Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

Shmonova, Ekaterina A.; Voloshina, O. V.; Ovsienko, M. V.; Smirnov, Sergey V.; Nolde, Dmitry E.; Doroshenko, V. G.

doi:10.1371/journal.pone.0231560

Cited by 9 publications

(25 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The decrease of the PCA formation rate towards the end of the cultivation could be attributed to the reduced activity of 3-dehydroshikimate dehydratase (QsuB). Previous in vitro analysis of the substrate-saturated QsuB enzyme from C. glutamicum showed non-competitive inhibition ( K I = 0.96 mM) at a concentration of 0.6 mM PCA [45]. Further in vitro analysis of this enzyme showed its highest activity in presence of 1 mM Co 2+ and at a pH of 8.0 ± 8.4.…”

Section: Resultsmentioning

confidence: 91%

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose withCorynebacterium glutamicum

Labib

Goertz

Bruesseler

et al. 2021

Preprint

View full text Add to dashboard Cite

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of D-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1) from D-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-molPCA C-molXYL-1, which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on D-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 ± 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a three-chamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

show abstract

Section: Resultsmentioning

confidence: 91%

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose withCorynebacterium glutamicum

Labib

Goertz

Bruesseler

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Multiple DSD variants from fungi, Acinetobacter species, Bacillus species, and Pseudomonas species have been identified. Earlier studies have reported the use of DSD from Corynebacterium glutamicum , Podospora pauciseta , and Klebsiella pneumonia ( Hansen et al, 2009 ; Curran et al, 2013 ; Shmonova et al, 2020 ; Table 1 ). In this study, the P. putida DSD gene was cloned and expressed in E. coli .…”

Section: Discussionmentioning

confidence: 99%

“…The synthesis is constrained in two different ways: (i) the PEP flux to the phosphotransferase system (PTS) ( Rodriguez et al, 2014 ), and (ii) the feedback inhibition of the three DAHP synthase enzymes AroG, AroF and AroH by phenylalanine, tyrosine and tryptophan, respectively ( Johansson and Lidén, 2006 ). Further limitations of PCA production through the shikimate pathway are ascribed to the product toxicity to the cells and inhibition of the DSD enzyme, resulting in termination of the process ( Pugh et al, 2014 ; Shmonova et al, 2020 ). Addition of 1.5 g/L PCA to Escherichia coli culture has been shown to completely impair cell growth ( Pugh et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

“…The toxicity of PCA could be due to its insertion in the core of the cell membrane, increasing the membrane fluidity and decreasing the structural integrity, as has been observed for other aromatic compounds ( Ramos et al, 2002 ). Studies on DSD of Corynebacterium glutamicum have shown the enzyme to be competitively and non-competitively inhibited by PCA (inhibition constants Ki ∼ 0.38 mM and Ki’ ∼ 0.96 mM, respectively) ( Shmonova et al, 2020 ). It is possible to minimize the inhibition by in situ removal of the product, which would have an additional benefit of shifting the reaction equilibrium toward product formation ( Sayed et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

“…This report presents a study on PCA production through dehydration of 3-dehydroshikimate using recombinant Pseudomonas putida DSD in an E. coli strain engineered for phenylalanine overproduction by removing the enzymes AroF and AroG, that are inhibited by tyrosine and phenylalanine, respectively, as well as the essential enzymes in the tyrosine and tryptophan biosynthesis pathway ( Fox et al, 2008 ; Peek et al, 2017 ; Shmonova et al, 2020 ). The physiological and metabolic effect of PCA production was further evaluated to identify the probable mechanism involved.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Enhanced Protocatechuic Acid Production From Glucose Using Pseudomonas putida 3-Dehydroshikimate Dehydratase Expressed in a Phenylalanine-Overproducing Mutant of Escherichia coli

Örn

Sacchetto

Niel

et al. 2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Protocatechuic acid (PCA) is a strong antioxidant and is also a potential platform for polymer building blocks like vanillic acid, vanillin, muconic acid, and adipic acid. This report presents a study on PCA production from glucose via the shikimate pathway precursor 3-dehydroshikimate by heterologous expression of a gene encoding 3-dehydroshikimate dehydratase in Escherichia coli. The phenylalanine overproducing E. coli strain, engineered to relieve the allosteric inhibition of 3-deoxy-7-phosphoheptulonate synthase by the aromatic amino acids, was shown to give a higher yield of PCA than the unmodified strain under aerobic conditions. Highest PCA yield of 18 mol% per mol glucose and concentration of 4.2 g/L was obtained at a productivity of 0.079 g/L/h during cultivation in fed-batch mode using a feed of glucose and ammonium salt. Acetate was formed as a major side-product indicating a shift to catabolic metabolism as a result of feedback inhibition of the enzymes including 3-dehydroshikimate dehydratase by PCA when reaching a critical concentration. Indirect measurement of proton motive force by flow cytometry revealed no membrane damage of the cells by PCA, which was thus ruled out as a cause for affecting PCA formation.

show abstract

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum

Labib

Görtz

Brüsseler

et al. 2021

Biotech & Bioengineering

View full text Add to dashboard Cite

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of D-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L −1 ) from D-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol D-xylose, which corresponds to 38% of the maximum theoretical yield. Under growthdecoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when D-glucose and D-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

show abstract

Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

Cited by 9 publications

References 33 publications

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose withCorynebacterium glutamicum

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose withCorynebacterium glutamicum

Enhanced Protocatechuic Acid Production From Glucose Using Pseudomonas putida 3-Dehydroshikimate Dehydratase Expressed in a Phenylalanine-Overproducing Mutant of Escherichia coli

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum

Contact Info

Product

Resources

About