Glutamicum Strain Research Articles

Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes.

Indole is produced in nature by diverse organisms and exhibits a characteristic odor described as animal, fecal, and floral. In addition, it contributes to the flavor in foods, and it is applied in the fragrance and flavor industry. In nature, indole is synthesized either from tryptophan by bacterial tryptophanases (TNAs) or from indole-3-glycerol phosphate (IGP) by plant indole-3-glycerol phosphate lyases (IGLs). While it is widely accepted that the tryptophan synthase α-subunit (TSA) has intrinsically low IGL activity in the absence of the tryptophan synthase β-subunit, in this study, we show that Corynebacterium glutamicum TSA functions as a bona fide IGL and can support fermentative indole production in strains providing IGP. By bioprospecting additional bacterial TSAs and plant IGLs that function as bona fide IGLs were identified. Capturing indole in an overlay enabled indole production to titers of about 0.7 g L–1 in fermentations using C. glutamicum strains expressing either the endogenous TSA gene or the IGL gene from wheat.

A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics.

Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titers during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel L-lysine transporter, belonging to a previously uncharacterized EamA superfamily, which is further in vivo characterized as L-lysine exporter using Xenopus oocyte expression system as well as Escherichia coli host. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced yield, titer, and the specific production of L-lysine in an industrial Corynebacterium glutamicum strain by 7.8%, 9.5%, and 12%, respectively. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titers and productivity of toxicity-limited bioprocesses.

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

Fermentative Production of Halogenated Tryptophan Derivatives with Corynebacterium glutamicum Overexpressing Tryptophanase or Decarboxylase Genes.

The aromatic amino acid l‐tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in Corynebacterium glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L−1 7‐Cl‐indole and 23 mg L−1 7‐Br‐indole were attained. Tryptamine production led to a much higher titer of 2.26 g L−1 upon expression of AADC from B. atrophaeus. AADC enzymes were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L−1 7‐Br‐tryptamine with a volumetric productivity of 8.3 mg L−1 h−1 in a fed‐batch fermentation.

Efficient production of lysine from genetically modified Corynebacterium glutamicum by feedback inhibition resistant strain

Lysine is an essential amino acid required for the synthesis of proteins. Lysine deficiency leads to numerous irreversible damages to the brain and other body organs. It plays a vital role in the treatment of osteoporosis and Herpes Simplex Virus (HSV). Lysine is commonly produced through fermentation. However, its efficient production is still a bottle neck. The wild strains of microorganisms are unable to produce sufficient amino acid. The current study was designed to enhance the lysine production through feedback inhibition resistant strains. N-methyl-N-nitro-N-nitrosoguanidine (NTG) was utilized to mutate the Corynebacterium glutamicum strain B391 to increase the production of Lysine. C. glutamicum was used as fermenting agent and cultivated in molasses-based media. S-β-aminoethyl-L-cysteine (AEC) lysine analogue resistant mutants were selected. It was observed that 30 g/L and 55 g/L of lysine were produced by the parent and mutant strains, respectively. The increase in lysine production was observed with 10% of sugar concentration in 100 mL of molasses media (6.5 pH, and temperature 30°C) with inoculum size of 8%. Furthermore, two different molasses media with different ingredients (termed M1 and M2) were used for the lysine production. The lysine production M-2 was observed more prominent.

Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma-aminobutyric acid.

Corynebacterium glutamicum has been used as a sustainable microbial producer for various bioproducts using cheap biomass resources. In this study, a high GABA-producing C. glutamicum strain was constructed by chromosomal editing. Lactobacillus brevis-derived gadB2 was introduced into the chromosome of C. glutamicum ATCC 13032 to produce gamma-aminobutyric acid and simultaneously blocked the biosynthesis of lactate and acetate. GABA transport and degradation in C. glutamicum were also blocked to improve GABA production. As precursor of GABA, l-glutamic acid synthesis in C. glutamicum was enhanced by introducing E. coli gdhA encoding glutamic dehydrogenase, and the copy numbers of gdhA and gadB2 were also optimized for higher GABA production. The final C. glutamicum strain CGY705 could produce 33.17 g/L GABA from glucose in a 2.4-L bioreactor after 78 h fed-batch fermentation. Since all deletion and expression of genes were performed using chromosomal editing, fermentation of the GABA-producing strains constructed in this study does not need supplementation of any antibiotics and inducers. The strategy used in this study has potential value in the development of GABA-producing bacteria.

