Metabolic engineering of Corynebacterium glutamicum for l-tyrosine production from glucose and xylose

Kurpejović, Eldin; Burgardt, Arthur; Bastem, Gülsüm Merve; Junker, Nora; Wendisch, Volker F.; Akbulut, Berna Sarıyar

doi:10.1016/j.jbiotec.2022.12.005

Cited by 7 publications

(5 citation statements)

References 57 publications

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“…In addition, the yield of phenylalanine from xylose was also expectedly higher, reaching 63.1 ± 3.1 mg/g versus just 28.4 ± 2.8 mg/g from glucose. These relative outcomes are consistent with past reports (Kurpejović et al, 2023;Vargas-Tah et al, 2015). As suggested by our FBA results (Figure 1b), the increased titer and yield of phenylalanine on xylose was likely a result of increased supply of E4P (predicted as the predominant, limiting precursor).…”

Section: Comparing Phenylalanine Production By Nst74 Using Glucose Xy...supporting

confidence: 93%

Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

Liu,

Machas,

Mhatre

et al. 2023

Biotech & Bioengineering

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Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co‐utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose‐to‐glucose flux ratio. A mutant copy of the xylose‐specific activator (XylR) was then introduced into the phenylalanine‐overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose−xylose co‐utilization. Carbon contribution analysis through 13C‐fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose−xylose mixture; a threefold increase over NST74. Then, using biomass‐derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9‐fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L‐h). This study presents a novel strategy for enhancing phenylalanine production through the co‐utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

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Section: Comparing Phenylalanine Production By Nst74 Using Glucose Xy...supporting

confidence: 93%

Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

Liu,

Machas,

Mhatre

et al. 2023

Biotech & Bioengineering

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“…The optimized OPH-NH 3 medium was tested for amino acid production by different metabolically engineered C. glutamicum strains. Categorization of the metabolic precursors for amino acid biosynthesis guided our strain selection: ʟ-arginine (α-ketoglutarate) using strain ARG5 ( Jensen et al, 2015 ), ʟ-lysine (oxaloacetate/aspartate) by GRLys1 Δ sugR Δ ldhA ( Pérez-García et al, 2016 ), ʟ-serine (3-phosphoglycerate) by SER1 ( Stolz et al, 2007 ), ʟ-valine (pyruvate) using strain VAL1 ( Lange et al, 2003 ), and ʟ-tyrosine (shikimate) by strain AROM3 ( Kurpejović et al, 2023 ). The strains were cultivated in OPH-NH 3 medium [80 vol% OPH with 300 mM NH 3 (added before pH neutralization with KOH) and 200 mM phosphate buffer].…”

Section: Resultsmentioning

confidence: 99%

Utilization of orange peel waste for sustainable amino acid production by Corynebacterium glutamicum

Junker,

Sariyar Akbulut,

Wendisch

2024

Front. Bioeng. Biotechnol.

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Oranges are the most processed fruit in the world–it is therefore apparent that the industrial production of orange juice generates large quantities of orange peel as a by-product. Unfortunately, the management of the orange peel waste leads to economic and environmental problems. Meanwhile, the use of sustainable raw materials for the production of bulk chemicals, such as amino acids, is becoming increasingly attractive. To address both issues, this study focused on the use of orange peel waste as a raw material for media preparation for the production of amino acids by engineered Corynebacterium glutamicum. C. glutamicum grew on pure orange peel hydrolysate (OPH) and growth was enhanced by the addition of a nitrogen source and a pH buffer. Inhibitory effects by the combination of high concentrations of OPH, (NH4)2SO4, and MOPS buffer in the wild-type strain (WT), were overcome in the tyrosine-producing engineered C. glutamicum strain AROM3. Genetic modifications that we identified to allow for improved growth rates under these conditions included the deletions of the vanillin dehydrogenase gene vdh, the ʟ-lactate dehydrogenase gene ldhA and the 19 genes comprising cluster cg2663-cg2686. A growth inhibiting compound present in high concentrations in the OPH is 5-(hydroxymethyl)furfural (HMF). We identified vdh as being primarily responsible for the oxidation of HMF to its acid 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), as the formation of HMFCA was reduced by 97% upon deletion of vdh in C. glutamicum WT. In addition, we showed that growth limitations could be overcome by adjusting the media preparation, using a combination of cheap ammonia water and KOH for pH neutralization after acidic hydrolysis. Overall, we developed a sustainable medium based on orange peel waste for the cultivation of C. glutamicum and demonstrated the successful production of the exemplary amino acids ʟ-arginine, ʟ-lysine, ʟ-serine, ʟ-valine and ʟ-tyrosine.

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“…During the construction of the p -CA-producing C. glutamicum variants, the translation efficiency of the prephenate dehydratase gene pheA was reduced by replacing the canonical ATG start codon with GTG, avoiding PHE auxotrophy. A similar strategy was pursued for TYR production with C. glutamicum [ 53 ]. In this case, the start codon was changed to the least preferred TTG, which severely reduced the expression of pheA and rendered the expensive supplementation of 2 mM PHE necessary to restore growth.…”

Section: Discussionmentioning

confidence: 99%

Microbial synthesis of the plant natural product precursor p-coumaric acid with Corynebacterium glutamicum

Mutz,

Kösters,

Wynands

et al. 2023

Microb Cell Fact

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Background Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose. Results Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation. Conclusions The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants.

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Metabolic engineering of Corynebacterium glutamicum for l-tyrosine production from glucose and xylose

Cited by 7 publications

References 57 publications

Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

Utilization of orange peel waste for sustainable amino acid production by Corynebacterium glutamicum

Microbial synthesis of the plant natural product precursor p-coumaric acid with Corynebacterium glutamicum

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