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Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration

BIOTECHNOLOGY ADVANCES 2023年第1期63卷 108102页

作者： Nielsen, Jochem R. Weusthuis, Ruud A. Huang, Wei E. Univ Oxford Dept Engn Sci Oxford OX1 3PJ England Wageningen Univ & Res Dept Bioproc Engn NL-6700 AA Wageningen Netherlands

Enzymes need to be efficient, robust, and highly specific for their effective use in commercial bioproduction. These properties can be introduced using various enzyme engineering techniques, with random mutagenesis and directed evolution (DE) often being chosen when there is a lack of structural information-or mechanistic un- derstanding-of the enzyme. The screening or selection step of DE is the limiting part of this process, since it must ideally be (ultra)-high throughput, specifically target the catalytic activity of the enzyme and have an accurately quantifiable metric for said activity. Growth-coupling selection strategies involve coupling a desired enzyme activity to cellular metabolism and therefore growth, where growth (rate) becomes the output metric. redox cofactors (NAD+/NADH and NADP+/NADPH) have recently been identified as promising target molecules for growth coupling, owing to their essentiality for cellular metabolism and ubiquitous nature. redox cofactor oxidation or reduction can be disrupted through metabolic engineering and the use of specific culturing con-ditions, rendering the cell inviable unless a 'rescue' reaction complements the imposed metabolic deficiency. Using this principle, enzyme variants displaying improved cofactor oxidation or reduction rates can be selected for through an increased growth rate of the cell. In recent years, several E. coli strains have been developed that are deficient in the oxidation or reduction of NAD+/NADH and NADP+/NADPH pairs, and of non-canonical redox cofactor pairs NMN+/NMNH and NCD+/NCDH, which provides researchers with a versatile toolbox of enzyme engineering platforms. A range of redox cofactor dependent enzymes have since been engineered using a variety of these strains, demonstrating the power of using this growth-coupling technique for enzyme engi-neering. This review aims to summarize the metabolic engineering involved in creating strains auxotrophic for the reduced or oxidized state of redox c

关键词： Growth -coupling Metabolic engineering redox cofactor Enzyme engineering redox cofactor auxotrophy Non -canonical redox cofactor

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A semisynthetic, multicofactor artificial metalloenzyme retains independent site activity

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JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 2025年第1期30卷 13-23页

作者： Wertz, Ashlee E. Rosenkampff, Ilmari Ibouanga, Philippe Huber, Matthias Hess, Corinna R. Ruediger, Olaf Shafaat, Hannah S. Ohio State Univ Dept Chem & Biochem 100 W 18th Ave Columbus OH 43210 USA Max Planck Inst Chem Energy Convers Stiftstr 34-36 D-45470 Mulheim An Der Ruhr Germany Tech Univ Munich Catalysis Res Ctr Bioinorgan Chem D-85748 Garching Germany Univ Paris Saclay Inst Chim Mol & Materiaux Orsay CNRS F-91405 Orsay France Univ Regensburg Fac Chem & Pharm D-93053 Regensburg Germany Univ Calif Los Angeles Dept Chem & Biochem 607 Charles E Young Dr East Los Angeles CA 90095 USA

Native metalloenzymes are unparalleled in their ability to perform efficient small molecule activation reactions, converting simple substrates into complex products. Most of these natural systems possess multiple metallocofactors to facilitate electron transfer or cascade catalysis. While the field of artificial metalloenzymes is growing at a rapid rate, examples of artificial enzymes that leverage two distinct cofactors remain scarce. In this work, we describe a new class of artificial enzymes containing two different metallocofactors, incorporated through bioorthogonal strategies. Nickel-substituted rubredoxin (NiRd), which is a structural and functional mimic of [NiFe] hydrogenases, is used as a scaffold. Incorporation of a synthetic bimetallic inorganic complex based on a macrocyclic biquinazoline ligand (MMBQ) was accomplished using a novel chelating thioether linker. Neither the structure of the NiRd active site nor the MMBQ were altered upon attachment, and each site retained independent redox activity. Electrocatalysis was observed from each site, with the switchability of the system demonstrated through the use of catalytically inert metal centers. This MMBQ-NiRd platform offers a new avenue to create multicofactor artificial metalloenzymes in a robust system that can be easily tuned both through modifications to the protein scaffold and the synthetic moiety, with applications for redox catalysis and tandem reactivity.