Transcriptional Regulation of the Creatine Utilization Genes of Corynebacterium glutamicum ATCC 14067 by AmtR, a Central Nitrogen Regulator.

In the genus Corynebacterium, AmtR is a key component of the nitrogen regulatory system, and it belongs to the TetR family of transcription regulators. There has been much research on AmtR structure, functions, and regulons in the type strain C. glutamicum ATCC 13032, but little research in other C. glutamicum strains. In this study, chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq) was performed to identify the AmtR regulon in C. glutamicum ATCC 14067. Ten peaks were obtained in the C. glutamicum ATCC 14067 genome including two new peaks related to three operons (RS_01910-RS_01915, RS_15995, and RS_16000). The interactions between AmtR and the promoter regions of the three operons were confirmed by electrophoretic mobility shift assays (EMSAs). The RS_01910, RS_01915, RS_15995, and RS_16000 are not present in the type strain C. glutamicum ATCC 13032. Sequence analysis indicates that RS_01910, RS_01915, RS_15995, and RS_16000, are related to the degradation of creatine and creatinine; RS_01910 may encode a protein related to creatine transport. The genes RS_01910, RS_01915, RS_15995, and RS_16000 were given the names crnA, creT, cshA, and hyuB, respectively. Real-time quantitative PCR (RT-qPCR) analysis and sfGFP (superfolder green fluorescent protein) analysis reveal that AmtR directly and negatively regulates the transcription and expression of crnA, creT, cshA, and hyuB. A growth test shows that C. glutamicum ATCC 14067 can use creatine or creatinine as a sole nitrogen source. In comparison, a creT deletion mutant strain is able to grow on creatinine but loses the ability to grow on creatine. This study provides the first genome-wide captures of the dynamics of in vivo AmtR binding events and the regulatory network they define. These elements provide more options for synthetic biology by extending the scope of the AmtR regulon.

Production of Cinnamaldehyde through Whole-Cell Bioconversion from trans-Cinnamic Acid Using Engineered Corynebacterium glutamicum.

Cinnamaldehyde (CAD) has various applications in foods and pharmaceuticals and has gained prominence as a potent nematicide in agricultural research owing to its nematicidal activity. However, conventional methods of CAD production, including extraction from plants or organic chemical synthesis, are environmentally hazardous and limit its utilization for downstream applications. Here, we engineered Corynebacterium glutamicum as a whole-cell biocatalyst for the efficient bioconversion of trans-cinnamic acid (t-CA) into CAD. An expression module of Mycobacterium phlei carboxylic acid reductase was constructed for the conversion of t-CA to CAD. Additionally, the putative dehydrogenase-related genes (dkgA, adhC, and cg1176) responsible for the conversion of CAD to cinnamyl alcohol were deleted from the engineered C. glutamicum strain to prevent the loss of CAD. Furthermore, as the conversion is NADPH-dependent, we investigated the conversion efficiency by exchanging the putative promoter region for the zwf gene, which encodes glucose-6-phosphate dehydrogenase, with a strong promoter to increase the NADPH pool. Finally, a bioconversion platform using C. glutamicum as a whole-cell biocatalyst was developed by deleting the vdh gene, which is involved in the reverse conversion of CAD to t-CA. Taken together, a 100% conversion yield of 1.1 g/L CAD from 1.2 g/L t-CA was obtained within 30 min.

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.

