBa_K5303109	Composite	pTarget-adE	Dongxun Li
BBa_K5303110	Composite	pTarget-apE	Dongxun Li
BBa_K5303111	Composite	pTarget-baE	Dongxun Li
BBa_K5303112	Composite	pTarget-tsE	Dongxun Li

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.

Hosts	Pathway	Substrates	Titer(g/L)	References
E.coli	C4	Glucose/Glycine	11.5	[3]
		Glucose/Glycine/Amber Acid	7.3	[4]
		Glucose/Glycine	30.7	[5]
	C5	Glucose	11.4	[6]
		Glucose	2.81	[7]
C. glutamicum	C4	Glucose/Glycine	18.5	[8]
	C5	Glucose	1.79	[9]
		Glucose	3.16	[10]

Table. 1 Research progresses for 5-ALA biosynthesis in engineered microorganisms. The highest 5-ALA titer of 30.7 g/L was obtained utilizing E. coli as the chassis strain.

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.

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, researchs 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.

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.

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).