5-ALA was confirmed as the common precursor of tetrapyrrole compounds and was found in many organisms. Two natural 5-ALA biosynthesis pathways are known to date. One is the C4 pathway (Shemin pathway), which was firstly reported by Shemin and Russell (1953). It exists in animals, yeast, some protozoa and purple non-sulfur photosynthetic bacteria. In this pathway, glycine and succinyl-CoA are condensed to 5-ALA under the catalysis of 5-aminolevulinic acid synthase (ALAS), with pyridoxal 5′-phosphate (PLP) as the cofactor (Figure 1) (Sasaki et al., 2002). Succinyl-CoA is synthesized by methylmalonyl-CoA mutase, which utilizes vitamin B₁₂ as an essential co-factor (38). The other is the C5 pathway (Beale pathway), which starts from the discovery of 5-ALA in Chlorella vulgaris by Beale (Beale, 1970). Glutamate produced via the TCA cycle acts as the substrate of the C5 pathway (Kang et al., 2012). The C5 pathway is mainly present in higher plants, algae, and many bacteria. The pathway starts with the ligation of tRNA and glutamate to generate L-glutamyl–tRNA, catalyzed by glutamyl–tRNA synthetase (GluTS) (Figure 1). The NADPH-dependent glutamyl-tRNA reductase (GluTR) reduces the carboxyl group of Glu–tRNA to a formyl group, which enables the conversion of L-Glu–tRNA into L-glutamic acid 1-semialdehyde (GSA). In the last step, 5-ALA is created through transamination by glutamate-1-semialdehyde aminotransferase (GSA-AM) (Moser et al., 2001). The enzymes involved in this pathway, GluTS, GluTR and GSA-AM, are encoded by the genes gltX, hemA (homonymic with the ALAS gene of the C4 pathway) and hemL, respectively. However, a few microorganisms have both C4 and C5 pathways with Euglena gracilis being a well-known example (Weinstein and Beale, 1983).

Tetrapyrrole compounds are produced from 5-ALA in specific downstream pathways and are important for physiological and metabolic activities of organisms. The expression levels of downstream genes affect enzymes activity and transcription of the 5-ALA biosynthesis pathway as well as cell growth. In other words, regulated expression of key genes in downstream pathways is conducive to further increase 5-ALA production.

ALAD is the first enzyme in the biosynthetic pathways of heme, chlorophyll and other essential porphyrins. They all start from 5-ALA in the downstream pathway and ALAD has a close relationship with final accumulation of 5-ALA. The metabolic intermediate protoporphyrinogen IX inhibits the expression of ALAD and further influences the accumulation of 5-ALA (Figure 1) (Zhang J. L. et al., 2015; Zhang et al., 2016). Many studies have attempted to improve the concentration of 5-ALA by inhibiting the expression of ALAD to reduce the intracellular utilization of 5-ALA (Ding et al., 2017; Yu et al., 2015). As ALAD is an important enzyme catalyzing the first step in the synthesis of many essential porphyrins, the complete loss of ALAD activity has a negative impact on cell growth. The replacements of the initiation codon or promoter to regulate ALAD expression at the transcriptional and translational levels is another validated measure to realize the balance between cell growth and 5-ALA biosynthesis. After altering the most preferred initiation codon AUG into GUG in E. coli, the production of 5-ALA significantly increased from 1.31 to 1.76 g/L (Ding et al., 2017). Promoter engineering is another effective way to directly downregulate ALAD expression at the transcriptional level by replacing its promoter with constitutive promoters and stationary-phase promoters (Figure 3). A strain in which the stationary-phase promoter fliCp was introduced displayed superior performance, and the final production of 5-ALA was 2.68 g/L (increased by 11% over the control strain) (Zhang J. L. et al., 2019). Additionally, a glycine-OFF riboswitch from Clostridium pasteurianum, Apt2#82, was utilized to dynamically downregulate ALAD expression in the presence of glycine, resulting in an 11% improvement of 5-ALA production in recombinant E. coli (Zhou et al., 2019) (Figure 3). It is reported that the antisense RNA (asRNAs) of hemB could decrease its’ mRNA level by 50%, and the production of 5-ALA was increased by 17.6% (Ge F. L. et al., 2021) (Figure 3). In a recent research, CRISPR interference (CRISPRi) was used to repress the expression of hemB, and the titer of 5-ALA increased 3.7 fold compared to the control (Miscevic et al., 2021). However, weakening the activity of ALAD cannot guarantee the elevation of production (Xie et al., 2003; Zhang J. L. et al., 2019), implying that ALAD has an appropriate range of activity and advanced research should be carried on.

