Metabolic engineering of Escherichia coli for secretory production of free haem

Zhao, Xin; Choi, Kyeong Rok; Lee, Sang Yup

doi:10.1038/s41929-018-0126-1

Cited by 91 publications

(123 citation statements)

References 37 publications

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“…As expected, the growth trend and HBA titer of the strain with hemE knockdown increased compared with the control strain. In contrast with the instability of the antisense RNA strategy [12], the heme levels of strains constructed via RBS engineered remained stable until the end of fermentation. Our results thus confirmed the feasibility of using RBS engineering to control the metabolic flux of the heme branch.…”

Section: Discussionmentioning

confidence: 92%

“…However, these two competing pathways cannot be completely knocked out because they are essential. It has been reported that antisense RNA and small RNA strategies are effective approaches for knocking down the heme branch, but the effect was unstable and the inhibition level cannot be finely controlled [6,12]. To overcome these shortcomings, we used the CRISPR/cas9 system to edit the E. coli genome and decreased the effects of the heme and siroheme branches by reducing the initial translation rates of hemE and cysG, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…A reverse-phase Agilent TC-C18 column (5 μ m, 4.6 mm × 250 mm) was used and the absorbance at 400 nm was monitored. The elution method was modified according to a previous report [12]. For mobile phase, 30% methanol with 0.1% trifluoroacetic acid was used for the first 1 min with a flow rate of 0.8 mL min −1 .…”

Section: Methodsmentioning

confidence: 99%

“…and eliminating feedback inhibition in Bacillus megaterium [11]. Zhao et al successfully increased the yield of free heme in E. coli by screening overexpression targets in the heme biosynthesis pathway, regulating the metabolic flux in heme biosynthesis, and disrupting the putative heme degradation enzyme encoded by the yfeX gene [12].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

et al. 2020

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Background: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B 12. The introduction of a heterologous de novo vitamin B 12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B 12 , such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B 12 production. Results: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L −1 , respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L −1 , representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. Conclusions: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L −1 in E. coli. This lays the foundation for high-yield production of vitamin B 12 in E. coli and will hopefully accelerate its industrial production.

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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

et al. 2020

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“…Allergies and antibiotic contamination have caused widespread public concern for food quality and safety [16]. Plant-based protein, such as soy protein, is restricted due to allergies from β-conglycinin and soy globulin [17], and reducing the content of these allergens is needed to avoid health risks in future food production. Many studies have shown that the proteases papain and pepsin can hydrolyze soybean allergens and relieve allergenicity [18].…”

Section: Food Safetymentioning

confidence: 99%

Current progress and prospects of enzyme technologies in future foods

Pang

Yin

Zhang

et al. 2020

Syst Microbiol and Biomanuf

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Enzyme technologies are widely used in the food industry due to their advantages of high efficiency, specificity, and safety. Recently, "future foods" is emerging as a new research hotspot with healthier foods that are more nutritious, delicious, and sustainable; however, these foods still have problems with texture, nutrition, and flavor. Advances in enzyme technology have enabled the development of new tools and approaches to better manipulate food textures and nutritional aspects. In this review, we summarize enzyme technology applications in future food production, focusing on food texture, safety, and flavors. Furthermore, we discuss the prospects of enzyme-based technologies for future food production, including the modification of enzyme activities, the development of suitable food-grade hosts for enzyme production, and the optimization synergistic multi-enzyme systems.

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Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

Zhou

et al. 2022

Advanced Science

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By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins.

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Metabolic engineering of Escherichia coli for secretory production of free haem

Cited by 91 publications

References 37 publications

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies

Current progress and prospects of enzyme technologies in future foods

Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

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