关键词： Hydrogenase redox cofactor Electron transfer Bioorthogonal coupling Electrocatalysis

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Stabilization of the catalytically active structure of a molybdenum-dependent formate dehydrogenase depends on a highly conserved lysine residue

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FEBS JOURNAL 2025年第12期292卷 3165-3179页

作者： Li, Feilong Lienemann, Michael Aalto Univ Dept Bioprod & Biosyst Espoo Finland VTT Tech Res Ctr Finland Ltd Espoo Finland

Molybdenum-dependent formate dehydrogenases (Mo-FDHs) reversibly catalyze the interconversion of CO2 and formate, and therefore may be utilized for the development of innovative energy storage and CO2 utilization concepts. Mo-FDHs contain a highly conserved lysine residue in the vicinity of a catalytically active molybdenum (Mo) cofactor and an electron-transferring [4Fe-4S] cluster. In order to elucidate the function of the conserved lysine, we substituted the residue Lys44 of Escherichia coli formate dehydrogenase H (EcFDH-H) with structurally and chemically diverse amino acids. Enzyme kinetic analysis of the purified EcFDH-H variants revealed the Lys-to-Arg substitution as the only amino acid exchange that retained formate oxidation catalytic activity, amounting to 7.1% of the wild-type level. Ultraviolet-visible (UV-Vis) spectroscopic analysis indicated that >90% of the [4Fe-4S] cluster was lost in the case of EcFDH-H variants -K44E and -K44M, whereas the cluster occupancy of the K44R variant decreased by merely 4.5%. Furthermore, the K44R substitution resulted in a slight decrease in its melting temperature and a significant formate affinity decrease, apparent as a 32-fold K-m value increase. Consistent with these findings, molecular dynamics simulations predicted an increase in the backbone and cofactor mobility as a result of the K44R substitution. These results are consistent with the conserved lysine being essential for stabilizing the catalytically active structures in EcFDH-H and may support engineering efforts on Mo-FDHs to design more efficient biocatalysts for CO2 reduction.

关键词： CO2 utilization lysine molybdenum-dependent formate dehydrogenase redox cofactor site-directed mutagenesis

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Enzymatic synthesis and characterization of nicotinamide riboside pseudouridine diphosphate

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TETRAHEDRON LETTERS 2025年 155卷

作者： Hu, Yinghan Wang, Xueying Huang, Yanzhe Guo, Xiaojia Xue, Haizhao Pfeiffer, Martin Nidetzky, Bernd Zhao, Zongbao K. Chinese Acad Sci Dalian Inst Chem Phys Dalian 116023 Peoples R China Univ Chinese Acad Sci Beijing 100049 Peoples R China Dalian Univ Technol Sch Bioengn Dalian 116024 Peoples R China Graz Univ Technol Inst Biotechnol & Biochem Engn A-8010 Graz Austria Austrian Ctr Ind Biotechnol A-8010 Graz Austria

Nicotinamide adenine dinucleotide (NAD) analogs are of great interests for chemical and biological applications. In this work, a novel NAD analog, nicotinamide riboside pseudouridine diphosphate (NpUD) was synthesized via coupling of pseudouridine triphosphate and nicotinamide mononucleotide catalyzed by a mutant of nicotinic acid mononucleotide adenylyltransferase. It was found that NpUD is more stable than NAD against a common pyrophosphatase NudC found in Escherichia coli. Interestingly, NpUD could function as redox cofactor for several oxidoreductases, among them a mutant methanol dehydrogenase showed 1.9-fold higher activity with NpUD than that with NAD. Overall, the results offer opportunities to explore NpUD in other applications.

关键词： NAD analog Pseudouridine triphosphate Enzymatic synthesis redox cofactor

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Biocatalytic Production of 1,2,4-Butanetriol beyond a Titer of 100 g/L: Boosting by Intermediates

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ACS SUSTAINABLE CHEMISTRY & ENGINEERING 2023年第17期11卷 6592-6599页

作者： Sutiono, Samuel Zachos, Ioannis Paschalidis, Leandros Pick, Andre Burger, Jakob Sieber, Volker Tech Univ Munich Chair Chem Biogenic Resources Campus Straubing Biotechnol & Sustainabil D-94315 Straubing Germany Tech Univ Munich SynBioFoundryTUM D-94315 Straubing Germany CarboCode Germany GmbH Byk Gulden Str 2 D-78467 Constance Germany DSM R&D Ctr Dortmund Otto Hahn Str 15 D-44227 Dortmund Germany Tech Univ Munich Lab Chem Proc Engn Campus Straubing Biotechnol & Sustainabil D-94315 Straubing Germany CASCAT GmbH D-94315 Straubing Germany

