Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission

Wang, Qian; Xu, Jiasheng; Sun, Zhiqiang; Luan, Yaqi; Liu, Ying; Wang, Junshu; Liang, Quanfeng; Qi, Qingsheng

doi:10.1016/j.ymben.2018.08.003

Cited by 65 publications

(62 citation statements)

References 43 publications

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“…Furthermore, we additionally introduced pZA‐AES into MGΔgltA‐Mv, but this only led to a slight increase in mevalonate production (Figure 3b). We also introduced pZA‐XPK into MGΔgltA‐Mv according to a previous report (Wang et al, 2018). However, pZA‐XPK had no positive effect on MGΔgltA‐Mv after 66 hr cultivation (data not shown).…”

Section: Resultsmentioning

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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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%

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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“…The MVA production by these strains did not show an obvious positive correlation with the zwf promoter strength. Since the MVA yield did not represent the ux distribution between the EMP pathway and the PPP, 13 C-MFA was performed to detected the metabolic ux distribution in strains BW-P08 BF and BW-P10 BF (which had high MVA titers) and the control strain BW-P BF. The ux ratio between the PPP and the EMP pathway in strains BW-P08 BF and BW-P10 BF was much higher than that in strain BW-P BF (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Suitable ux distribution between the EMP pathway and PPP has a positive effect on the production of target compounds. In the EP-bi do pathway, the theoretical optimal carbon split to the EMP pathway and PPP in the EP-bi do route for MVA production is 1:6 [13]; therefore, the maximum carbon theoretical conversion rate of the EP-bi do pathway is 86% (mol/mol). In our previously constructed EP-bi do pathway, the carbon split ratio was 0.43:0.57.…”

Section: Enhancement Of Ppp Ux By Increasing Zwf Expressionmentioning

confidence: 99%

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Fine and dynamic tuning the glycolytic flux ratio of an artificial carbon saving pathway for high yield of mevalonate in Escherichia coli

Liu¹,

Xian²,

Xu³

et al. 2020

Preprint

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Background In natural Escherichia coli, glucose is mainly metabolized via the Embden-Meyerhoff-Parnas (EMP) pathway. However, in the metabolic process of conversion of pyruvate to acetyl-CoA, one-third of the carbon is lost at CO2. To decrease the loss of glucose in the metabolic process and enhance the carbon conversion efficiency production of desired products by E. coli, we constructed a carbon saving pathway, EP-bifido pathway. As the balance of energy and reducing power was not optimal, we use synthetic biology methods to precisely and dynamically adjust the EMP pathway and pentose phosphate pathway (PPP) flux to improve the production of mevalonate (MVA) via the EP-bifido pathway. ResultHere, we enhanced the MVA titer and yield in E. coli in two ways. First, the promoter of the first gene of the PPP, zwf, was replaced with a set of promoters of different strength to enhance PPP flux for NADPH supply. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels and production routes. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, the expression of pfkA was suppressed at a certain time by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system to avoid the growth defect caused by pfkA direct knock-out. The resulting MVA yield of strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. ConclusionThis is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

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“…This could be due to insufficient reducing equivalents generated via NOG. In addition, a bifid shunt, which also involves Pkt, has been introduced in E. coli under the same biotechnological principle of NOG 11 . The EP-bifido pathway has enabled the increase in production of acetyl-CoA from acetyl phosphate (AcP) and has been applied to enhance the levels of acetyl-CoA–derived chemicals.…”

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confidence: 99%

A hybrid Embden–Meyerhof–Parnas pathway provides a synthetic link between sugar and phosphate metabolism

Lee

Cho

Woo

2020

Preprint

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16The fundamental Embden-Meyerhoff-Paranas (EMP) pathway for sugar catabolism, 17 anabolism, and energy metabolism has been reconstituted with non-oxidative glycolysis 18 (NOG). Although carbon conservation was achieved via NOG, the energy metabolism 19 was significantly limited. Herein, we showed the construction of a hybrid EMP that 20 replaced the first phase of the EMP in Corynebacterium glutamicum with NOG and 21 revealed a metabolic link of carbon and phosphorus metabolism. In accordance with 22 synthetic glucose kinase activity and phosphoketolase on the hybrid EMP, cell growth 23 was completely recovered in the C. glutamicum pfkA mutant strain where the first phase 24 of EMP was eliminated. Notably, we have revealed a phosphate-replenishing pathway 25 that involved trehalose biosynthesis for the generation of inorganic phosphate (Pi) sources 26 in the hybrid EMP when external Pi supply was limited. Thus, the re-designed hybrid 27 EMP pathway with balanced carbon and phosphorus states provides an efficient 28 microbial platform for biochemical production. 29 30 31Recently, carbon conservation in sugar metabolism has been successfully achieved using 49 synthetic design with Pkt and a carbon rearrangement cycle to generate 3 acetyl-CoA per 50 fructose 6-phosphate (F6P) molecule 9 . Non-oxidative glycolysis (NOG) has provided a 51 biotechnological basis for increasing the theoretical yield by reducing CO2 evolution. To fulfill 52 carbon conservation in Escherichia coli, adaptive evolution has been performed in a minimal 53 glucose medium after rational metabolic engineering 10 , which results in slower growth of the 54 NOG strains than their parental strain. This could be due to insufficient reducing equivalents 55 generated via NOG. In addition, a bifid shunt, which also involves Pkt, has been introduced in 56

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Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO2 emission

Cited by 65 publications

References 43 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

Fine and dynamic tuning the glycolytic flux ratio of an artificial carbon saving pathway for high yield of mevalonate in Escherichia coli

A hybrid Embden–Meyerhof–Parnas pathway provides a synthetic link between sugar and phosphate metabolism

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