Iterative conformational dynamics-guided protein engineering reshapes biocatalyst properties for efficient and cost-effective cytidine 5ʹ-monophosphate production

Li, Zonglin; Lou, Longwei; Sun, Chuanqi; Xu, Wentian; Li, Zhimin

doi:10.1016/j.cej.2021.131597

Cited by 14 publications

(19 citation statements)

References 33 publications

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“…Then, the recombinant plasmids were transformed into E. coli Rosetta (DE3) for Sl UMPK and Da NDK production. Expression and purification of the enzymes were as described previously (Li et al, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…SlUMPK and DaNDK production. Expression and purification of the enzymes were as described previously (Li et al, 2021).…”

Section: Construction Of Expression Systemmentioning

confidence: 99%

“…Nucleoside kinase was derived from the thermophilic bacterium Phorcysia thermohydrogeniphila ( Pt NK). Based on the previous research (Li et al, 2021), a semirational design was used to obtain a variant with a 13.5‐fold increase in activity and a 7.6‐fold increase in catalytic efficiency ( k cat / K M ) compared to the wild type. Uridylate kinase was obtained from an acidophilic bacterium Sulfurovum lithotrophicum ( Sl UMPK), which is insensitive to UTP inhibition but has poor thermostability.…”

Section: Introductionmentioning

confidence: 99%

“…UTP is also involved in the synthesis of glycogen, has a specific role in immune metabolism, and benefits the respiratory system of the organism (Ding et al, 2020). In this paper, we established a four-enzyme-coupled pathway for in vitro production of UTP from uridine (Figure 1) and used Plasmids pET28a-rbsK, encoding the PtNK, and pET28a-ppk, encoding the PPK from S. lithotrophicum (SlPPK) were available in our laboratory (Li et al, 2021;Sun et al, 2021). All chemicals were purchased from Titan.…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, with the cross‐application of computer science, computational chemistry, and other disciplines in protein engineering, there is an increasing number of means to address the challenge and a spurt of success stories. For example, reconstructing ancestral sequences by inferring phylogenetic relationships between modern homologs and applying different amino acid substitution models is considered a powerful approach to screen for enhanced thermostability or unique active proteins (Furukawa et al, 2020; Gumulya et al, 2018; Selberg et al, 2021; Spence et al, 2021); iterative conformational dynamics analysis (ICDA) based on molecular dynamics (MD) simulations can continuously improve the reliability of key amino acids used to construct mutant libraries, thereby increasing the likelihood of obtaining target variants, even if the protein models used in the simulations do not achieve the accuracy of real experimental structures (Heo et al, 2019; Li et al, 2021); and remodeling of protein surface charges successfully alters the pH adaptation or thermostability of enzymes (Han et al, 2021; Huang et al, 2021; Nakamura et al, 2021; Wang et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Sun

Lou

et al. 2022

Biotech & Bioengineering

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The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway‐related substances. Here, we constructed a four‐enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2‐fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2‐fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two‐stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.

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Section: Methodsmentioning

confidence: 99%

“…SlUMPK and DaNDK production. Expression and purification of the enzymes were as described previously (Li et al, 2021).…”

Section: Construction Of Expression Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Sun

Lou

et al. 2022

Biotech & Bioengineering

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Engineering of the Lrp/AsnC‐type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L‐cysteine biosynthesis pathway in Escherichia coli

Zhou,

Li,

Zhong

et al. 2024

Biotech & Bioengineering

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L‐cysteine is an important sulfur‐containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high‐throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L‐cysteine‐related genes one by one. To overcome this limitation, in this study, a biosensor‐based approach for multilevel biosynthetic pathway optimization of L‐cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant‐based biosensor with improved specificity and an 8.71‐fold increase in dynamic range. Using the developed biosensor, we performed high‐throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29‐fold increase in L‐cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L‐cysteine, which enhances transcriptional activation. Overall, our biosensor‐based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

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Reaction-kinetic model-guided biocatalyst engineering for dual-enzyme catalyzed bioreaction system

Cheng

Zhou

Chen

et al. 2023

Chemical Engineering Journal

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Iterative conformational dynamics-guided protein engineering reshapes biocatalyst properties for efficient and cost-effective cytidine 5ʹ-monophosphate production

Cited by 14 publications

References 33 publications

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Engineering of the Lrp/AsnC‐type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L‐cysteine biosynthesis pathway in Escherichia coli

Reaction-kinetic model-guided biocatalyst engineering for dual-enzyme catalyzed bioreaction system

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