scholarly journals Development of a vanillate biosensor for the vanillin biosynthesis pathway in E. coli

2018 ◽  
Author(s):  
Aditya M. Kunjapur ◽  
Kristala L. J. Prather
Keyword(s):  
Metabolic Engineering ◽  
Dynamic Range ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
Homo Sapiens ◽  
Biosynthesis Pathway ◽  
Dynamic Regulation ◽  
E Coli ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

AbstractGenetically encoded small molecule sensors can facilitate metabolic engineering by enabling high-throughput detection of metabolite concentrations, directed evolution of host and pathway enzymes, and dynamic regulation. The engineered de novo vanillin biosynthesis pathway assembled in Escherichia coli is industrially relevant and ideal for biosensor deployment given that the pathway requires only three heterologous enzyme-catalyzed reactions, generates naturally occurring metabolites, and may benefit from dynamic regulation. However, pathway flux is stalled and diverted by the activity of the Homo sapiens catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To confront this challenge, we constructed and applied a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using components from a previously characterized E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We characterized sensor substrate specificity and found that this construct and an evolved variant are remarkably selective, exhibiting no detectable response to the regioisomer byproduct isovanillate. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases. We identified eight that appear to have greater desired activity than the originally used variant, including three previously uncharacterized O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Some enzymic syntheses of 15N-L-aspartic acid and 15N-L-glutamic acid

1969 ◽  
Vol 47 (3) ◽  
pp. 411-415 ◽  
Author(s):  
J. A. Zintel ◽  
A. J. Williams ◽  
R. S. Stuart
Keyword(s):  
Glutamic Acid ◽  
Aspartic Acid ◽  
Fumaric Acid ◽  
Aspartate Aminotransferase ◽  
Efficient Utilization ◽  
E Coli ◽  
Acid Dehydrogenase ◽  
Acid Catalyzed ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Some methods for the preparation of 15N-L-aspartic acid and 15N-L-glutamic acid using enzyme catalyzed reactions are described. 15N-L-Aspartic acid is prepared by the addition of ammonia to fumaric acid, catalyzing the reaction with aspartase which was partially purified from E. coli. 15N-L-Glutamic acid with a high 15N/14N ratio is prepared by transamination with 15N-L-aspartic acid catalyzed by aspartate aminotransferase. 15N-L-Glutamic acid with a low 15N/14N ratio is prepared by a glutamic acid dehydrogenase catalyzed exchange of L-glutamic acid with 15N-ammonia. These enzymes are commercially available. Since efficient utilization of 15N is obtained, aspartic acid or glutamic acid may be prepared with any desired 15N/14N ratio.

scholarly journals Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 288
Author(s):  
Habiba Kausar ◽  
Ghazala Ambrin ◽  
Mohammad K. Okla ◽  
Walid Soufan ◽  
Abdullah A. Al-Ghamdi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Real Time ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Regulatory Element ◽  
Flux Analysis ◽  
Bacterial Cells ◽  
Biosynthesis Pathway ◽  
Real Time Monitoring ◽  
E Coli

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Vitamin B ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Optimization Of Fermentation ◽  
Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

scholarly journals An In Vivo Biocatalytic Cascade Featuring an Artificial Enzyme Catalyzed New-to-Nature Reaction

2021 ◽  
Author(s):  
Linda Ofori Atta ◽  
Zhi Zhou ◽  
Gerard Roelfes
Keyword(s):  
Carbonyl Compounds ◽  
Artificial Enzymes ◽  
Catalytic Residue ◽  
Artificial Enzyme ◽  
E Coli ◽  
Iminium Ion ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Benzaldehyde Derivatives

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism, making reactions possible that have no equivalent in nature. Here, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme catalyzed new-to-nature reaction. The artificial enzyme in this study is a pAF containing evolved variant of the Lactococcal multidrug resistance Regulator, designated LmrR_V15pAF_RMH, which efficiently converts in vivo produced benzaldehyde derivatives into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial enzyme catalyzed reactions are an important step towards achieving a hybrid metabolism.

