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

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 growth-decoupled 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.

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum.

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicuml-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37g/l 2-methyl-1-butanol and 2.76g/l 3-methyl-1-butanol in defined medium within 48h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Microaerobic growth-decoupled production of α-ketoglutarate and succinate from xylose in a one-pot process using Corynebacterium glutamicum.

BACKGROUND: Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacterium glutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process. METHODS AND RESULTS: C. glutamicum WMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C. glutamicum WMB2evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4 mmol L-1 (11.45 g L-1 ) α-ketoglutarate and 96.2 mmol L-1 (11.36 g L-1 ) succinate. CONCLUSION: The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.

Alone at last! - Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum.

Corynebacterium glutamicum can grow on d-xylose as sole carbon and energy source via the five-step Weimberg pathway when the pentacistronic xylXABCD operon from Caulobacter crescentus is heterologously expressed. More recently, it could be demonstrated that the C. glutamicum wild type accumulates the Weimberg pathway intermediate d-xylonate when cultivated in the presence of d-xylose. Reason for this is the activity of the endogenous dehydrogenase IolG, which can also oxidize d-xylose. This raised the question whether additional endogenous enzymes in C. glutamicum contribute to the catabolization of d-xylose via the Weimberg pathway. In this study, analysis of the C. glutamicum genome in combination with systematic reduction of the heterologous xylXABCD operon revealed that the hitherto unknown and endogenous dehydrogenase KsaD (Cg0535) can also oxidize α-ketoglutarate semialdehyde to the tricarboxylic acid cycle intermediate α-ketoglutarate, the final enzymatic step of the Weimberg pathway. Furthermore, heterologous expression of either xylX or xylD, encoding for the two dehydratases of the Weimberg pathway in C. crescentus, is sufficient for enabling C. glutamicum to grow on d-xylose as sole carbon and energy source. Finally, several variants for the carbon-efficient microbial production of α-ketoglutarate from d-xylose were constructed. In comparison to cultivation solely on d-glucose, the best strain accumulated up to 1.5-fold more α-ketoglutarate in d-xylose/d-glucose mixtures.

The myo-inositol/proton symporter IolT1 contributes to d-xylose uptake in Corynebacterium glutamicum.

Corynebacterium glutamicum has been engineered to utilize d-xylose as sole carbon and energy source. Recently, a C. glutamicum strain has been optimized for growth on defined medium containing d-xylose by laboratory evolution, but the mutation(s) attributing to the improved-growth phenotype could not be reliably identified. This study shows that loss of the transcriptional repressor IolR is responsible for the increased growth performance on defined d-xylose medium in one of the isolated mutants. Underlying reason is derepression of the gene for the glucose/myo-inositol permease IolT1 in the absence of IolR, which could be shown to also contribute to d-xylose uptake in C. glutamicum. IolR-regulation of iolT1 could be successfully repealed by rational engineering of an IolR-binding site in the iolT1-promoter. This minimally engineered C. glutamicum strain bearing only two nucleotide substitutions mimics the IolR loss-of-function phenotype and allows for a high growth rate on d-xylose-containing media (µmax = 0.24 ±â€¯0.01 h-1).

Production of d-xylonic acid using a non-recombinant Corynebacterium glutamicum strain.

It was found that Corynebacterium glutamicum ΔiolR devoid of the transcriptional regulator IolR accumulates high amounts of d-xylonate when cultivated in the presence of d-xylose. Detailed analyses of constructed deletion mutants revealed that the putative myo-inositol 2-dehydrogenase IolG also acts as d-xylose dehydrogenase and is mainly responsible for d-xylonate oxidation in this organism. Process development for d-xylonate production was initiated by cultivating C. glutamicum ΔiolR on defined d-xylose/d-glucose mixtures under batch and fed-batch conditions. The resulting yield matched the theoretical maximum of 1 mol mol-1 and high volumetric productivities of up to 4 g L-1 h-1 could be achieved. Subsequently, a novel one-pot sequential hydrolysis and fermentation process based on optimized medium containing hydrolyzed sugarcane bagasse was developed. Cost-efficiency and abundance of second-generation substrates, good performance indicators, and enhanced market access using a non-recombinant strain open the perspective for a commercially viable bioprocess for d-xylonate production in the near future.

Miniaturized and automated adaptive laboratory evolution: Evolving Corynebacterium glutamicum towards an improved d-xylose utilization.

Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCDCc, which is capable of utilizing d-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2evo was obtained, showing a specific growth rate of 0.26h-1 on d-xylose as sole carbon and energy source. WMB2evo grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved d-xylose utilization.