The combinatorial perturbation of multiple genes in the downstream pathway has attracted widespread attention, with most studies focusing on engineered E. coli. Among several genes in the downstream pathway, the upregulation of hemD and hemF facilitated the accumulation of 5-ALA, while the overexpression of hemB, hemG and hemH had no effect (Zhang J. L. et al., 2019). The expression of hemD and hemF with moderate intensity, as well as overexpression of hemA and hemL increased the production of 5-ALA in recombinant E. coli LADF-6 to 3.25 g/L, 3.78-fold higher than the parental strain (Zhang J. L. et al., 2015). Recent studies indicated that the iron regulator small RNA ryhB distinctly downregulates the transcription of hemB and hemH, perturbing the accumulation of heme and 5-ALA (Figure 1) (Li et al., 2014). Subsequently, further studies investigated the transcriptional regulatory mechanism of ryhB in relation to other genes of the heme synthesis pathway. It was demonstrated that both overexpression of hemD and hemF downregulated the transcription of ryhB, while overexpression of hemB resulted in an unexpected increase (Zhang et al., 2016). In addition, a heme-responsive regulatory system (Figure 3) was designed and constructed by CRISPRi to dynamically and precisely control the downstream pathways for 5-ALA synthesis (Zhang et al., 2020a). In this work, a H149D mutant was obtained by semi-rational design with site saturated mutagenesis of HrtR from Lactococcus lactis, and used as a biosensor to dynamically regulate the expression of hemC and hemH in the downstream pathways, which resulted in a 5-ALA titer of up to 5.35 g/L 5-ALA in batch-cultures of E. coli (Zhang et al., 2020a).

Thus, the biosynthesis of 5-ALA interacts with heme synthesis in downstream pathways and the deeper regulation is complex and ambiguous. Therefore, the simple deletion or overexpression of genes may not be able to produce a high yield of 5-ALA, and more elaborate regulatory strategies need to be developed to achieve a balance between cell growth and 5-ALA biosynthesis.

The supply of cofactors immediately impacts the production of metabolic products and the modification in cofactor engineering can accelerate the yield of targeted products, such as acetoin and 2,3-butanediol (Yang et al., 2017). During the synthesis of 5-ALA, the conversion of substances and the activation of enzymes mostly relies on cofactors such as pyridoxal PLP, coenzyme A, NADPH/NADP, etc. The supply of sufficient cofactors is of great significance to ensure the flow of electrons, activation of enzymes and maintenance of their stability. Currently, studies on cofactor supply mainly focus on the strengthening of the pyridoxal 5′-phosphate and coenzyme A supply by exogenous addition and metabolic pathway engineering, and also few involves the metabolic engineering of ATP and NAD(P)H (Figure 2).

High-level synthesis of target products may be harmful to the microbial host cells and further inhibit the production of the target product to some extent. 5-ALA is one of the typical representatives, which easily result in the accumulation of reactive oxygen species (ROS) that has toxic effects on cells. It is therefore critical to improve 5-ALA production by accelerating intracellular 5-ALA transfer to the extracellular environment, alleviating the cytotoxicity.

Because its physical properties are close to those of proteinogenic amino acids, different transport proteins with the ability to transport amino acids and their analogues have been used to improve the production of 5-ALA (Figure 2) (Moser et al., 2001). The serine/threonine transporter RhtA has been widely used in numerous recombinant strains and exhibited excellent results in increasing the extracellular 5-ALA concentration. Kang et al. firstly applied the rhtA gene in 5-ALA synthesis and successfully demonstrated its feasibility for further improvement of production (Kang et al., 2011a). Recently, overexpressing rhtA with a T7 system composed of orthogonal T7RNA polymerase and the T7 promoter increased 5-ALA production 40-fold in E. coli (Tan S. I. et al., 2019). Introduction of rhtA into the high-yield recombinant C. glutamicum AL-OI** increased production of 5-ALA by 28% over the control (Ko et al., 2019). Interestingly, the expression level of rhtA was upregulated 17-fold in the high-yield E. coli Transetta GTR/GBP co-expressing HemA1 and pgr7 from A. thaliana (Zhao and Zhai, 2019). The timely and effective transport of 5-ALA outside the cell is of great significance for the continuous synthesis of 5-ALA. It is undeniable that RhtA is not the only protein that can transport 5-ALA. However, only few export proteins have been found, and the discovery of transporters remains a great challenge.

