Melanie Mindt and Lenny Ferrer as shared first authors, Dirk Bosch, Katarina Cankar and Volker F. Wendisch have published a study on de novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum. INDIE partners Bielefeld University, Wageningen Plant Research and  Axxence Aromatic GmbH contributed to this publication. In this work, a C. glutamicum shikimate accumulating base strain was engineered for tryptophan production and a titre of 2.14 ± 0.02 g L^-1 tryptophan was achieved. Next, de novo indole production from glucose was enabled by expressing P. rettgeri tnaA and upon process optimisation a titre of 1.38 ± 0.04 g L^-1 indole was achieved. The process was accelerated by decoupling growth from production increasing the volumetric productivity about 4-fold to 0.08 g L^-1 h^-1.

Mindt M, Ferrer L, Bosch D, Cankar K, Wendisch VF (2023) De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol https://doi.org/10.1007/s00253-023-12397-4 

Katarina Cankar, Nadja Henke and Volker F. Wendisch from the INDIE partners Wageningen Plant Research and Bielefeld University summarized how Corynebacterium glutamicum has been engineered for the sustainable production of functional food additives and ingredients in their recent review. A number of compounds were covered including carotenoid pigments, while one focus detailed aromatic flavour and fragrance compounds including vanillin and indole. Differences and similarities of the metabolic engineering strategies to achieve de novo production of vanillin and indole such as abolishing degradation of aldehyde intermediates in the former process and bioprospecting IGL and TNA enzymes used for indole production in the latter process were discussed.

Cankar K, Henke NA, Wendisch VF (2023) Functional food additives/ingredients production by engineered Corynebacterium glutamicum. Syst Microbiol Biomanuf 3:110-121. https://doi.org/10.1007/s43393-022-00141-4.

Marko Petek, Maja Zagorščak and Andrej Blejec as shared first authors, Živa Ramšak, Anna Coll, Špela Baebler, and Kristina Gruden from the INDIE team of National Institute of Biology have published an article on managing life science research data with pISA-tree. In life science research projects, large amounts of diverse data are generated, thus managing them in a findable, accessible, interoperable and reusable (FAIR) manner is a big challenge. To fast-track discoveries, wider implementation of open data management following FAIR principles is imperative. pISA-tree data management framework is as simple as possible, and as detailed as necessary, and enables synchronisation with the FAIRDOMHub repository. Behind the Paper

Petek, M., Zagorščak, M., Blejec, A. et al. pISA-tree - a data management framework for life science research projects using a standardised directory tree. Sci Data (2022) 9, 685. https://doi.org/10.1038/s41597-022-01805-5

Lenny Ferrer and Melanie Mindt as shared first authors, Maria Suarez-Diez, Tatjana Jilg, Maja Zagorscak, Jin-Ho Lee, Kristina Gruden, Volker F. Wendisch and Katarina Cankar have published a study on the production of indole via bacterial tryptophan synthase alpha subunit and plant indole-3-glycerol phosphate lyase enzymes in Corynebacterium glutamicum. Authors from the INDIE partners Wageningen Plant Research, Bielefeld University, Axxence Aromatic GmbH, National Institute of Biology and Laboratory of Systems and Synthetic Biology at Wageningen University and Research contributed to the publication in collaboration with Jin-Ho Lee from Kyungsung University. 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.

Ferrer L, Mindt M, Suarez-Diez M, Jilg T, Zagorscak M, Lee J-H, Gruden K, Wendisch VF, Cankar K (2022) Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes. J. Agric. Food Chem. 70:5634-5645.  https://doi.org/10.1021/acs.jafc.2c01042

Melanie Mindt and Katarina Cankar have led a study on the biotransformation of L-tryptophan to indole by recombinant Corynebacterium glutamicum as cell factory for flavor and fragrance applications. Authors from the INDIE partners Wageningen Plant Research, Axxence Aromatic GmbH, Bielefeld University, and Laboratory of Systems and Synthetic Biology at Wageningen University and Research contributed to the publication. The bioconversion production process established in this study provides an attractive route for sustainable indole production from tryptophan in C. glutamicum. Industrially relevant indole titers were achieved within 24 h and indole was concentrated in the organic layer as a pure product after the fermentation.

Mindt M, Kashkooli AB, Suarez-Diez M, Ferrer L, Jilg T, Bosch D, Martins dos Santos V, Wendisch VF and Cankar K (2022) Production of indole by Corynebacterium glutamicum microbial cell factories for flavor and fragrance applications. Microb. Cell. Fact. (2022) 21:45. https://doi.org/10.1186/s12934-022-01771-y

Melanie Mindt and Volker F. Wendisch from the INDIE teams of Wageningen Plant Research and Bielefeld University contributed to a side project by Anastasia Kerbs and Lynn Schwardmann from Bielefeld University on fermentative production of N-methyl-L-phenylalanine. This N-functionalized aromatic amino acid is used a constituent of peptides enabling passive blood–brain barrier permeation. In part, this work benefitted from the INDIE expertise on metabolic engineering of the shikimate pathway.

Kerbs A, Mindt M, Schwardmann L and Wendisch VF (2021) Sustainable production of N-methylphenylalanine by reductive methylamination of phenylpyruvate using engineered Corynebacterium glutamicum. Microorganisms 9: 824. doi: https://doi.org/10.3390/microorganisms9040824

Tatjana Walter, Arman Beyraghdar Kashkooli, Katarina Cankar and Volker F. Wendisch from the INDIE teams of Bielefeld University and Wageningen Plant Research published on the physiological response of Corynebacterium glutamicum to indole together with Kareen H. Veldmann, Susanne Götker, Tobias Busche, Christian Rückert and Jannik Paulus from Bielefeld University. The work combined growth and RNAseq experiments coupled with adaptive laboratory evolution (ALE), genome sequencing to reveal candidate genes and reverse genetics to identify genes causing indole resistance. Indole was shown to reduce iron in the cultivation medium by a chemical reaction. Moreover, a transcriptional regulator of the iolT2-rhcM2D2 operon was identified and named IhtR for 1,2,4-trihydroxybenzene, hydroquinone, and indole responsive repressor. The insight into chemical and gene regulatory effects by indole will guide efforts by the INDIE project to develop a fermentative indole production process.

