Challenges and opportunities of bioprocessing 5-aminolevulinic acid using genetic and metabolic engineering: a critical review

Abstract5-Aminolevulinic acid (5-ALA), a non-proteinogenic five-carbon amino acid, has received intensive attentions in medicine due to its approval by the US Food and Drug Administration (FDA) for cancer diagnosis and treatment as photodynamic therapy. As chemical synthesis of 5-ALA performed low yield, complicated processes, and high cost, biosynthesis of 5-ALA via C4 (also called Shemin pathway) and C5 pathway related to heme biosynthesis in microorganism equipped more advantages. In C4 pathway, 5-ALA is derived from condensation of succinyl-CoA and glycine by 5-aminolevulic acid synthase (ALAS) with pyridoxal phosphate (PLP) as co-factor in one-step biotransformation. The C5 pathway involves three enzymes comprising glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (HemA), and glutamate-1-semialdehyde aminotransferase (HemL) from α-ketoglutarate in TCA cycle to 5-ALA and heme. In this review, we describe the recent results of 5-ALA production from different genes and microorganisms via genetic and metabolic engineering approaches. The regulation of different chassis is fine-tuned by applying synthetic biology and boosts 5-ALA production eventually. The purification process, challenges, and opportunities of 5-ALA for industrial applications are also summarized.

Redirection of metabolic flux in Shewanella oneidensis MR-1 by CRISPRi and modular design for 5-aminolevulinic acid production

Programming non-canonical organisms is more attractive due to the prospect of high-value chemical production. Among all, Shewanella oneidensis MR-1 possesses outstanding heme synthesis ability and is well-known for electron transfer, thus has high potential in microbial fuel cell and bioremediation. However, heme, as the final product of C4 and C5 pathways, is regulated by heme cluster for the high-value 5-aminolevulinic acid (ALA) for cancer photodynamic therapy, which has never been explored in MR-1. Herein, the heme metabolism in MR-1 was firstly optimized for ALA production. We applied CRISPR interference (CRISPRi) targeted on the genes to fine-tune carbon flux in TCA cycle and redirected the carbon out-flux from heme, leading to a significant change in the amino acid profiles, while downregulation of the essential hemB showed a 2-fold increasing ALA production via the C5 pathway. In contrast, the modular design including of glucokinase, GroELS chaperone, and ALA synthase from Rhodobacter capsulatus enhanced ALA production markedly in the C4 pathway. By integrating gene cluster under dual T7 promoters, we obtained a new strain M::TRG, which significantly improved ALA production by 145-fold. We rewired the metabolic flux of MR-1 through this modular design and successfully produced the high-value ALA compound at the first time.

Glutamate-1-semialdehyde aminotransferase from Sulfolobus solfataricus*

Glutamate-1-semialdehyde aminotransferase (GSA-AT) from the extremely thermophilic bacterium Sulfolobus solfataricus has been purified to homogeneity and characterized. GSA-AT is the last enzyme in the C5 pathway for the conversion of glutamate into the tetrapyrrole precursor Δ-aminolaevulinate (ALA) in plants, algae and several bacteria. The active form of GSA-AT from S. solfataricus seems to be a homodimer with a molecular mass of 87 kDa. The absorption spectrum of the purified aminotransferase is indicative of the presence of pyridoxamine 5´-phosphate (PMP) cofactor, and the catalytic activity of the enzyme is further stimulated by addition of PMP. 3-Amino-2,3-dihydrobenzoic acid is an inhibitor of the aminotransferase activity. The N-terminal amino acid sequence of GSA-AT from S. solfataricus was found to share significant similarity with the eukaryotic and eubacterial enzymes. Evidence is provided that ALA synthesis in S. solfataricus follows the C5 pathway characteristic of plants, algae, cyanobacteria and many other bacteria.

Charging of Both, Plastidal tRNAgln and tRNAglu with Glutamate and Subsequent Amidation of the Misacylated tRNAgln by a Glutamyl-tRNA Amidotransferase in the Unicellular Green Alga Scenedesmus obliquus, Mutant C-2A'

, 1995 C5-Pathway, Glutamyl-tRNAglu-Synthetase (E.C.6.1.1.17), Misacylation of tR N A glu, Amidotransferase, Scenedesmus obliquus In a previous paper we described the purification of a glutamyl-tRNA synthetase from the unicellular green alga Scenedesmus obliquus, m utant C-2A'. We now dem onstrate that, firstly, this enzyme is capable of mischarging plastidal tR N A gln from barley with glutamate, as well as it regularly charges the plastidal tR N A glu from Scenedesmus. Secondly, we show that the mischarged glutamyl-tRNAgln is subsequently am idated by a glutamyl-tRNA am idotransfer­ ase to form the glutaminyl-tRNAglri required for plastidal protein biosynthesis. This phenom ­ enon could already be dem onstrated for higher plant chloroplasts, mitochondria, cyanobact­ eria and gram-positive bacteria, as far as investigated. As recently shown the applied glutamyl-tRNA synthetase from Scenedesmus is a plastidal enzyme. In this paper we prove by treatm ent with m onobromobim ane and cyanogen bromide that the "regular" substrate of the enzyme, tR N A glu from Scenedesmus, is a plastidal tRNA with the plastid-specific sulfur modification in the anticodon. In the case of cyanogen bromide treatm ent, a total inactivation of the tRNA was achieved, revealing the presence of a sulfur modification in the plastid-tRNA glu anticodon.

Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the synthesis of 5-aminolaevuline acid via the C5 pathway (hemA and hemL) and uroporphyrinogen III (hemB, hemC and hemD). B. subtilis HemA protein (molecular mass 50 kDa) was overexpressed in hemA mutant of both Escherichia coli and B. subtilis. A mutant B. subtilis HemA protein with a Cys to Tyr change at position 105 was also overexpressed. Both wild-type and mutant HemA proteins migrated as oligomers (molecular mass greater than or equal to 230 kDa) on gel-filtration columns. All column fractions containing wild-type HemA protein had glutamyl-tRNA reductase activity. No glutamyl-tRNA reductase activity was found with the mutant HemA protein. It is concluded that the B. subtilis hemA gene product is identical to, or part of, the glutamyl-tRNA reductase of the C5 pathway.

Inhibition of Bacteriochlorophyll Biosynthesis by Gabaculin (3-Amino, 2,3-dihydrobenzoic Acid) and Presence of an Enzyme of the C5-Pathway of δ-Aminolevulinate Synthesis in Chloroflexus aurantiacus

Biosynthesis of bacteriochlorophyll c and a in the thermophilic phototrophic prokaryote. Chloroflexus aurantiacus Ok-70-fl, was strongly inhibited by the antibiotic gabaculin (3-amino 2,3- dihydrobenzoic acid), an inhibitor of the glutamate-C5-pathway of ö-aminolevulinate (ALA) synthesis. The key enzyme of the Shemin-pathway of ALA formation, ALA synthase (EC 2.3.1.37), was not detected in cell extracts of Chi. aurantiacus. However, the extracts catalyzed ALA formation from glutamate 1-semialdehyde, a reaction being highly sensitive to gabaculin.