A second strategy for facilitating the transport of 5-ALA relies on changing the permeability of the cell wall. Penicillin inhibits the synthesis of cell wall components by restraining the activity of penicillin-binding proteins and further benefits the accumulation of 5-ALA (Laneelle et al., 2013; Ramzi et al., 2015). However, the exogenous addition of penicillin not only increases the production cost but is also not environmentally friendly. Our previous study directly influenced the synthesis of the cell wall by deleting the non-essential high molecular-weight penicillin-binding protein genes pbp1a, pbp1b, and pbp2b individually, and all were positive for the production of 5-ALA with increases of 13.53, 29.47, and 22.22%, respectively (Feng et al., 2016), demonstrating the vital role of cell wall permeability for extracellular 5-ALA accumulation.

The production of 5-ALA in vivo is limited by low volumetric productivity, byproduct losses and complex fermentation processes. Moreover, the producer strains tend to be exposed to the toxic effects of ROS, which affects the normal growth of the cells (Zeng et al., 2015). To address these limitations, cell-free metabolic engineering that utilizes purified enzymes or crude cell extracts to synthesize 5-ALA was proposed (Dudley et al., 2015).

In an early study using cell-free extracts of recombinant E. coli with the addition of succinate, glycine, LA and ATP, the production of 5-ALA reached 22.6 mM (2.96 g/L) in vitro (Van Der Werf and Zeikus, 1996). By contrast, the same recombinant strain from which the cell extract was prepared was only able to produce 2.25 mM (0.30 g/L) 5-ALA in vivo, which illustrates the great potential for high-level 5-ALA production in vitro. Limited by the relatively lower enzyme activities of cell extracts and complex engineered of recombinant strains, researchers were continually looking for more efficient and commercially viable approaches for 5-ALA synthesis. With the purification of the key enzymes in the metabolic pathway and elucidation of their structure and mechanism (Brown et al., 2018), it is possible to produce 5-ALA using a cell-free multi-enzyme catalysis system. Meng et al. (Meng et al., 2016) firstly constructed a cell-free multi-enzyme catalysis system based on the C4 pathway using the substrates succinic acid, glycine, and polyphosphate, catalyzed by the three enzymes, ALAS, succinyl-CoA synthase and polyphosphate kinase. Although the final titer of 5-ALA reached only 5.4 mM (0.71 g/L), the study demonstrated the possibility of cell-free multienzymatic synthesis. Subsequently, a cell-free semi-permeable process based on the C5 pathway was realized using glucose, sodium polyphosphate, ATP, NADPH, glutamate and nine enzymes (Zhao et al., 2019). Similar to previous research, the yield of 5-ALA was very low and the cost was very high, but this cell-free semi-permeable system firstly realized the recycling of enzymes and high-value cofactors for 5-ALA biosynthesis. In spite of the challenges faced by current studies, the considerable potential to realize commercial in vitro production by cell-free metabolic engineering cannot be ignored.

Ehrlich’s reagent was firstly used for the quantification of 5-ALA. It produces an adduct, that can be detected colorimetrically at 553 nm (Mauzerall and Granick, 1956; Tomokuni and Ogata, 1972). This method involves cell collection, extraction of intracellular components, and the Ehrlich reaction, which requires about 30 min for each sample (Mauzerall and Granick, 1956; Tomokuni and Ogata, 1972). Ehrlich’s reagent, as a classic detection method, has been widely accepted and used to measure the 5-ALA content, but the method suffers from severe sample background interference, in addition to being time-consuming and laborious.

