Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

ABSTRACT A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.

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Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0267-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Pei Xu ◽

Xiao-Ling Wu ◽

Min-Hua Zong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Rational Design ◽

Glucuronic Acid ◽

Specific Activity ◽

Value Added ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glucaric Acid ◽

Triple Mutant ◽

Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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Combinatorial synthetic pathway fine-tuning and cofactor regeneration for metabolic engineering of Escherichia coli significantly improve production of D-glucaric acid

New Biotechnology ◽

10.1016/j.nbt.2020.03.004 ◽

2020 ◽

Vol 59 ◽

pp. 51-58 ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Xiao-Yang Ou ◽

Ying-Jie Zeng ◽

Min-Hua Zong ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Cofactor Regeneration ◽

Fine Tuning ◽

Glucaric Acid ◽

Synthetic Pathway ◽

Improve Production

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Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

Enzyme Research ◽

10.4061/2010/597010 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Sakuko Ueshima ◽

Hisashi Muramatsu ◽

Takanori Nakajima ◽

Hiroaki Yamamoto ◽

Shin-ichiro Kato ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Syringae ◽

Homology Search ◽

Hydroxyl Group ◽

Enzymatic Reactions ◽

Reaction Products ◽

Gene Encoding ◽

Genes Encoding ◽

Single Operon

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

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Recombineering Using RecTE from Pseudomonas syringae

Applied and Environmental Microbiology ◽

10.1128/aem.00911-10 ◽

2010 ◽

Vol 76 (15) ◽

pp. 4960-4968 ◽

Cited By ~ 64

Author(s):

Bryan Swingle ◽

Zhongmeng Bao ◽

Eric Markel ◽

Alan Chambers ◽

Samuel Cartinhour

Keyword(s):

Escherichia Coli ◽

Pseudomonas Syringae ◽

Recombination Frequency ◽

Quantitative Assay ◽

Tomato Dc3000 ◽

Linear Dna ◽

Dna Oligonucleotides ◽

Double Stranded Dna ◽

Genes Encoding ◽

Genomic Recombination

ABSTRACT In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

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Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Journal of Bacteriology ◽

10.1128/jb.00586-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1565-1573 ◽

Cited By ~ 38

Author(s):

Sang-Hwal Yoon ◽

Tae Seok Moon ◽

Pooya Iranpour ◽

Amanda M. Lanza ◽

Kristala Jones Prather

Keyword(s):

Agrobacterium Tumefaciens ◽

Pseudomonas Syringae ◽

Glucuronic Acid ◽

Glucaric Acid ◽

Michaelis Constant ◽

Turnover Number ◽

Reading Frame ◽

E Coli ◽

Uronate Dehydrogenase

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat = 1.9 × 102 s−1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

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Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01065-09 ◽

2009 ◽

Vol 75 (13) ◽

pp. 4660-4660 ◽

Cited By ~ 5

Author(s):

Tae Seok Moon ◽

Sang-Hwal Yoon ◽

Amanda M. Lanza ◽

Joseph D. Roy-Mayhew ◽

Kristala L. Jones Prather

Keyword(s):

Escherichia Coli ◽

Recombinant Escherichia Coli ◽

Glucaric Acid ◽

Synthetic Pathway

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Faculty Opinions recommendation of IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1002791.46105 ◽

2002 ◽

Author(s):

Regine Hengge

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Cluster Assembly ◽

Genes Encoding ◽

Assembly Proteins

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Faculty Opinions recommendation of Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1003128.30060 ◽

2001 ◽

Author(s):

Koreaki Ito

Keyword(s):

Escherichia Coli ◽

Signal Recognition Particle ◽

Genetic Screen ◽

Signal Recognition ◽

Genes Encoding

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β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization

Microorganisms ◽

10.3390/microorganisms9010055 ◽

2020 ◽

Vol 9 (1) ◽

pp. 55

Author(s):

María Florencia Eberhardt ◽

José Matías Irazoqui ◽

Ariel Fernando Amadio

Keyword(s):

Bacterial Species ◽

Value Added ◽

Raw Material ◽

Activity Levels ◽

Treatment Technology ◽

Catalytic Domains ◽

Stabilization Ponds ◽

Cazy Database ◽

Genes Encoding ◽

Value Added Products

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. β-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding β-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with β-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-β-galactoside and lactose. The activity levels of one of these novel β-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic β-galactosidases from diary stabilization ponds’ metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries’ by-products.

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