Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

Kang, Zhen; Yang, Weijun; Gu, Pengfei; Wang, Qian; Qi, Qingsheng

doi:10.1016/j.ymben.2011.05.003

Cited by 126 publications

(111 citation statements)

References 44 publications

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“…If optimal hemoprotein expression is required, our heme-overexpression system could be improved by tuning the expression level of the HEM genes. Similarly, although feeding ALA is a straightforward method for increasing precursor availability for the heme biosynthetic pathway, the cost would be prohibitive on an industrial scale, and overexpression of the ALA biosynthetic pathway might be a preferable solution (44). Finally, this coupled strategy of optimizing an enzyme and its host could be applied iteratively, for example, by finding newly beneficial enzyme mutations that take advantage of the increased heme supply or identifying the next limiting stress once the host has sufficient heme.…”

Section: Discussionmentioning

confidence: 99%

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Michener

Nielsen

Smolke

2012

Proc. Natl. Acad. Sci. U.S.A.

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Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.systems biology | yeast | CYP102A1 | cytochrome P450

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Section: Discussionmentioning

confidence: 99%

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Michener

Nielsen

Smolke

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…6) (20). Finally, we overexpressed the heterologous nonspecific ALA exporter RhtA from E. coli together with the R. capsulatus ALAS (hemA) (7). This modification increased the ALA titer from 12.46 g/liter to 14.7 g/liter in 16 h, resulting in a high productivity of 0.92 g/liter/h (Fig.…”

Section: Figmentioning

confidence: 99%

“…Previously, we developed a strategy to produce ALA in recombinant E. coli via the C 5 pathway. Through overexpression of heterologous stabilized HemA from Salmonella arizona and HemL from E. coli, with the concomitant expression of an ALA exporter, the recombinant E. coli produced 4.13 g/liter ALA in modified minimal medium, using glucose as the sole carbon source (7). However, because the C 5 pathway involves a number of enzymatic activities, utilizes ATP and NADPH as cofactors, and is dependent on tRNA-Glu, its complicated relationships with energy metabolism, oxidation-reduction states, and protein synthesis make subsequent strain improvement rather difficult (8,9).…”

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confidence: 99%

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

Yang

Liu

Cheng

et al. 2016

Appl Environ Microbiol

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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...

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“…19 Even so, due to the addition of precursor glycine and succinic acid, it is still more attractive to produce ALA directly from glucose with a metabolic engineering strategy. [20][21] Previously, it has been demonstrated that the key enzyme GluTR of the C5 pathway is feed-back inhibited by heme. [22][23] Further studies confirmed that N-terminal domain of GluTR plays an important effect on the protein stability and insertion of 2 lysine residues with positive charge to the third and forth positions at the Nterminus of GluTR improved protein stabilization.…”

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confidence: 99%

“…24 Accordingly, overexpression of the GluTR mutant with GSA-AT substantially enhanced ALA production. 20 Then, based on the fact that ALA dehydratase is feedback inhibited by the intermediate protoporphyrinogen IX, 25 both cell growth and ALA production was further improved with addition of Fe 2+ in the minimal medium. 26 However, the stability of GluTR was still a rate-limiting step for achieving high titer of ALA.…”

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confidence: 99%

N-terminal engineering of glutamyl-tRNA reductase with positive charge arginine to increase 5-aminolevulinic acid biosynthesis

et al. 2016

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Five-Aminolevulinic acid (ALA), the universal precursor of all tetrapyrroles, has various applications in medicine and agriculture industries. Glutamyl-tRNA reductase (GluTR) as the first key enzyme of C5 pathway is feedback regulated by heme, and its N-terminus plays a critical role on its stability control. Here, the GluTR N-terminus was engineered by inserting different numbers of positively charged lysine and arginine residues. The results confirmed that insertion of lysine or arginine residues (especially one arginine residue) behind Thr2 significantly increased the stability of GluTR. By co-expression of the GluTR variant R1 and the glutamate-1-semialdehyde aminotransferase, ALA production was improved 1.76-fold to 1220 mg/L. The GluTR variant R1 constructed here could be used for engineering the C5 pathway to enhance ALA and other products.

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Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

Cited by 126 publications

References 44 publications

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

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

N-terminal engineering of glutamyl-tRNA reductase with positive charge arginine to increase 5-aminolevulinic acid biosynthesis

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