Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli

Yu, Tzu-Hsuan; Yi, Ying-Chen; Shih, I-Tai; Ng, I‐Son

doi:10.1007/s12010-019-03178-9

Cited by 26 publications

(25 citation statements)

References 29 publications

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“…As presented in this study, our strategies in engineering the TCA cycle for improved succinyl‐CoA production generated higher levels of 5‐ALA (via Shemin pathway) compared to previous strategies in which carbon flux was directed toward α‐ketoglutarate precursor for 5‐ALA biosynthesis via the C5 pathway (Noh, Lim, Park, Seo, & Jung, 2017). While similar studies generated comparable 5‐ALA titers (Cui et al, 2019; Yu, Yi, Shih, & Ng, 2019; Zhang et al, 2019), our study showed higher 5‐ALA yields particularly when no structurally related carbons were supplemented. Future strain engineering for 5‐ALA biosynthesis via the Shemin pathway could include tuning of carbon flux via the glyoxylate shunt by manipulation of aceA expression which showed physiological advantages over iclR inactivation (Noh et al, 2017).…”

Section: Discussionsupporting

confidence: 46%

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Miscevic

Mao

Kefale

et al. 2020

Biotech & Bioengineering

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Herein, we report the development of a microbial bioprocess for high-level production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (~30 g L −1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L −1 (36.9% of the theoretical maximum yield) and 6.93 g L −1 (50.9% of the theoretical maximum yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

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Section: Discussionsupporting

confidence: 46%

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Miscevic

Mao

Kefale

et al. 2020

Biotech & Bioengineering

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“…Glycine is arepresentative amino acid that is widely used as af ood and feed additive,s upplement and chemical feedstock. [33][34][35][36][37][38] As conventional amino acid manufacturing relies on raw materials and fuels obtained from petroleum and toxic reagents, [4,39,40] this report provides asustainable route to alternate the existing processes.Moreover, we clearly demonstrated that our electrochemical approach enables utilization of astable carboxylic acid, oxalic acid, and nitrate for amine synthesis,w hereas existing chemical (non-electrochemical) reductive amination methods require reactive aldehydes/ketones and amines.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical Synthesis of Glycine from Oxalic Acid and Nitrate

et al. 2021

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In manufacturing C−N bond‐containing compounds, it is an important challenge to alternate the conventional methodologies that utilize reactive substrates, toxic reagents, and organic solvents. In this study, we developed an electrochemical method to synthesize a C−N bond‐containing molecule avoiding the use of cyanides and amines by harnessing nitrate (NO3−) as a nitrogen source in an aqueous electrolyte. In addition, we utilized oxalic acid as a carbon source, which can be obtained from electrochemical conversion of CO2. Thus, our approach can provide a route for the utilization of anthropogenic CO2 and nitrate wastes, which cause serious environmental problems including global warming and eutrophication. Interestingly, the coreduction of oxalic acid and nitrate generated reactive intermediates, which led to C−N bond formation followed by further reduction to an amino acid, namely, glycine. By carefully controlling this multireduction process with a fabricated Cu–Hg electrode, we demonstrated the efficient production of glycine with a faradaic efficiency (F.E.) of up to 43.1 % at −1.4 V vs. Ag/AgCl (current density≈90 mA cm−2).

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“…One is the C4 pathway (Shemin pathway), involving the formation of 5-ALA from succinyl-CoA (a C4-compound) and glycine via a single step decarboxylating condensation reaction, catalyzed by 5-ALA synthase (ALAS) (Warnick and Burnham 1971 ). This C4 pathway occurs in mammals, fungi and some photosynthetic bacteria (Kang et al 2012 ; Liu et al 2014 ; Li et al 2014 ; Yu et al 2020 ). The second route, the C5 pathway found in most bacteria and all plants, is an upstream metabolism of the heme biosynthesis (Schon et al 1986 ; Sasaki et al 2002 ).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, great efforts had been made to construct recombinant E. coli and C. glutamicum by expressing an exogenous 5-ALA synthetase (ALAS) gene in order to improve 5-ALA production via the C4 pathway (Li et al 2014 ; Yang et al 2016 ; Ding et al 2017 ; Chen et al 2020 ; Yu et al 2020 ; Miscevic et al 2021 ). So far, the production of 5-ALA has been significantly improved through fermentation process optimization, pathway engineering or tolerance engineering.…”

Section: Introductionmentioning

confidence: 99%

Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

et al. 2021

AMB Expr

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Abstract5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.

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Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli

Cited by 26 publications

References 29 publications

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Electrochemical Synthesis of Glycine from Oxalic Acid and Nitrate

Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

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