Metabolic engineering of mevalonate-producing Escherichia coli strains based on thermodynamic analysis

Nagai, Hikaru; Masuda, Ami; Toya, Yoshihiro; Mizutani, Fumio; Shimizu, Hiroshi

doi:10.1016/j.ymben.2018.02.012

Cited by 22 publications

(17 citation statements)

References 53 publications

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“…We also constructed a gnd ‐deficient strain (MGΔgnd) to function as a branch point between the ED pathway and the PP pathway. Enhancing the ED pathway is a promising approach to increase mevalonate production (Nagai et al, 2018). The titer of mevalonate using the gnd ‐deficient strain MGΔgnd‐MV was only slightly increased (5.49 g/L) compared to wild‐type production (Figure 2b), which corresponds to previous reports.…”

Section: Resultsmentioning

confidence: 99%

“…Masuda et al (2017) improved mevalonate production by limiting nutrients in the medium and through cultivation at the stationary phase. Nagai et al (2018) also suggested that the Entner‐Doudoroff pathway (ED pathway) is more suitable for mevalonate production compared to the Embden‐Meyerhof‐Parnas Pathway (EMP pathway) according to the results of metabolic and thermodynamic analyses. Wang et al (2018) constructed the “EP‐bifido pathway” by introducing the fxpk gene from Bifidobacterium adolescentis into E. coli to increase the supply of acetyl‐CoA and produce a high yield (64.7 mol%) of mevalonate.…”

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Satowa

Fujiwara

Uchio

et al. 2020

Biotech & Bioengineering

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Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value‐added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate‐producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl‐CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA‐MV, increased levels of intracellular acetyl‐CoA up to sevenfold higher than the wild‐type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β‐glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Satowa

Fujiwara

Uchio

et al. 2020

Biotech & Bioengineering

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“…Candidates for the components of the push and pull strategy are commonly selected based on many studies on the related enzymes but there were a few articles on the biliverdin biosynthesis pathway in C. glutamicum . Therefore, we primally introduced in vitro thermodynamic analysis of all enzymes that were related to the pathway [37, 38]. This analysis provided information on the thermodynamic stability of enzymes using the calculation of Gibbs free energy.…”

Section: Discussionmentioning

confidence: 99%

Systems metabolic engineering of Corynebacterium glutamicum for the bioproduction of biliverdin via protoporphyrin independent pathway

Seok

Lee

et al. 2019

J Biol Eng

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Background Biliverdin, a prospective recyclable antioxidant and one of the most important precursors for optogenetics, has received growing attention. Biliverdin is currently produced by oxidation of bilirubin from mammalian bile using chemicals. However, unsustainable procedures of extraction, chemical oxidation, and isomer separation have prompted bio-based production using a microbial cell factory. Results In vitro thermodynamic analysis was performed to show potential candidates of bottleneck enzymes in the pathway to produce biliverdin. Among the candidates, hemA and hemL were overexpressed in Corynebacterium glutamicum to produce heme, precursor of biliverdin. To increase precursor supply, we suggested a novel hemQ -mediated coproporphyrin dependent pathway rather than noted hemN -mediated protoporphyrin dependent pathway in C. glutamicum . After securing precursors, hmuO was overexpressed to pull the carbon flow to produce biliverdin. Through modular optimization using gene rearrangements of hemA , hemL , hemQ , and hmuO , engineered C. glutamicum BV004 produced 11.38 ± 0.47 mg/L of biliverdin at flask scale. Fed-batch fermentations performed in 5 L bioreactor with minimal medium using glucose as a sole carbon source resulted in the accumulation of 68.74 ± 4.97 mg/L of biliverdin, the highest titer to date to the best of our knowledge. Conclusions We developed an eco-friendly microbial cell factory to produce biliverdin using C. glutamicum as a biosystem. Moreover, we suggested that C. glutamicum has the thermodynamically favorable coproporphyrin dependent pathway. This study indicated that C. glutamicum can work as a powerful platform to produce biliverdin as well as heme-related products based on the rational design with in vitro thermodynamic analysis. Electronic supplementary material The online version of this article (10.1186/s13036-019-0156-5) contains supplementary material, which is available to authorized users.

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“…Recently, activation of the Entner–Doudoroff (ED) pathway in genetically engineered strains of Escherichia coli has been shown to enhance glucose consumption, and isopropanol [ 15 ], mevalonate [ 16 ], and isobutanol [ 17 ] production. In these studies, the ED pathway that can supply NADPH was considered to be a better pathway in terms of carbon yield than the pentose phosphate (PP) pathway that can also supply NADPH, since the generation of NADPH in the PP pathway is accompanied by CO 2 generation in the 6-phosphogluconate dehydrogenase (6PGDH) reaction (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…1 ). It was also expected to bypass the rate-limiting steps of the EMP pathway, including the reactions catalyzed by 6-phosphofructokinase (PFK) and fructose-bisphosphate aldolase [ 16 ]. Although these studies have clearly indicated that activation of the ED pathway enhances glucose consumption, the mechanism underlying this phenomenon is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Coexistence of the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways enhances glucose consumption of ethanol-producing Corynebacterium glutamicum

Jojima

Igari

Noburyu

et al. 2021

Biotechnol Biofuels

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Background It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner–Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden–Meyerhof–Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways to coexist. Results Three genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A 13C-labeling experiment using [1-13C] glucose indicated a distinctive 13C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD+ ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed. Conclusions The increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.

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Metabolic engineering of mevalonate-producing Escherichia coli strains based on thermodynamic analysis

Cited by 22 publications

References 53 publications

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Systems metabolic engineering of Corynebacterium glutamicum for the bioproduction of biliverdin via protoporphyrin independent pathway

Coexistence of the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways enhances glucose consumption of ethanol-producing Corynebacterium glutamicum

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