Metabolic engineering of Corynebacterium glutamicum for de novo production of 3-hydroxycadaverine

Functionalization of amino acids and their derivatives opens up the possibility to produce novel compounds with additional functional groups, which can expand their application spectra. Hydroxylation of polyamide building blocks might allow crosslinking between the molecular chains by esterification. Consequently, this can alter the functional properties of the resulting polymers. C. glutamicum represents a well-known industrial workhorse and has been used extensively to produce lysine and lysine derivatives. These are used as building blocks for chemical and pharmaceutical applications. In this study, it was shown for the first time that C3-hydroxylated cadaverine can be produced de novo by a lysine overproducing C. glutamicum strain. The lysine hydroxylase from Flavobacterium johnsoniae is highly specific for its natural substrate lysine and, therefore, hydroxylation of lysine precedes decarboxylation of 4-hydroxylysine (4-HL) to 3-hydroxycadaverine (3-HC). For optimal precursor supply, various cultivation parameters were investigated identifying the iron concentration and pH as major effectors on 4-HL production, whereas the supply with the cosubstrate 2-oxoglutarate (2-OG) was sufficient. Deletion of the gene coding for the lysine exporter LysE suggested that the exporter may also be involved in the export of the structurally similar 4-HL. With the optimised setting for 4-HL production, the pathway was extended towards 3-HC by decarboxylation. Three different genes coding for lysine/4-HL decarboxylases, LdcC and CadA from E. coli and DCFj from F. johnsoniae, were expressed in the 4-HL producing strain and compared regarding 3-HC production. It was shown in a semi-preparative biocatalysis that all three decarboxylases can accept 4-HL as substrate with varying efficiencies. In vivo, LdcC supported 3-HC production best with a final titer of 11 mM. To improve titers a fed-batch cultivation in 1 L bioreactor scale was performed and the plasmid-based overexpression of ldcC was induced after 24 h resulting in the highest titer of 8.6 g L-1 (74 mM) of 3-hydroxycadaverine reported up to now.

Construction of an IS-Free Corynebacterium glutamicum ATCC 13 032 Chassis Strain and Random Mutagenesis Using the Endogenous ISCg1 Transposase.

Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a “zero-background” host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.

Rational reformation of Corynebacterium glutamicum for producing L-lysine by one-step fermentation from raw corn starch.

This article focuses on engineering Corynebacterium glutamicum to produce L-lysine efficiently from starch using combined method of "classical breeding" and "genome breeding." Firstly, a thermo-tolerable L-lysine-producing C. glutamicum strain KT45-6 was obtained after multi-round of acclimatization at high temperature. Then, amylolytic enzymes were introduced into strain KT45-6, and the resultant strains could use starch for cell growth and L-lysine production except the strain with expression of isoamylase. In addition, co-expression of amylolytic enzymes showed a good performance in starch degradation, cell growth and L-lysine production, especially co-expression of α-amylase (AA) and glucoamylase (GA). Moreover, L-lysine yield was increased by introducing AA-GA fusion protein (i.e., strain KT45-6S-5), and finally reached to 23.9 ± 2.3g/L in CgXIIIPM-medium. It is the first report of an engineered L-lysine-producing strain with maximum starch utilization that may be used as workhorse for producing amino acid using starch as the main feedstock. KEY POINTS: • Thermo-tolerable C. glutamicum was obtained by temperature-induced adaptive evolution. • The fusion order between AA and GA affects the utilization efficiency of starch. • C. glutamicum with starch utilization was constructed by optimizing amylases expression.

Enhanced production of \u03b3-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation

BackgroundBio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics.ResultsUnder aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture.ConclusionsOur results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.Graphical

Transcriptome analysis of L-leucine-producing Corynebacterium glutamicum under the addition of trimethylglycine.