Many platform chemicals such as 1,2,4-butanetriol (BTO) are still derived from petrochemicals. BTO as a versatile compound with applications ranging from pharmaceutical synthons to plasticizers is of great interest to be manufactured from renewable resources. Albeit biosynthetic pathways to produce BTO from pentoses were proposed two decades ago, no studies have reported production at high yields and titers typically demanded by chemical industries. In this work, we aimed to tackle this challenge by combining several strategies. Selection of suitable enzymes and reaction optimization in a 500 mu L lab scale allowed us to achieve a titer of 1.2 M (125 g/L) (S)-BTO from 180 g/L D-xylose with a yield >97%. By the addition of an intermediate of the cascade, we could reduce up to 90% of the original redox cofactor used while still maintaining a space-time yield (STY) of 3.7 g/L/h. By applying the same approach, which we term "intermediate boosting", we could push the STY to 9.4 g/L/h. After having identified byproduct formation as a possible bottleneck, we increased production of (S)-BTO further to 1.6 M (170 g/L), approaching the toxicity level of BTO at 200 g/L that microorganisms can handle. We demonstrated, however, that our enzymes were still functional at 300 g/L BTO. Finally, we proposed several strategies to further increase the titer and yield of BTO as a feasible alternative to the petroleum-based synthetic route. This work highlights the importance of a combinatorial approach to boost the enzymatic biosynthesis of chemicals.

关键词： biotransformation biocatalyst sustainability pentose chemicals redox cofactor intermediate boosting

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Modular Synthesis of New Pyrroloquinoline Quinone Derivatives

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SYNTHESIS-STUTTGART 2022年第6期55卷 1000-1006页

作者： Janssen, Rachel Vetsova, Violeta A. Putz, Dominik Mayer, Peter Daumann, Lena J. J. Ludwig Maximilians Univ Munchen Dept Chem Butenandtstr 5-13 Haus D-81377 Munich Germany

Pyrroloquinoline quinone (PQQ) is an important cofactor of methanol dehydrogenases and glycose dehydrogenases. In addition, isolated PQQ is used as a central component in sensors and biomimetic complexes. The synthesis of PQQ derivatives is of interest for developing new alcohol oxidation catalysts and redox sensors. This work describes a modular synthesis for derivatives of PQQ bearing methyl and ketone groups instead of carboxylic acid moieties. These modifications reduce the possible coordination sites of PQQ for metal ions outside the protein environment.

关键词： redox cofactor pyrroloquinoline quinone alcohol oxidation pincer ligand natural product derivatives

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High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

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METABOLIC ENGINEERING 2022年第0期73卷 158-167页

作者： Last, Daniel Hasan, Mahmudul Rothenburger, Linda Braga, Daniel Lackner, Gerald Leibniz Inst Nat Prod Res & Infect Biol Hans Knoll Inst Jr Res Grp Synthet Microbiol Beutenberg str 11a D-07745 Jena Germany Leibniz Inst Aging Fritz Lipmann Inst Core Facil Flow Cytometry Beutenberg str 11 D-07745 Jena Germany

Coenzyme F-420 is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F-420-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F-420 levels are produced in microorganisms, F-420 availability is a serious bottleneck for research and application. Recent advances in our understanding of the F-420 biosynthesis enabled heterologous overproduction of F-420 in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F(420 )biosynthesis in E. coli. However, it still led to the production of low amounts of F-420 and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module ( "core ") addressed parts of the riboflavin biosynthesis pathway and F-O synthase for the conversion of GTP to the stable F-420 intermediate F-O. The enzymes of the second module ( "decoration ") were chosen to turn F-O into F-420. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F-420-derived fluorescence. This approach yielded the highest titer of coenzyme F-420 produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F-420-dependent bioprocesses as well as applications of F-420-dependent enzymes in biocatalysis.

关键词： Coenzyme F-420 redox cofactor Deazaflavin Ribosome binding site library Metabolic engineering Metabolomics

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Metabolic reprogramming and machine learning-guided cofactor engineering to boost nicotinamide mononucleotide production in Escherichia coli

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BIORESOURCE TECHNOLOGY 2025年 426卷 132350页

作者： Xiong, Bo Yang, Tianrui Zhang, Zixiong Li, Xiang Yu, Huan Wang, Lian You, Zixuan Peng, Wenbin Jin, Luyu Song, Hao Tianjin Univ State Key Lab Synthet Biol Tianjin 300072 Peoples R China Tianjin Univ Sch Chem Engn & Technol Tianjin 300072 Peoples R China Northeastern Univ Coll Life & Hlth Sci Shenyang 110169 Peoples R China

Nicotinamide mononucleotide (NMN) is a bioactive compound in NAD(P)+ metabolism, which exhibits diverse pharmaceutical interests. However, enhancing NMN biosynthesis faces the challange of competing with cell growth and disturbing intracellular redox homeostasis. Herein, we boosted NMN production in Escherichia coli by reprogramming central carbon metabolism with a machine learning (ML)-guided cofactor engineering strategy. Engnieering NMN biosynthesis-related pathway directed carbon flux toward NMN with the NADPH level increased by 73 %, which, although enhanced NMN titer (2.45 g/L), impaired cell growth. A quorum sensing (QS)-controlled cofactor engineering system was thus contructed and optimized by ML models to address redox imbalance, which led to 3.04 g/L NMN with improved cell growth. The final strain S344 produced 20.13 g/L NMN in fed-batch fermentation. This study showed that perturbation on cofactor level is a crucial limiting factor for NMN biosynthesis, and proposed a novel ML-guided strategy to manipulate intracellular redox state for efficient NMN production.