Walter T, Veldmann KH, Götker S, Busche T, Rückert C, Kashkooli AB, Paulus J, Cankar K and Wendisch VF (2020) Physiological Response of Corynebacterium glutamicum to indole. Microorganisms 8: 1945. https://doi.org/10.3390/microorganisms8121945

A team of 10 authors with Tatjana Walter, Katarina Cankar, Lenny Ferrer, Melanie Mindt and Volker F. Wendisch from the INDIE teams of Bielefeld University and Wageningen Plant Research published on fermentative production of N-methylanthranilate together with Nour Al Medani, Arthur Burgardt, Anastasia Kerbs and Joe Max Risse from Bielefeld University and Jin-Ho Lee from Kyungsung University. Product formation relied on the heterologous SAM-dependent N-methyltransferase from the plant herb-of-grace (Ruta graveolens). Moreover, strain engineering enabled optimized provision of anthranilate as precursor. This is a milestone of the INDIE project.

Walter T, Al Medani N, Burgardt A, Cankar K., Ferrer L, Kerbs A, Lee J-H, Mindt M, Risse JR and Wendisch VF (2020) Fermentative N-methylanthranilate production by engineered Corynebacterium glutamicum. Microorganisms 8: 866. https://doi.org/10.3390/microorganisms8060866

Volker F. Wendisch from the INDIE team of Bielefeld University and Prof. Jin-Ho Lee from Kyungsung University, Busan, South Korea, contributed to the book "Corynebacterium glutamicum – Biology and Biotechnology" edited by M. Inui and K. Toyoda, by reviewing C. glutamicum metabolic engineering from the classical, genetic engineering, systems biology eras to the era of synthetic biology for strain development and by forecasting the impact of the most recent methods such as CRISPR technology and adaptive laboratory evolution.

Wendisch VF, Lee J-H (2020) Metabolic engineering in Corynebacterium glutamicum. In: Corynebacterium glutamicum – Biology and Biotechnology. Inui M, Toyoda K (Eds); Microbiology Monographs. Springer Nature Switzerland. pp. 287-322. https://doi.org/10.1007/978-3-030-39267-3_10

Melanie Mindt, Tatjana Walter and Volker F. Wendisch from the INDIE teams of Bielefeld University and Wageningen Plant Research and Pierre Kugler reviewed N-functionalization of amines by biocatalysis and fermentation. Strains overproducing precursors can be equipped with N-functionalizing enzymes such as imine or ketimine reductases, opine or amino acid dehydrogenases, N-hydroxylases, N-acyltransferase or polyamine synthetases for overproduction of building blocks used for the preparation of bioactive molecules, such as N-methyl-L-alanine, N-methyl-L-glutamate, or L-theanine, a flavor-enhancing compound.

Mindt M, Walter T, Kugler P and Wendisch VF (2020) Microbial engineering for production of N-functionalized amino acids and amines. Biotechnol. J. 15: 1900451. doi: https://doi.org/10.1002/biot.201900451

Volker F. Wendisch from the INDIE team of Bielefeld University contributed to the book "Minimal Cells: Design, Construction, Biotechnological Applications" edited by A. R. Lara and G. Gosset, by reviewing how chassis cells can be developed such that they are as fit as wild type regarding a chosen target function, e.g. growth in glucose minimal medium, while having to invest less to maintain gene functions not required under theses conditions. Some biotechnological applications are given. In the project INDIE we are using strains with reduced genome contents.

Wendisch VF (2020) Genome-Reduced Corynebacterium glutamicum Fit for Biotechnological Applications. In: Lara A., Gosset G. (eds) Minimal Cells: Design, Construction, Biotechnological Applications. Springer, Cham. doi: https://doi.org/10.1007/978-3-030-31897-0_4

Volker F. Wendisch from the INDIE team of Bielefeld University and Elvira Sgobba reviewed design, construction and application of synthetic microbial consortia to sustainable biotechnological production. Examples included production of aromatic compounds such as phenylpropanoids from sugars. Strategies to divide labour between two or more species in a synthetic microbial consortium to allow to access complex substrates or for synthesis via complex linear or converging multi-step routes were highlighted. The review adresses challenges and implications for adaptive laboratory evolution for future improvement of synthetic consortia.

Sgobba E and Wendisch VF (2020) Synthetic microbial consortia for small molecule production. Curr. Opin. Biotechnol. 62:72-79. doi: https://doi.org/10.1016/j.copbio.2019.09.011

Volker F. Wendisch from the INDIE team of Bielefeld University reviewed recent advances of metabolic engineering for amino acid production. Prospects driven by technological push and market pull altering this field are exemplified for specialty proteinogenic amino acids, cyclic amino acids and omega-amino acids. The review also covers amino acid derivatives that may be obtained by - amongst others - hydroxylation, halogenation and N-methylation.

Wendisch VF (2019) Metabolic engineering advances and prospects for amino acid production. Metab. Eng. 58: 17-34. doi: https://doi.org/10.1016/j.ymben.2019.03.008

Axxence produces natural flavour ingredients using plants as crude enzyme sources. The following video shows the biocatalytic production process for the manufacturing of natural 2,6-nonadienal from unsaturated fatty acids using cucumber as the crude enzyme source.