Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fed-batch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1, 0.52 mg g−1, and 0.25 mg L−1 h−1, respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures.

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

Microorganisms ◽

10.3390/microorganisms9040824 ◽

2021 ◽

Vol 9 (4) ◽

pp. 824

Author(s):

Anastasia Kerbs ◽

Lynn Schwardmann ◽

Melanie Mindt ◽

Volker F. Wendisch

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Corynebacterium Glutamicum ◽

Xanthomonas Campestris ◽

De Novo ◽

Xylose Isomerase ◽

Sustainable Production ◽

Volumetric Productivity ◽

Genes Encoding ◽

Branched Chain

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.

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Production of Cadaverine in Recombinant Corynebacterium Glutamicum Overexpressing Lysine Decarboxylase (ldcC) and Response Regulator dr1558

10.21203/rs.3.rs-764745/v1 ◽

2021 ◽

Author(s):

Soong-bin Kang ◽

Jong-il choi

Keyword(s):

Corynebacterium Glutamicum ◽

Phosphoenolpyruvate Carboxykinase ◽

Deinococcus Radiodurans ◽

Response Regulator ◽

Fold Increase ◽

Lysine Biosynthesis ◽

Lysine Decarboxylase ◽

Fed Batch ◽

Fed Batch Fermentation ◽

Polymerase Chain

Abstract In this study, the response regulator DR1558 from Deinococcus radiodurans was overexpressed in recombinant Corynebacterium glutamicum with lysine decarboxylase (ldcC). The recombinant C. glutamicum strain overexpressing dr1558 and ldcC produced 5.9 g/L of cadaverine by flask cultivation, whereas the control strain overexpressing only ldcC produced 4.5 g/L of cadaverine. To investigate the mechanism underlying the effect of DR1558, the expression levels of genes related to central metabolism and lysine-biosynthesis were analyzed by quantitative-real time polymerase chain reaction. The results showed that phosphoenolpyruvate carboxykinase (pck) was downregulated, and pyruvate kinase (pyk) and other lysine biosynthesis genes were upregulated. Furthermore, in fed-batch fermentation, C. glutamicum coexpressing dr1558 produced 25.14 g/L of cadaverine, a 1.25-fold increase in concentration relative to the control. These results suggested that the heterologous expression of dr1558 may improve the production of biorefinery products by recombinant C. glutamicum.

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Side-by-Side Comparison of Clean and Biomass-Derived, Impurity-Containing Syngas as Substrate for Acetogenic Fermentation with Clostridium ljungdahlii

Fermentation ◽

10.3390/fermentation6030084 ◽

2020 ◽

Vol 6 (3) ◽

pp. 84

Author(s):

Alba Infantes ◽

Michaela Kugel ◽

Klaus Raffelt ◽

Anke Neumann

Keyword(s):

Biomass Gasification ◽

Product Formation ◽

Inhibitory Effects ◽

Fed Batch ◽

Gaseous Mixtures ◽

Clostridium Ljungdahlii ◽

Acetogenic Bacteria ◽

Fed Batch Fermentation ◽

Total Productivity ◽

The Impact

Syngas, the product of biomass gasification, can play an important role in moving towards the production of renewable chemical commodities, by using acetogenic bacteria to ferment those gaseous mixtures. Due to the complex and changing nature of biomass, the composition and the impurities present in the final biomass-derived syngas will vary. Because of this, it is important to assess the impact of these factors on the fermentation outcome, in terms of yields, productivity, and product formation and ratio. In this study, Clostridium ljungdahlii was used in a fed-batch fermentation system to analyze the effect of three different biomass-derived syngases, and to compare them to equivalent, clean syngas mixtures. Additionally, four other clean syngas mixtures were used, and the effects on product ratio, productivity, yield, and growth were documented. All biomass-derived syngases were suitable to be used as substrates, without experiencing any complete inhibitory effects. From the obtained results, it is clear that the type of syngas, biomass-derived or clean, had the greatest impact on product formation ratios, with all biomass-derived syngases producing more ethanol, albeit with lesser total productivity.

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Sublethal acid stress and uncoupling effects on cell growth and product formation in Xanthomonas campestris cultures

Biochemical Engineering Journal ◽

10.1016/s1369-703x(02)00070-0 ◽

2002 ◽

Vol 12 (3) ◽

pp. 181-192 ◽

Cited By ~ 5

Author(s):

M.Eugénia Esgalhado ◽

Ana Teresa Caldeira ◽

J.Carlos Roseiro ◽

A.Nick Emery

Keyword(s):

Cell Growth ◽

Xanthomonas Campestris ◽

Acid Stress ◽

Product Formation

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Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3418-3428.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3418-3428 ◽

Cited By ~ 163

Author(s):

Hideo Kawaguchi ◽

Alain A. Vertï¿½s ◽

Shohei Okino ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Xylose Isomerase ◽

Mineral Medium ◽

Gene Encoding ◽

Xylose Consumption ◽

E Coli ◽

High Copy Number Plasmid ◽

Sugar Mixture ◽

Pentose Sugar

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertï¿½s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

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Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.3100-3107.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 3100-3107 ◽

Cited By ~ 53

Author(s):

S. Guillouet ◽

A. A. Rodal ◽

G.-H. An ◽

P. A. Lessard ◽

A. J. Sinskey

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Carbon Balance ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Original Activity ◽

Threonine Dehydratase ◽

Fourfold Increase ◽

Overexpressing Strain

ABSTRACT The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by theilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and atdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.

