Natural 5-Aminolevulinic Acid: Sources, Biosynthesis, Detection and Applications

Abstract

5-Aminolevulinic acid (5-ALA) is the key precursor for the biosynthesis of tetrapyrrole compounds, with wide applications in medicine, agriculture and other burgeoning fields. Because of its potential applications and disadvantages of chemical synthesis, alternative biotechnological methods have drawn increasing attention. In this review, the recent progress in biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts are summarized. The research progress on 5-ALA biosynthesis via the C4/C5 pathway in microbial cells is emphasized, and the corresponding biotechnological design strategies are highlighted and discussed in detail. In addition, the detection methods and applications of 5-ALA are also reviewed. Finally, perspectives on potential strategies for improving the biosynthesis of 5-ALA and understanding the related mechanisms to further promote its industrial application are conceived and proposed.

Keywords: 5-aminolevulinic acid; application; biosynthetic pathway; detection; metabolic engineering.

FIGURE 1

Biosynthetic pathways and downstream pathways of 5-aminolevulinic acid. The figure is divided into three parts: central carbon metabolic pathways, biosynthetic pathways and downstream pathways. The dashed green line indicates positive regulation, the dashed red line indicates feedback inhibition. The genes in green or red represent the enzymes that are positive or negative for 5-aminolevulinic acid accumulation, respectively. The polygons represent transcriptional regulators and red or blue represent positive/negative regulation. PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen decarboxylase; CPO, coproporphyrinogen oxidase; PPO, protoporphyrinogen oxidase; FECH, ferrochelatase; FHY3, Far-red Elongated Hypocotyl 3; FAR1, Far-red Impaired Response 1; mitochondrial ClpX (mtClpX); 3PG, 3-phosphoglycerate.

FIGURE 2

Genetic manipulations in metabolic engineering strategies for 5-aminolevulinic acid biosynthesis. The genes in green or red represent the enzymes that should be overexpressed or inactivated to accelerate 5-ALA accumulation, respectively. The red cross indicates that the pathways are disrupted, the dashed green line indicates positive regulation, and the dashed red line indicates feedback inhibition. pdxH, encoding pyridoxal 5-phosphate synthase; pdxY, pyridoxal kinase; Cgl0788-Cgl0789, pyridoxal 5′-phosphate synthase gene; gapA, encoding glyceraldehyde 3-phosphate dehydrogenase; serA, encoding 3-phosphoglycerate dehydrogenase; serB, encoding phosphoserine phosphatase; serC, encoding phosphoserine aminotransferase; glyA, encoding serine hydroxymethyl transferase; coaA, encoding pantothenate kinase; dfp, encoding dephospho-CoA kinase; coaD, encoding pantetheine-phosphate adenylyltransferase; coaE, encoding dephospho-CoA kinase; ppc, encoding phosphoenolpyruvate carboxylase; pyc, encoding pyruvate carboxylase; pck, encoding phosphoenolpyruvate carboxykinase; gltA, encoding citrate synthase; ldhA, encoding L-lactate dehydrogenase; pqo, encoding pyruvate:menaquinone oxidoreductase; pta, encoding phosphotransacetylase; ackA, encoding acetate kinase; cat, encoding acetyl-CoA:CoA transferase; ACO2, encoding aconitase; icd, encoding isocitrate dehydrogenase; sucAB, encoding α-oxoglutarate dehydrogenase; odhI, encoding α-oxoglutarate dehydrogenase inhibitor; sucCD, encoding succinyl-CoA synthetase; aceA, encoding isocitrate lyase; aceB, encoding malate synthase; iclR, encoding the transcriptional regulator of glyoxylate cycle genes aceBAK; gdhA, encoding glutamate dehydrogenase; gltX, encoding glutamyl-tRNA synthetase; hemA, encoding glutamyl-tRNA reductase; hemL, encoding glutamate-1-semialdehyde aminotransferase; pgr7, encoding hem1 stimulator protein; hemA, encoding 5-aminolevulinate synthase; rhtA, encoding serine/threonine transporter; lysE, encoding lysine/arginine transporter; putP, encoding L-proline transporter; Ncgl1221, encoding glutamate transporter; agxt, encoding glyoxylate aminotransferase from Homo sapiens. G6P, glucose-6-phosphate; 3PP, 3-phosphoserine; GAP, glyceraldehyde 3-phosphate; DPG, 1,3-bisphosphoglyceric acid; PEP, phosphoenolpyruvate; PYR, pyruvic acid; AcCoA, acetyl-CoA; AHP, 3-hydroxy-1-aminoacetone phosphate; DXP, deoxyxylulose 5-phosphate; PNP, pyridoxine 5′-phosphate; PL, pyridoxal; Rup, ribulose 5-phosphate; PAN, pantothenate; 4PPAN, 4′-phosphopantothenate; PPC, 4′-phosphopantethenine; PPA, dephpspho-CoA; CoA, coenzyme A; ADK, adenylate kinase; HK, hexokinase; ZWF, glucose-6-phosphate dehydrogenase; PGL, phosphogluconolactonase; GND, 6-phosphogluconate dehydrogenase; R5P, D-ribulose 5-phosphate.

FIGURE 3

Strategies for tuning the expression and regulation of genes. Tuning gene expression and regulation requires a balance between cell growth and a specific biological activity in order to transform the original system within the microbial cells into one that generates the target product. The strategies include several methods, such as CRISPRi, Riboswitch engineering, Synthetic asRNA engineering, RBS engineering. Promoter engineering, 5′-UTR engineering and RBS library can be used to optimize the expression of the target genes, and thereby increase the biosynthesis of 5-ALA. In riboswitch engineering, a glycine-OFF riboswitch was utilized to dynamically downregulate ALAD expression in the presence of glycine, while small amounts of glycine allow hemB to be expressed normally (Zhou et al., 2019). Using CRISPRi technology, a heme-responsive regulatory system was developed to control the concentration of heme dynamically and precisely. At low heme concentration, the regulatory system hinders the expression of CRISPRi (Zhang et al., 2020a).