5-ALA is stable only under acidic conditions. Currently, it is mostly sold in the form of hydrochloride and there is need for a simpler approach. The conversion of chemicals into easily detectable signals such as color or fluorescence has been a powerful tool for de novo metabolite monitoring and in vitro small molecule detection. Chung et al. (2005) proposed a combination of hemA with a green fluorescent protein gene (egfp) to realize the characterization and investigation of extracellular 5-ALA. Although the concentration of 5-ALA is reflected in the fluorescence intensity, the precise concentration of 5-ALA still depends on Ehrlich’s reagent. Recently, a fluorescence-based whole-cell system was developed for the simultaneous detection and production of 5-ALA in recombinant E. coli (Tan S. I. et al., 2019). In this method, a constitutive 5-ALA production strain with R. sphaeroides HemA (RshemA) and a fusion of super-folder-green fluorescent protein was established with an optimized promoter, medium, and fusion sites. The de novo quantification of intracellular and extracellular 5-ALA by monitoring the fluorescence of sfGFP fused to RshemA was also explored using dual promoters and dual plasmids (Tan S. I. et al., 2019). Additionally, to increase the understanding of the regulatory mechanism of 5-ALA in higher organisms, matrix isopotential synchronous fluorescence spectrometry and the first derivative technique were combined for the direct determination of δ-ALA in urine samples (Ajmal et al., 2019). The fluorescent derivatization of 5-ALA was based on the Hantzsch reaction. Matrix-isopotential synchronous fluorescence spectra were recorded along a matrix-isopotential trajectory, which combines the points of equal intensity in theoretical 3D contours. Maximum sensitivity and appropriate selectivity were ensured by the study of the experimental variables in the d-ALA bands, centered at its excitation and emission maxima (392.6 and 464.5 nm) (Ajmal et al., 2019). This method was comparable to conventional high performance liquid chromatography-fluorescence, with a detection limit of 5.4 ppb and lower linear-range limit of 18 ppb. These methods have improved effectiveness, enabling real-time detection with outstanding sensitivity, which is of great significance for ALA synthesis, detection and studies of the underlying metabolic mechanism.

Compared with a microbial fermentation broth with high 5-ALA content, samples with low 5-ALA concentrations such as blood, urine or tissue require more sensitive detection technology. The LC–MS/MS method was developed to simultaneously determine 5-ALA concentrations in fluids or tissues after solid-phase extraction, butanol derivatization, and quantification by selective reaction monitoring using ¹³C₅, ¹⁵N-ALA and 2,4-¹³C₂-PBG internal standards (Zhang et al., 2011; Dogan et al., 2019). The assay was highly sensitive for 5-ALA (LLOQ = 0.05-0.005 µM), and required ∼4 h from extraction to results. Later, a HPLC and GC–MS test method for 5-ALA detection was also developed (Ren et al., 2018; Hara et al., 2019) to obtain the real 5-ALA levels in microbial fermentation broth. Although these methods are sensitive and practical, more research is still needed to improve 5-ALA detection and decrease the assay costs.

To date, 5-ALA has been demonstrated to act as a metabolic intermediate and also as a growth regulator in plant cultivation. It is regarded as a plant growth promoting hormone since many studies found that it could regulate the growth and development of higher plants. Instead of only focusing on the effects of 5-ALA on plant, more and more researchers began to use omics to explain its mechanism (Zheng et al., 2021). The role of 5-ALA in stimulating physiochemical processes in higher plants under stress, regulating plant growth and development in seed germination, vegetative growth and fruit coloring has been reviewed by Wu et al., in 2019 (Wu et al., 2018). Here, we mainly discussed the latest studies on its role in plant growth as well as the amelioration of the effects caused by various abiotic stresses.

5-ALA can selectively kill weeds without affecting monocotyledonous wheat and corn crops (Rebeiz et al., 1984). Therefore, a large amount of 5-ALA is used in plants or pests to exert the effects of herbicides and insecticides (Sasikala et al., 1994). For example, two different types of agricultural herbicides have been produced based on the principle that 5-ALA is necessary for plant life and cannot be synthesized in the dark. “Norfluron” can inhibit Glu-tRNA gene expression and prevent 5-ALA synthesis, resulting in a lack of 5-ALA leading to plant death (Kumar et al., 1999). Acifluorfen-methyl is a product that deregulates 5-ALA synthesis leading to a large accumulation of 5-ALA, thereby inhibiting the synthesis of heme and porphyrin compounds (Gullner and Dodge, 2000). However, higher concentrations required in herbicides and insecticides made it difficult to popularize the application or 5-ALA in this field based on current prices.

