Enhancing β-Carotene Production in Escherichia coli by Perturbing Central Carbon Metabolism and Improving the NADPH Supply

Wu, Yuanqing; Ping, Yan; Li, Yang; Liu, Xuewei; Wang, Zhiwen; Chen, Tao; Zhao, Xiao‐Fan

doi:10.3389/fbioe.2020.00585

Cited by 39 publications

(29 citation statements)

References 55 publications

(82 reference statements)

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“…Thus, cofactor engineering is a useful strategy for enhancing the efficiency of metabolic pathways and maximising the metabolic flux toward target products [ 40 ]. Generally, increasing the NADPH supply in an organism involves reducing the NADPH consumption through competing pathways, thereby enhancing the regeneration of endogenous NADPH and conversion of NADH to NADPH [ 41 ]. This study investigated the effects of enhancing endogenous cofactor production and supplementing exogenous cofactor regeneration on MK-7 synthesis.…”

Section: Resultsmentioning

confidence: 99%

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

et al. 2022

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Background Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. Results We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. Conclusions In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

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Section: Resultsmentioning

confidence: 99%

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

et al. 2022

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“…Genetic engineering approaches such as the insertion of functional genes such as crtI , crtE , crtY , and crtYB enhance the production of β-carotene and block the expression of repressor genes like crgA which blocks the synthesis of β-carotene. These approaches have been applied to red yeast and fungus so far (Zhang et al 2016 ; Wu et al 2020 ). There are various environmental conditions such as organism-based culture condition, optimization of light intensity, salt stress, pH, and carbon sources that can be optimized for enhanced production of β-carotene.…”

Section: Discussionmentioning

confidence: 99%

Disease Prevention and Treatment Using β-Carotene: the Ultimate Provitamin A

Anand

Mohan

Bharadvaja

2022

Rev. Bras. Farmacogn.

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Humans being unable to synthesize beta-carotene, the provitamin A, depend on external sources as its supplement. Health benefits and dietary requirements of beta-carotene are interrelated. This orange-red coloured pigment has been enormously examined for its capacity to alleviate several chronic diseases including various types of cancer, cystic fibrosis, as well as COVID-19. However, this class of phytoconstituents has witnessed a broad research gap due to several twin conclusions that have been reported. Natural sources for these compounds along with their extraction methods have been mentioned. The current communication aims at contributing to the global scientific literature on beta-carotene’s application in prevention and treatment of lifestyle diseases. Graphical abstract

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“…The design of cascade reactions using scaffolds can be applied to the MEP pathway for the efficient production of retinal. Furthermore, increasing the level of co-factors such as ATP and NADPH, which play important roles as donors of phosphate and electrons, respectively, are crucial for metabolic pathways, so that they can be used to increase the efficiency of enzymes involved in the MVA pathway for carotenoid production [76]. Levels of NADPH can be increased by enhancing metabolic flux through the pentose phosphate pathway via knock out of genes encoding glucose 6-phosphate isomerase and PEP carboxylase [77].…”

Section: Future Perspectives Of Bco In Synthetic Bacteriamentioning

confidence: 99%

Molecular Properties of β-Carotene Oxygenases and Their Potential in Industrial Production of Vitamin A and Its Derivatives

et al. 2022

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β-Carotene 15,15′-oxygenase (BCO1) and β-carotene 9′,10′-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. β-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

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Enhancing β-Carotene Production in Escherichia coli by Perturbing Central Carbon Metabolism and Improving the NADPH Supply

Cited by 39 publications

References 55 publications

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

Disease Prevention and Treatment Using β-Carotene: the Ultimate Provitamin A

Molecular Properties of β-Carotene Oxygenases and Their Potential in Industrial Production of Vitamin A and Its Derivatives

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