A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

Abstract

5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. IMPORTANCEIn this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.A s the common precursor of tetrapyrroles such as porphyrin, heme, vitamin B 12 , and chlorophyll, 5-aminolevulinic acid (ALA) has been reported to be effective in tumor-localizing and photodynamic therapy for various diseases (1-3). ALA can also be used as a selective biodegradable herbicide and insecticide or an adversity resistance and growth-accelerating agent in agriculture (4, 5).In living organisms, two kinds of metabolic pathways have been described for ALA biosynthesis (Fig. 1). One is the C 5 pathway, which occurs in algae, higher plants, and many bacteria, including Escherichia coli and archaea. The C 5 pathway involves the following three enzymatic activities: glutamyl-tRNA synthetase (GluRS) (encoded by gltX), a NADPH-dependent glutamyl-tRNA reductase (HemA, encoded by hemA), and a glutamate-1-semialdehyde aminotransferase (HemL, encoded by hemL). The other is the C 4 pathway, which is present in birds, mammals, yeast, and purple non-sulfur-photosynthetic bacteria. In this pathway, ALA is formed through one-step catalysis by 5-aminolevulinic acid synthase (ALAS), which condenses glycine and succinyl-coenzyme A (CoA), an intermediate of the tricarboxylic acid (TCA) cycle.In E. coli, the native pathway for ALA biosynthesis is the C 5 pathway, which is tightly regulated by feedback inhibition of the end product heme (6). Previously, we developed a strategy to produce ALA in recombinant E. coli vi...

Enhancing 5‐aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli

“…ALA has recently received significant attention due to its numerous applications. To improve the production level of ALA, extensive efforts have been invested in screening novel enzymes (Lou et al, ; Zhang et al, ), balancing metabolic pathways (Ding, Weng, Du, Chen, & Kang, ; Feng et al, ; Noh, Lim, Park, Seo, & Jung, ), enhancing product export (Kang et al, ; Yang et al, ), and optimizing fermentation conditions (Yang et al, , ). However, to the best of our knowledge, no reports have thus far focused on improving ALA production by tolerance engineering.…”

“…P. Yang et al engineered C. glutamicum by expressing a heterologous ALAS and a nonspecific ALA exporter and deactivating the succinyl‐coenzyme A synthetase. The recombinant strain produced 14.7 g/L ALA from glucose and glycine by a two‐step fermentation (P. Yang et al, ). However, the two‐step fermentation strategy that requires cultivating, collecting, and resuspending cells in a new buffer seems challenging for large‐scale production.…”

Improved fermentative production of gamma‐aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose

“…By contrast, production of the glutamate-derived compound aminolevulinic acid was improved by addition of the glutamate trigger penicillin (Ramzi et al, 2015) or by deletion of the penicillin-binding protein genes pbp1a, pbp1b, and pbp2b (Feng et al, 2016). It has to be noted that glutamate is the main fermentation product in 5-ALA production which also requires addition of high concentrations of glycine as precursor (Feng et al, 2016;Ramzi et al, 2015;Yang et al, 2016).…”

Efficient bioproduction of 5-aminolevulinic acid, a promising biostimulant and nutrient, from renewable bioresources by engineered Corynebacterium glutamicum

“…By strengthening this biosynthetic pathway, 5-ALA production was achieved [14][15][16][17][18][19][20], but the highest titer and productivity were only 5.25 g/L and 0.16 g/L h, respectively [20]. To improve the 5-ALA production level, the exogenous C4 pathway for 5-ALA biosynthesis originated from photosynthetic bacteria was introduced into E. coli and C. glutamicum by expressing the 5-ALA synthetase (ALAS) catalyzing the condensation of succinyl-CoA and glycine to 5-ALA. Several strategies have been applied to further enforce the C4 biosynthetic route, such as enzyme screening [21][22][23][24][25][26], pathway engineering [27][28][29][30][31], tolerance engineering [32], and fermentation process optimization [27,33]. By reinforcing the native antioxidant defense system in an ALAS-expressing E. coli strain to combat with the reactive oxygen species generated by 5-ALA, Zhu et al obtained the highest 5-ALA titer (11.5 g/L) of one-step fermentation [32].…”