The abiotic stresses caused by environment, such as drought, salinity, high or low temperature, cause great losses to crop yields and social and economic aspects, and 5-ALA is helpful to alleviate these problems effectively. With the foliage application of 5-ALA at 75 mg/L, the activities of catalase, ascorbate peroxidase and superoxide dismutase of sunfower (Helianthus annuus L.) enhanced by 157.1, 90 and 80% under drought stress than no 5-ALA addition (Sher et al., 2021). It is reported that exogenous addition of a suitable concentration of 5-ALA can effectively alleviate weak light stress in tobacco seedlings (Li et al., 2019) and damages induced by UV-B in Cajanus cajan L. seedlings (Gupta and Prasad, 2021). Moreover, 5-ALA has shown a significant effect on alleviating abiotic stress caused by low temperature (Yan et al., 2020) and salinity (Islam et al., 2021; Wu et al., 2021) and so on in plants, which were summarized in detail in a recent review (Rhaman et al., 2021).

Many studies have investigated fluorescence-guided resection of malignant tumors based on 5-ALA and protoporphyrin IX accumulation in tumors. This approach has become a well-established technique to facilitate greater extent of resection resulting in improved progression free survival (Stummer et al., 1998; Stummer et al., 2000; Pichlmeier et al., 2008; Stummer and Kamp, 2009; Rapp et al., 2012; Hadjipanayis et al., 2015; Guyotat et al., 2016). Additionally, 5-ALA was used with varying success in tumors such as meningiomas, medulloblastomas, ependymomas, and metastatic carcinoma (Millesi et al., 2016;Zhang C. et al., 2019; Wainwright et al., 2019). Other applications in medicine are being constantly developed, in addition to the applications related to cancer. Recently, there have been some excellent reviews covering the research progress on 5-ALA-based treatment of tumors (Wainwright et al., 2019; Zhang C. et al., 2019), and will not be repeated here.

Photodynamic therapy (PDT) with 5-ALA has been demonstrated as an effective treatment for severe acne in medical and cosmetic field (Itoh et al., 2000; Gold and Goldman, 2004), and recent studies have further researched the concentration of 5-ALA administration in different cases (Serini et al., 2019; Zhang et al., 2020b). Additionally, 5-ALA or its derivatives and ferric citrate compounds also are the main ingredient of disease prevention and improvement agent. A recent research demonstrated that 5-ALA combined with sodium ferric citrate could effectively improve aerobic capacity and voluntary exercise training achievement in older women over 75 years (Masuki et al., 2016; Ichihara et al., 2021). 5-ALA was also developed as a combined therapeutic-diagnostic agent in dentistry based on the strong dual modality of 5-ALA as lethal to cariogenic bacteria through photodynamic inactivation and enhancing LIF intensity for identification of dental caries (Lashkari et al., 2019). However, 5-ALA has a complex mechanism of action in the body, and the metabolic mechanism of 5-ALA conversion is still unclear. These unresolved issues led to a more prudent acceptance process of related products containing 5-ALA.

It was found that adding appropriate amounts of 5-ALA to animal feed can significantly improve their growth. In invertebrate aquaculture, dietary administration of 5-ALA effectively improved the immune system, ATP levels and acute hepatopancreatic necrosis disease resistance in pacific white shrimp (Direkbusarakom et al., 2021). Dankook University studied the effects of 5-ALA on the growth performance, blood status, and immune system of weaned piglets, sows, broilers and hens in South Korea. These results showed that adding appropriate amounts of 5-ALA to the diet can increase the hemoglobin concentration and iron in the serum, the levels of lymphocyte subsets, the quality of immune organs and the immune performance of these animals (Chen Y. J. et al., 2008; Chen Y. et al., 2008; Wang et al., 2009). Similarly, 5-ALA had the same effect on cows (Hendawy et al., 2019). Our own recent research work tried to efficiently enrich feed with 5-ALA by developing a solid-state fermentation process based on S. cerevisiae (Mao et al., 2020). In this work, 5-ALA not only plays a role in improving the animal’s growth performance and immune system, but yeast cells can be used as feed protein to provide nutrition for animals. In summary, it was concluded that 5-ALA could indeed be used as an animal feed additive to improve the iron status and immune response in livestock, which was also reviewed recently by Hendawy et al. recently (Hendawy et al., 2020).

