Enhancement of  <scp>l</scp>‐phenylalanine production in <i>Escherichia coli</i> by heterologous expression of <i>Vitreoscilla</i> hemoglobin

Wu, Weibin; Guo, Xiaolei; Zhang, Mingliang; Huang, Qing-Gen; Qi, Feng; Huang, Jianzhong

doi:10.1002/bab.1605

Cited by 22 publications

(13 citation statements)

References 32 publications

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“…To demonstrate this method, the optimization of a key enzyme of the chorismate pathway for the biosynthesis of L-tryptophan (Trp) was performed as an example. 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is a key enzyme for engineering microbes for efficient biosynthesis of aromatic amino acids (AAAs): Trp, phenylalanine (Phe), and tyrosine (Tyr) (Chen and Zeng, 2017; Kim et al., 2015; Wu et al., 2018). It is under strong feedback inhibition by the end products (Ogino et al., 1982; Sprenger, 2006).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Chen

Zeng

2019

Metabolic Engineering Communications

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Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intracellular conditions of the later production strain. This does not necessarily result in the identification of the best enzyme variant for in vivo use in the production strain. In this work, we propose a method which integrates CRISPR/Cas9-facilitated engineering of the target gene(s) with growth-coupled and sensor-guided in vivo screening (CGSS) for protein engineering and pathway optimization. The efficiency of the method is demonstrated for engineering 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase AroG, a key enzyme in the chorismate pathway for the synthesis of aromatic amino acids (AAAs), to obtain variants of AroG (AroG fbr ) with increased resistance to feedback inhibition of Phe. Starting from a tryptophan (Trp)-producing E. coli strain (harboring a reported Phe-resistant AroG variant AroG S180F ), the removal of all the endogenous DAHP synthases makes the growth of this strain dependent on the activity of an introduced AroG variant. The different catalytic efficiencies of AroG variants lead to different intracellular concentration of Trp which is sensed by a Trp biosensor (TnaC-eGFP). Using the growth rate and the signal strength of the biosensor as criteria, we successfully identified several novel Phe-resistant AroG variants (including the best one AroG D6G−D7A ) which exhibited higher specific enzyme activity than that of the reference variant AroG S180F at the presence of 40 mM Phe. The replacement of AroG S180F with the newly identified AroG D6G−D7A in the Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ± 1.02 g/L at 36 h) in a simple fed-batch fermentation.

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Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Chen

Zeng

2019

Metabolic Engineering Communications

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“…These had led to increased productivity with a decrease demand for aeration [89]. Recombinant E. coli strain PAPV (harboring aro F, phe A fbr, and tac‐vgd genes) in the aerobic fermentation process has shown more cell growth (22%) and higher l ‐phenylalanine production (16.6%) from more glucose consumption (5%), compared to the strain (harboring only aroF and pheA fbr in E. coli PAP) under oxygen‐limited conditions [92].…”

Section: Primary Metabolitesmentioning

confidence: 99%

Primary metabolites from overproducing microbial system using sustainable substrates

Srivastava

Akhtar

Verma

et al. 2020

Biotech and App Biochem

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Primary (or secondary) metabolites are produced by animals, plants, or microbial cell systems either intracellularly or extracellularly. Production capabilities of microbial cell systems for many types of primary metabolites have been exploited at a commercial scale. But the high production cost of metabolites is a big challenge for most of the bioprocess industries and commercial production needs to be achieved. This issue can be solved to some extent by screening and developing the engineered microbial systems via reconstruction of the genome‐scale metabolic model. The predicted genetic modification is applied for an increased flux in biosynthesis pathways toward the desired product. Wherein the resulting microbial strain is capable of converting a large amount of carbon substrate to the expected product with minimum by‐product formation in the optimal operating conditions. Metabolic engineering efforts have also resulted in significant improvement of metabolite yields, depending on the nature of the products, microbial cell factory modification, and the types of substrate used. The objective of this review is to comprehend the state of art for the production of various primary metabolites by microbial strains system, focusing on the selection of efficient strain and genetic or pathway modifications, applied during strain engineering.

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“…The dissolved oxygen (DO) in culture is considered an important factor for bacterial growth and l-phenylalanine productivity. To increase the DO level, co-expression of a hemoglobin gene from Vitreoscilla with aroF and pheA FBR was performed in E. coli CICC10245, which led to 21.9% more biomass and 16.6% more l-phenylalanine production, while only approximately 5% more glucose was consumed (Wu et al 2018). Another strategy to relieve the feedback inhibition of key enzymes is reduction of intracellular accumulation of l-phenylalanine.…”

Section: Other Engineering Targets For L-phenylalanine Productionmentioning

confidence: 99%

Metabolic engineering for the production of l-phenylalanine in Escherichia coli

Liu

Niu

et al. 2019

3 Biotech

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As one of the three proteinogenic aromatic amino acids, l-phenylalanine is widely applied in the food, chemical and pharmaceutical industries, especially in production of the low-calorie sweetener aspartame. Microbial production of l-phenylalanine has become attractive as it possesses the advantages of environmental friendliness, low cost, and feedstock renewability. With the progress of metabolic engineering, systems biology and synthetic biology, production of l-phenylalanine from glucose in Escherichia coli with relatively high titer has been achieved by improving the intracellular levels of precursors, alleviating transcriptional repression and feedback inhibition of key enzymes, increasing the export of l-phenylalanine, engineering of global regulators, and overexpression of rate-limiting enzymes. In this review, successful metabolic engineering strategies for increasing l-phenylalanine accumulation from glucose in E. coli are described. In addition, perspectives for further improvement of production of l-phenylalanine are discussed.

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Enhancement of l‐phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin

Cited by 22 publications

References 32 publications

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Primary metabolites from overproducing microbial system using sustainable substrates

Metabolic engineering for the production of l-phenylalanine in Escherichia coli

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