关键词： NMN Metabolic engineering Quorum sensing redox cofactor Machine learning models

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Biocatalytic production of (S)-1,2,4-butanetriol from D-xylose by whole cells of engineered Escherichia coli

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MOLECULAR CATALYSIS 2024年 562卷

作者： Ji, Yangyang Cui, Yunfeng Liu, Xiangtao Li, Jianjiong Yao, Peiyuan Zhao, Qing Wu, Qiaqing Zhu, Dunming Tianjin Univ Sci & Technol Sch Biotechnol State Key Lab Food Nutr & Safety Key Lab Ind Fermentat MicrobiolMinist Educ Tianjin 300457 Peoples R China Chinese Acad Sci Tianjin Inst Ind Biotechnol Natl Ctr Technol Innovat Synthet Biol Natl Engn Res Ctr Ind EnzymesKey Lab Engn Biol Lo Tianjin 300308 Peoples R China Chinese Acad Sci Tianjin Inst Ind Biotechnol Tianjin Engn Res Ctr Biocatalyt Technol Tianjin 300308 Peoples R China

( S )-1,2,4-butanotriol is an important four carbon platform compound with extensive applications in many fields. Traditional chemical methods involving harsh reaction conditions and poor selectivity are environmentally unfriendly. Biosynthetic methods have made significant progress, but still cannot meet the needs of industrial production. In this study, a whole -cell biocatalytic system have been developed for the efficient production of ( S )-1,2,4-butanotriol from D -xylose. D -xylose dehydrogenase (CcXylB), D -xylonate dehydratase (TaXylD), 2-keto acid decarboxylase (SaPDC) and aldehyde reductase (EqAdhP) with high activity were obtained by screening the libraries of these enzymes. The plasmid pRSFDuet-TaXylD-T7-CcXylB-SaPDC-T7-EqAdhP (P5) was constructed and introduced into E. coli BL21(DE3) to give the recombinant strain E. coli BL21(DE3)-P5, which could completely convert 200 g/L D -xylose to ( S )-1,2,4-butanotriol in 74% isolated yield and >99% ee under the optimized cultivation and reaction conditions. This study provides a high-performance biocatalyst with potential industrial application for the synthesis of ( S )-1,2,4-butanotriol from renewable resources.

关键词： (S)-1,2,4-butanetriol D -xylose Whole -cell biocatalysis redox cofactor Biotransformation

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Strategies and challenges with the microbial conversion of methanol to high-value chemicals

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BIOTECHNOLOGY AND BIOENGINEERING 2021年第10期118卷 3655-3668页

作者： Zhan, Chunjun Li, Xiaowei Yang, Yankun Nielsen, Jens Bai, Zhonghu Chen, Yun Jiangnan Univ Natl Engn Lab Cereal Fermentat Technol Wuxi 214122 Jiangsu Peoples R China Jiangnan Univ Sch Biotechnol Key Lab Ind Biotechnol Minist Educ Wuxi Jiangsu Peoples R China Jiangnan Univ Sch Biotechnol Key Lab Carbohydrate Chem & Biotechnol Minist Educ Wuxi Jiangsu Peoples R China Chalmers Univ Technol Dept Biol & Biol Engn Kemivagen 10 SE-41296 Gothenburg Sweden Chalmers Univ Technol Novo Nordisk Fdn Ctr Biosustainabil Gothenburg Sweden BioInnovat Inst Copenhagen N Denmark

As alternatives to traditional fermentation substrates, methanol (CH3OH), carbon dioxide (CO2) and methane (CH4) represent promising one-carbon (C1) sources that are readily available at low-cost and share similar metabolic pathway. Of these C1 compounds, methanol is used as a carbon and energy source by native methylotrophs, and can be obtained from CO2 and CH4 by chemical catalysis. Therefore, constructing and rewiring methanol utilization pathways may enable the use of one-carbon sources for microbial fermentations. Recent bioengineering efforts have shown that both native and nonnative methylotrophic organisms can be engineered to convert methanol, together with other carbon sources, into biofuels and other commodity chemicals. However, many challenges remain and must be overcome before industrial-scale bioprocessing can be established using these engineered cell refineries. Here, we provide a comprehensive summary and comparison of methanol metabolic pathways from different methylotrophs, followed by a review of recent progress in engineering methanol metabolic pathways in vitro and in vivo to produce chemicals. We discuss the major challenges associated with establishing efficient methanol metabolic pathways in microbial cells, and propose improved designs for future engineering.

关键词： energy supply intermediate recycling methanol methylotrophs redox cofactor

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