scholarly journals Construction of a third NAD+de novo biosynthesis pathway

2020 ◽  
Author(s):  
Yong Ding ◽  
Xinli Li ◽  
Geoff P. Horsman ◽  
Pengwei Li ◽  
Min Wang ◽  
...  
Keyword(s):  
De Novo ◽  
Wide Distribution ◽  
Chiral Amines ◽  
Biosynthesis Pathway ◽  
Cofactor Engineering ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Cell Factories ◽  
General Utility ◽  
Whole Cell Bioconversion

AbstractOnly two de novo biosynthetic routes to nicotinamide adenine dinucleotide (NAD+) have been described, both of which start from a proteinogenic amino acid and are tightly controlled. Here we establish a C3N pathway starting from chorismate in Escherichia coli as a third NAD+de novo biosynthesis pathway. Significantly, the C3N pathway yielded extremely high cellular concentrations of NAD(H) in E. coli. Its utility in cofactor engineering was demonstrated by introducing the four-gene C3N module to cell factories to achieve higher production of 2,5-dimethylpyrazine and develop an efficient C3N-based whole-cell bioconversion system for preparing chiral amines. The wide distribution and abundance of chorismate in most kingdoms of life implies a general utility of the C3N pathway for modulating cellular levels of NAD(H) in versatile organisms.

scholarly journals L-Carnitine Production Through Biosensor-Guided Construction of the Neurospora crassa Biosynthesis Pathway in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Pierre Kugler ◽  
Marika Trumm ◽  
Marcel Frese ◽  
Volker F. Wendisch
Keyword(s):  
Escherichia Coli ◽  
Neurospora Crassa ◽  
De Novo ◽  
Nitrogen Sources ◽  
Bioactive Compound ◽  
Biosynthesis Pathway ◽  
One Pot ◽  
E Coli ◽  
Renewable Feedstocks ◽  
Eukaryotic Microorganisms

L-Carnitine is a bioactive compound derived from L-lysine and S-adenosyl-L-methionine, which is closely associated with the transport of long-chain fatty acids in the intermediary metabolism of eukaryotes and sought after in the pharmaceutical, food, and feed industries. The L-carnitine biosynthesis pathway has not been observed in prokaryotes, and the use of eukaryotic microorganisms as natural L-carnitine producers lacks economic viability due to complex cultivation and low titers. While biotransformation processes based on petrochemical achiral precursors have been described for bacterial hosts, fermentative de novo synthesis has not been established although it holds the potential for a sustainable and economical one-pot process using renewable feedstocks. This study describes the metabolic engineering of Escherichia coli for L-carnitine production. L-carnitine biosynthesis enzymes from the fungus Neurospora crassa that were functionally active in E. coli were identified and applied individually or in cascades to assemble and optimize a four-step L-carnitine biosynthesis pathway in this host. Pathway performance was monitored by a transcription factor-based L-carnitine biosensor. The engineered E. coli strain produced L-carnitine from supplemented L-Nε-trimethyllysine in a whole cell biotransformation, resulting in 15.9 μM carnitine found in the supernatant. Notably, this strain also produced 1.7 μM L-carnitine de novo from glycerol and ammonium as carbon and nitrogen sources through endogenous Nε-trimethyllysine. This work provides a proof of concept for the de novoL-carnitine production in E. coli, which does not depend on petrochemical synthesis of achiral precursors, but makes use of renewable feedstocks instead. To the best of our knowledge, this is the first description of L-carnitine de novo synthesis using an engineered bacterium.

scholarly journals Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Xueying Wang ◽  
Yongjin J. Zhou ◽  
Lei Wang ◽  
Wujun Liu ◽  
Yuxue Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Nicotinic Acid ◽  
De Novo ◽  
Colorimetric Assay ◽  
Reduced Form ◽  
Content Type ◽  
E Coli ◽  
Nad Biosynthesis ◽  
Nicotinamide Mononucleotide

ABSTRACT NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

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