Engineering transcription factor BmoR for screening butanol overproducers

Yu, Huan; Chen, Zhenya; Wang, Ning; Yu, Shengzhu; Yan, Yajun; Huo, Yi-Xin

doi:10.1016/j.ymben.2019.08.015

Cited by 33 publications

(32 citation statements)

References 53 publications

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“…Innate transcriptional regulatory protein (TRP)-based biosensors are widely-used in natural product high-throughput screening [23][24][25][26] . However, under most circumstances, innate transcription factors responsive to targeted compounds are not available.…”

Section: Discussionmentioning

confidence: 99%

“…Balancing pathway enzyme activities has been proved critical for pathway efficiency 23,24 . The reaction catalyzed by TYO, the rate-limiting enzyme of the redesigned hydroxytyrosol pathway, generated ammonia and hydrogen peroxide 25,26 , thus, an optimal activity instead of extremely high activity would benefit the whole pathway efficiency. In vivo-directed evolution combined with high-throughput screening of the end product is a good strategy for optimizing this enzyme to achieve improved hydroxytyrosol biosynthetic efficiency, by avoiding the toxicity caused by intermediate accumulation.…”

Section: Discussionmentioning

confidence: 99%

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Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Yao

et al. 2020

Nat Commun

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Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second ratelimiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Yao

et al. 2020

Nat Commun

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“…As a transcriptional regulator, SolR can positively control sporulation and solvent production, so that the combination of inactivating SolR and overexpressing aad was used to improve cellular performance, increasing the production of butanol, acetone, and ethanol to 17.6 g/L, 8.2 g/L, and 2.2 g/L, respectively [103]. More importantly, other regulatory factors (such as SpoIIE, sigF, sigE, and sigG) have been identified as being involved with improving cell growth and solvent formation, showing that understanding the regulatory mechanism of the solventogenic shift is of great interest, but identification of the molecular triggering machinery still presents an obstacle [104].…”

Section: Strain Improvement By Metabolic Engineeringmentioning

confidence: 99%

Pathway dissection, regulation, engineering and application: lessons learned from biobutanol production by solventogenic clostridia

Huang

et al. 2020

Biotechnol Biofuels

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The global energy crisis and limited supply of petroleum fuels have rekindled the interest in utilizing a sustainable biomass to produce biofuel. Butanol, an advanced biofuel, is a superior renewable resource as it has a high energy content and is less hygroscopic than other candidates. At present, the biobutanol route, employing acetone-butanolethanol (ABE) fermentation in Clostridium species, is not economically competitive due to the high cost of feedstocks, low butanol titer, and product inhibition. Based on an analysis of the physiological characteristics of solventogenic clostridia, current advances that enhance ABE fermentation from strain improvement to product separation were systematically reviewed, focusing on: (1) elucidating the metabolic pathway and regulation mechanism of butanol synthesis; (2) enhancing cellular performance and robustness through metabolic engineering, and (3) optimizing the process of ABE fermentation. Finally, perspectives on engineering and exploiting clostridia as cell factories to efficiently produce various chemicals and materials are also discussed.

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“…LuxR, a TF that is involved in quorum sensing in many bacteria, has been engineered to respond to butanoyl-homoserine lactone at concentrations as low as 10 nM (Hawkins et al, 2007). Structural analysis and site-directed mutagenesis were used to engineer a BmoR mutant that has a wider detection range (0-100 mM) for intracellular isobutanol than the wild-type protein (Yu et al, 2019). Promoter binding sites can also influence the dynamic range of TFs.…”

Section: Monitoring Metabolites In Vivomentioning

confidence: 99%

“…Novel genomic editing and protein engineering tools have been applied to synthesize target products via TF-mediated activation of silent BGCs (Tong et al, 2015;Zhang et al, 2017;Grau et al, 2018). Because TFs are sensitive to their corresponding signal molecules, they can also be used to construct highly sensitive biosensors for use in high-throughput screening for improved strains (Yu et al, 2019). Protein engineering can be used to alter the ligand specificity of TFs such that they can detect new signaling molecules (Machado et al, 2019), furthering expanding their applications in metabolic engineering.…”

Section: Introductionmentioning

confidence: 99%

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review

Zhang

et al. 2020

Front. Bioeng. Biotechnol.

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Metabolic regulation of gene expression for the microbial production of fine chemicals, such as organic acids, is an important research topic in post-genomic metabolic engineering. In particular, the ability of transcription factors (TFs) to respond precisely in time and space to various small molecules, signals and stimuli from the internal and external environment is essential for metabolic pathway engineering and strain development. As a key component, TFs are used to construct many biosensors in vivo using synthetic biology methods, which can be used to monitor the concentration of intracellular metabolites in organic acid production that would otherwise remain "invisible" within the intracellular environment. TF-based biosensors also provide a highthroughput screening method for rapid strain evolution. Furthermore, TFs are important global regulators that control the expression levels of key enzymes in organic acid biosynthesis pathways, therefore determining the outcome of metabolic networks. Here we review recent advances in TF identification, engineering, and applications for metabolic engineering, with an emphasis on metabolite monitoring and high-throughput strain evolution for the organic acid bioproduction.

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Engineering transcription factor BmoR for screening butanol overproducers

Cited by 33 publications

References 53 publications

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Pathway dissection, regulation, engineering and application: lessons learned from biobutanol production by solventogenic clostridia

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review

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