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Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinantSaccharomyces cerevisiae

Yeast ◽

10.1002/yea.1072 ◽

2004 ◽

Vol 21 (2) ◽

pp. 141-150 ◽

Cited By ~ 29

Author(s):

K. L. Träff-Bjerre ◽

M. Jeppsson ◽

B. Hahn-Hägerdal ◽

M.-F. Gorwa-Grauslund

Keyword(s):

Aldose Reductase ◽

Reductase Activity ◽

Product Formation ◽

Xylose Consumption

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Co-Expression of Threonine Dehydratase and Acetolactate Synthase in Escherichia Coli for L-Isoleucine Production

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.989-994.997 ◽

2014 ◽

Vol 989-994 ◽

pp. 997-1002 ◽

Cited By ~ 1

Author(s):

Jian Wang ◽

Jia Kai Sun ◽

Qing Yang Xu

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Carbon Flux ◽

Batch Fermentation ◽

Acetolactate Synthase ◽

Fed Batch ◽

Threonine Dehydratase ◽

E Coli ◽

Fed Batch Fermentation ◽

Fermentation Period

Metabolic engineering ofCorynebacterium glutamicumhas sought to divert carbon into L-isoleucine. However, the fermentation period of this strain is long. TheC.glutamicumYILW strain (LeuL, AHVr, SGr, Leu-MEr) was previously derived by repeated compound mutagenesis which could accumulate 20.2 g/L L-isoleucine in a 5-L jar fermentor. Overexpression of the threonine dehydratase gene (ilvA) fromCorynebacterium glutamicumYILW and coexpression of threonine dehydratase and acetolactate synthase (ilvBN) from it were employed to divert carbon flux toward L-isoleucine. The strainE. coliTRFC with the expression ofilvA could accumulate L-isoleucine of 6.8 g/L without accumulation of any L-threonine by fed-batch fermentation in a 5-L jar fermentor. However, the production of L-isoleucine by the strainE.coliTRFC with the co-expression ofilvA andilvBN was decreased by 19.1%, and the production of L-valine was increased by 40% compared with that ofE. coliTRFC with the expression ofilvA.

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Pushing product formation to its limit: Metabolic engineering of Corynebacterium glutamicum for l-leucine overproduction

Metabolic Engineering ◽

10.1016/j.ymben.2013.12.001 ◽

2014 ◽

Vol 22 ◽

pp. 40-52 ◽

Cited By ~ 81

Author(s):

Michael Vogt ◽

Sabine Haas ◽

Simon Klaffl ◽

Tino Polen ◽

Lothar Eggeling ◽

...

Keyword(s):

Metabolic Engineering ◽

Corynebacterium Glutamicum ◽

Product Formation

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Kinetic Modeling and Sensitivity Analysis of Kinetic Parameters for L-Glutamic Acid Production Using Corynebacterium glutamicum

International Journal of Chemical Reactor Engineering ◽

10.2202/1542-6580.2038 ◽

2009 ◽

Vol 7 (1) ◽

Cited By ~ 3

Author(s):

S. Suresh ◽

Noor Salam Khan ◽

Vimal Chandra Srivastava ◽

Indra Mani Mishra

Keyword(s):

Experimental Data ◽

Sensitivity Analysis ◽

Kinetic Model ◽

Glutamic Acid ◽

Corynebacterium Glutamicum ◽

Negative Impact ◽

Product Formation ◽

Biochemical Engineering ◽

Glutamic Acid Production ◽

Engineering Journal

In the present work, a kinetic model has been developed for the batch fermentation of glucose for the production of L-glutamic acid (LGA) by Corynebacterium glutamicum. Parameters of the kinetic model have been determined by using the experimental data available in literature and the results have been compared by carrying out computer simulation. The kinetic model proposed in the present study provides better predictions for the growth of biomass, substrate consumption and LGA production as compared to the prediction reported by Bona and Moser (Bioprocess Engineering 17(2) (1997b) 121-125). The present model also fits the experimental data given by Zhang et al. (Enzyme and Microbial Technology, 22, 205-209, 1998) and Khan et al. (Biochemical Engineering Journal, 25, 173178, 2005), respectively. Sensitivity analysis was carried out to assess the validity of the developed model, and to reveal the kinetic parameter that has maximum impact on LGA production. It is found that an increase in the non-growth associated product formation coefficient caused highest negative impact on LGA production.

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