It has been widely reported that the addition of trimethylglycine (betaine) decreases osmotic pressure inhibition for cell growth, leading to increased production of amino acids. However, the underlying mechanism is unclear. To determine the global metabolic differences that occur under the addition of trimethylglycine, transcriptome analysis was performed. Transcriptome analysis of Corynebacterium glutamicum JL1211 revealed that 272 genes exhibited significant changes under trimethylglycine addition. We performed Gene Ontology (GO) and KEGG enrichment pathway analyses on these differentially expressed genes (DEGs). Significantly upregulated genes were mainly involved in the regulation of ABC transporters, especially phosphate transporters and sulfur metabolism. The three phosphate transporter genes pstC, pstA and pstB were upregulated by 13.06-fold, 29.80-fold and 30.49-fold, respectively. Notably, the transcriptional levels of the cysD, cysN, cysH and sir genes were upregulated by 81.5-fold, 57.3-fold, 77.6-fold and 125.4-fold, respectively, consistent with assimilatory sulfate reduction under the addition of trimethylglycine. The upregulation of ilvBN and leuD genes might result in increased L-leucine formation. The data indicated changes in the transcriptome of C. glutamicum with trimethylglycine treatment, thus providing a mechanism supporting the application of trimethylglycine in the production of L-leucine and other amino acids by C. glutamicum strains.

Enhancing β-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering.

To directly produce β-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of β-alanine production was constructed in this study. To do this, the biosynthetic pathway of β-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase β-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing β-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate β-alanine. To further increase β-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and β-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3g/L of β-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32g/L) in shake-flask fermentation and produced 56.5 ± 3.2g/L of β-alanine with a productivity of 0.79g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of β-alanine that improves the efficiency of β-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of β-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to β-alanine. • A 56.5-g/L β-alanine production in fed-batch fermentation was achieved.

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress.

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Improve gamma-aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic flux

Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals, livestock, food additives, and so on. It is important to develop Corynebacterium glutamicum strains that can efficiently produce gamma-aminobutyric acid from glucose. In this study, production of gamma-aminobutyric acid in C. glutamicum CGY700 was improved by construction of CO2 anaplerotic reaction and overexpression of citrate synthase. The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck. The expression of ppc and gltA were controlled by promoters Ptac and PtacM, and the optimal mode of PtacM-ppc-Ptac-gltA was determined. Simultaneously, the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted, and glnA2 encoding glutamine synthase was overexpressed in the chromosome. The final strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake flask cultivation and 58.33 g/L gamma-aminobutyric acid via Fed-Batch fermentation with a yield of 0.30 g/g glucose. CGY-PG-304 was constructed by genome editing; therefore, it is stable and not necessary to add any antibiotics and inducer during fermentation.

Safety evaluation of the food enzyme d-psicose 3-epimerase from the genetically modified Corynebacterium glutamicum strain FIS002.

This assessment addresses a food enzyme preparation consisting of the immobilised intact but non‐viable cells of the genetically modified Corynebacterium glutamicum strain FIS002 by CJ‐Tereos Sweeteners Europe SAS. The production strain produces the food enzyme d‐fructose 3‐epimerase (d‐psicose 3‐epimerase; EC 5.1.3.30). The food enzyme preparation is used in processing fructose to produce a speciality carbohydrate d‐allulose (synonym d‐psicose). Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of d‐allulose, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,796 mg TOS/kg body weight (bw) per day, the highest dose tested. A search for similarity of the amino acid sequence of the enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood of such reactions to occur is low. The food enzyme preparation contains multiple copies of an antimicrobial resistance gene, which is considered a hazard. However, under the specific intended conditions of use described by the applicant, and based on the evidence showing the removal of TOS during the production of d‐allulose and the absence of recombinant DNA in the d‐allulose, the Panel concluded that the identified hazard associated with the food enzyme d‐psicose 3‐epimerase produced with the genetically modified C. glutamicum strain FIS002 will not result in a risk.

Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum.

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine–derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylA Xc B Cg (xylA from Xanthomonas campestris) and araBAD Ec from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcC Ec from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDA Ec from E. coli or with putrescine oxidase gene puo Rq from Rhodococcus qingshengii and patD Ec . Deletion of the l-glutamate dehydrogenase–encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcC Ec -patDA Ec ) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

Shikimate Metabolic Pathway Engineering in Corynebacterium glutamicum.

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.