According to the Comprehensive Enzyme Information System of BRENDA, there are 147 known organisms with 5-aminolevulinate synthesis ability, and we can assume that many natural hosts have not yet been developed to synthesize 5-ALA. In the future, high-yield production of 5-ALA can be achieved by fully screening natural hosts with innate advantages in 5-ALA synthesis. Furthermore, efficient 5-ALA synthesis can also be achieved through further metabolic engineering to transform the current model strains to give full play to their natural advantages. For example, C. glutamicum can naturally accumulate amino acids and lacks a glycine cleavage pathway, making it a highly attractive potential biosynthetic host (Jorgensen et al., 2012). It was shown that the activation of native antioxidant defense systems alleviated the deregulation of ROS metabolism due to 5-ALA accumulation and further improved 5-ALA tolerance and synthesis (Zhu et al., 2019). Therefore, hosts such as C. glutamicum and S. cerevisiae with the advantages of natural resistance to high concentrations of organic acids can be further explored as chassis cells for 5-ALA synthesis. Fully exploiting and utilizing the natural advantages of hosts to build a 5-ALA artificial cell factory may be an effective way to synthesize 5-ALA.

Although the synthesis and application of 5-ALA has been extensively studied, there are still some challenges that need to be resolved, such as the regulation and transport mechanisms in 5-ALA biosynthesis and the metabolic mechanisms related to 5-ALA application. First, the feedback regulation of ALAS or GluTR by heme exists in plants, animals and microorganisms, and influences the 5-ALA biosynthesis and host cell growth. In addition, feed-back regulation by downstream products in plant cells regulates the biosynthesis of 5-ALA in an opposite manner. However, the mechanism of feedback regulation by these factors and heme has not been elucidated. Secondly, the cellular transport capacity is also crucial for 5-ALA biosynthesis, which has been proved in previous research (Feng et al., 2016). However, Transport mechanisms of 5-ALA are not fully clear and need to be studied further. Detailed knowledge of the metabolic mechanisms of 5-ALA will resolve the safety issues and improve its industrial applications.

Recently, a growing fraction of research has transitioned towards employing a modular co-culture engineering strategy using multiple microbes growing together to facilitate a “divide-and-conquer” approach for chemical biosynthesis. Artificial microbial consortia have been constructed and applied to produce bio-based chemicals (Lu et al., 2019), natural products (Wang R. F. et al., 2020) and bioenergy (Jiang et al., 2019). According to the latest research progress on metabolic engineering for the synthesis of 5-ALA (Supplementary Table S1), the high-level production 5-ALA through the C4 pathway requires the exogenous addition of glycine in all cases. Moreover, our research group confirmed the effect of the addition of different glycine concentrations on the synthesis of 5-ALA in C. glutamicum (Zou et al., 2017), and the supply of glycine is the key limiting factor for 5-ALA synthesis in the C4 pathway (Ren et al., 2018). However, the addition of glycine not only increases the cost of the product, but the toxic effects of high glycine concentrations on the cells will also inhibit the growth of engineered bacteria and reduce the production efficiency. Moreover, 5-ALA is synthesized from the precursors glycine and succinyl-CoA in the C4 pathway, whose balanced supply is the key to the efficient synthesis of 5-ALA. To achieve precise control of the two precursors and the efficient synthesis of 5-ALA, synthetic microbial consortia can be constructed by placing the synthesis pathway modules of glycine and succinyl-CoA into different engineered strains. Based on the flexible optimization of the metabolic module within a single strain, combined with the adjustment of the inoculation volume and fermentation process of the microbial consortia, the artificial microbial consortia may be used as a potential method to synthesize 5-ALA. This approach can save costs and reduce the burden imposed on the cellular metabolism, solve the problem of flux optimization of nonlinear synthetic pathways, and achieve precise control of synthetic pathways.