Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways

Kogure, Takahisa; Suda, Masako; Hiraga, Kazumi; Inui, Masayuki

doi:10.1016/j.ymben.2020.11.007

Cited by 27 publications

(30 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…C. glutamicum shows stable growth to high cell densities (Riesenberg and Guthke, 1999;Pfeifer et al, 2017) and has been engineered for production of, e.g., N-functionalized amino acids (Mindt et al, 2020), diamines (Wendisch et al, 2018;Chae et al, 2020), alcohols (Jojima et al, 2015;Siebert and Wendisch, 2015), and organic acids (Wieschalka et al, 2013;Purwanto et al, 2018). With respect to production of molecules originating from the shikimate or isoprenoid pathways, metabolic engineering enabled production of terpenoids such as pinene (Kang et al, 2014), patchoulol (Henke et al, 2018), valencene (Frohwitter et al, 2014), or various carotenoids (Heider et al, 2014b,c;Henke et al, 2016) and of aromatic or aromatic-derived compounds such as muconic acid (Becker et al, 2018;Shin et al, 2018), phenylpropanoids (Kallscheuer et al, 2016(Kallscheuer et al, , 2017, 4-aminobenzoate (Kubota et al, 2016), shikimate (Sato et al, 2020), protocatechuate (Kogure et al, 2020), 4-hydroxybenzyl alcohol (Kim et al, 2020), and pHBA (Kitade et al, 2018;Purwanto et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Burgardt

Moustafa

Persicke

et al. 2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

show abstract

Section: Introductionmentioning

confidence: 99%

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Burgardt

Moustafa

Persicke

et al. 2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…The strains constructed here also allowed for xylose-based production of NMePhe. Xylose is a major constituent of lignocellulose hydrolysates [66] and is an abundant and renewable feedstock for many biotechnological processes [67]. Xylose cannot be catabolized by Saccharomyces cerevisiae [66,68] Zymomonas mobilis [69] or C. glutamicum [50,51].…”

Section: Discussionmentioning

confidence: 99%

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

et al. 2021

View full text Add to dashboard Cite

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

show abstract

“…glutamicum MB001(DE3) prophage-free derivative of ATCC 13032 with chromosomal expression of the T7 RNA polymerase gene under control of P lacUV5 (IPTG-inducible) [15] DelAro 5 C7 P O6 -iolT1 MB001(DE3) derivative with in-frame deletions of cg0344-cg0347, cg2625-cg2640, cg1226, cg0502, and cg3349-cg3354 combined with an exchange of the native promoter of the citrate synthase gene gltA by the dapA promoter variant C7 as well as two point mutations in the promoter of the inositol transporter gene iolT1 that abolishes repression of iolT1 by the regulator protein IolR [16] DelAro 5 C7 P O6 -iolT1 Δpyk derivative of DelAro 5 C7 P O6 -iolT1 with in-frame deletion of pyk (cg2291) coding for the pyruvate kinase This study PCA GLC DelAro 5 C7 P O6 -iolT1 derivative harboring the plasmids pMKEx2_aroF*-qsuB and pEKEx3_tkt [16] PCA GLC Δpyk PCA GLC derivative with in-frame deletion of pyk (cg2291) encoding for the pyruvate kinase This study PCA tamicum is capable of utilizing PCA as sole carbon and energy source [19,20] and in both approaches followed by Okai and co-workers, the natural PCA catabolism of C. glutamicum was not inactivated. Most recently, a high-titer production process for PCA from -glucose was presented [21]. For reaching the reported titer of 82.7 g L −1 PCA a two-step fermentation approach was applied.…”

Section: Strainmentioning

confidence: 99%

“…A production titer of 1.1 g L −1 PCA was reported after 96 h of fed-batch cultivation using a mini-jar fermenter containing a complex medium [17]. Most recently, by following a two-step fed-batch approach, a much higher titer of 82.7 g L −1 PCA was realized [21]. In this approach, the engineered strain was first grown to high cell density using complex medium, and then the biotransformation step was started from 10 % (w v −1 ) of the harvested biocatalyst.…”

Section: Development Of a One-pot Pca Production Processmentioning

confidence: 99%

“…Noteworthy, C. glutamicum is capable of utilizing PCA as sole carbon and energy source [19, 20] and in both approaches followed by Okai and co-workers, the natural PCA catabolism of C. glutamicum was not inactivated. Most recently, a high-titer production process for PCA from d -glucose was presented [21]. For reaching the reported titer of 82.7 g L −1 PCA a two-step fermentation approach was applied.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways

Cited by 27 publications

References 52 publications

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

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

Contact Info

Product

Resources

About