High-level production of l-homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering

Zhang, Yu; Wei, Minhua; Zhao, Genhai; Zhang, Wenjie; Li, Yingzi; Lin, Bor‐Shyh; Li, Yanjun; Xu, Qingyang; Chen, Ning; Zhang, Chenglin

doi:10.1016/j.biortech.2021.124814

Cited by 38 publications

(42 citation statements)

References 37 publications

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“…The dynamic control of gene expression has been suggested to provide a promising approach for addressing this issue and controlling cell growth and production (C. Y. Zhang et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for efficient osmotic stress‐free production of compatible solute hydroxyectoine

et al. 2021

Biotech & Bioengineering

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Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/ yield, the presence of the byproduct ectoine, and the requirement of high salinity.Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based autoregulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.

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“…The dynamic control of gene expression has been suggested to provide a promising approach for addressing this issue and controlling cell growth and production (C. Y. Zhang et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for efficient osmotic stress‐free production of compatible solute hydroxyectoine

et al. 2021

Biotech & Bioengineering

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“…In the recent decades, public awareness toward green and sustainable biosynthesis is rapidly increasing worldwide due to non-renewable petroleum resources and environmental issues. [1][2][3][4] Many kinds of products have been achieved using microbial production, such as chemicals and natural products. [5,6] For industrial bioproduction, one important issue is the stability of the engineered strain, which can maintain excellent performance and maximize the productivity during fermentation.…”

Section: Introductionmentioning

confidence: 99%

“…Several strategies have been used for the e cient engineering of cell factories with well stability, including modi cation at genome-level [3,7] and maintaining the plasmid stability [8,9]. Genome modi cation can be achieved using genome editing tools, such as the CRISPR-Cas9 system.…”

Section: Introductionmentioning

confidence: 99%

“…[7] Generally, due to the overexpression of key genes, antibiotics and inducers need to be added during the process of fermentation to avoid plasmid losing and induce gene expression by recombinant plasmids. [3,10] It has been observed that existence of expression plasmids in recombinant cells entails a metabolic burden of cells by replication, transcription and protein production, which can cause the loss of plasmid during fermentation process. [1] Additionally, the demand for antibiotics supplementation could increase the costs and result in environmental contamination.…”

Section: Introductionmentioning

confidence: 99%

“…L-homoserine, as one of the AFAAs, is an attractive platform precursor for the biosynthesis of many important compounds, such as L-threonine, L-methionine, 1,4-butanediol, L-phosohinotheicin and butyrolactone. [3,18,19] Thus, L-homoserine has been attracted great interests and is widely used in food, agriculture, pharmaceutical, cosmetic product ingredient, and animal-feed additive. [2,3,14] Constructing an e cient and sustainable L-homoserine producing cell factory with high titer and stability is imperative for the fairly increasing market capacity demand and application of L-homoserine.…”

Section: Introductionmentioning

confidence: 99%

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Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Sun

Tao

Sui

et al. 2021

Preprint

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Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.

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Targeting metabolic driving and minimization of by‐products synthesis for high‐yield production of D‐pantothenate in Escherichia coli

Zhang

Wang

et al. 2021

Biotechnology Journal

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Background: D-Pantothenate (DPA) is an important functional chemical that has been widely applied in healthcare, cosmetics, animal food, and feed industries. Methods and Results:In this study, a high-yield DPA-producing strain was constructed by metabolic engineering strategies with targeting metabolic driving and by-products minimization. The metabolic driving force of push and pull was firstly obtained to improve the production of DPA via enrichment of precursor pool and synthetic pathway, accumulating 4.29 g L −1 DPA in shake flask fermentation. To eliminate the metabolic pressure on DPA production, an amino throttling system was proposed and successfully attenuated the synthesis of four competitive amino acids by a single-step regulation of gdhA. Further minimization of acetate was carried out by pta deletion, and utilization of β-alanine was improved via enhancing its uptake system with producing 5.78 g L −1 DPA. Finally, the engineered strain produced 66.39 g L −1 DPA with β-alanine addition in fermentor under fed-batch fermentation. Conclusion:This study paved a foundation for the industrial production of DPA. K E Y W O R D Sβ-alanine, by-products, D-pantothenate, metabolic driving force, metabolic engineering INTRODUCTIOND-Pantothenate (DPA), also known as vitamin B5 (C 9 H 17 O 5 N), is a water-soluble vitamin that was discovered and extracted from animal liver to cure chicken pellagra, and was firstly synthesized in 1940. [1][2][3] DPA, the bioactive dextrorotatory (D) isomer, [4] is a key precursor used for biosynthesizing coenzyme A (CoA) and acyl carrier protein. [5,6] DPA, therefore, is widely used in healthcare, cosmetic, animal food, and feed industries. [7] DPA can be synthesized themselves in bacteria, fungi, archaea, and some plants, but animals can only obtain DPA from diet because of the absence of de novo biosynthetic pathway. [8,9] Abbreviations: AHAS, acetohydroxy acid synthase; CoA, coenzyme A; DPA, D-pantothenate;IPTG, isopropyl β-D-1-thiogalactopyranoside; PEP, phosphoenolpyruvate Consequently, the biosynthesis pathway of DPA in microorganisms and plants is worth noting for the development of antimicrobial, fungicidal, and herbicidal compounds. [10] In addition, DPA as a coenzyme participates in some enzyme reactions involved in protein metabolism and lipid metabolism. [11] Currently, DPA used for industrial application is mainly produced by chemical method via condensation of D-pantolactone and β-alanine in methanol or ethanol. D-Pantolactone is performed by chemical resolution or enzymatic catalysis from DL-pantolactone. However, chemical resolution, in addition to the usage of toxic chemical reagents, is laborious and expensive. Although enzymatic catalysis avoids the above disadvantages, the substrate DL-pantolactone is synthesized from the condensation of isobutyraldehyde and formaldehyde which are also not eco-friendly reagents. With the development of genetic

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High-level production of l-homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering

Cited by 38 publications

References 37 publications

Metabolic engineering of Escherichia coli for efficient osmotic stress‐free production of compatible solute hydroxyectoine

Metabolic engineering of Escherichia coli for efficient osmotic stress‐free production of compatible solute hydroxyectoine

Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Targeting metabolic driving and minimization of by‐products synthesis for high‐yield production of D‐pantothenate in Escherichia coli

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