Additional file 1 of High-efficiency production of 5-hydroxyectoine using metabolically engineered Corynebacterium glutamicum
Creators: name: Jungmann, Lukas
name: Hoffmann, Sarah Lisa
name: Lang, Caroline
name: De Agazio, Raphaela
name: Becker, Judith
name: Kohlstedt, Michael
name: Wittmann, Christoph
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Ecology

description: Additional file 1: Table S1. Primers used for plasmid construction. Figure S1. Phylogenetic analysis of the donor organisms used in this work. The phylogenetic tree of the donor organisms and the host C. glutamicum ATCC 13032 was compiled using the phyloT online tool (biobyte solutions GmbH, Heidelberg, Germany) and trees visualized with the iTOL online tool [85]. Figure S2. Screening for optimal 5-hydroxyectoine production in recombinant C. glutamicum. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD genes from Pseudomonas stutzeri A1501 (PST), Mycobacterium smegmatis ATCC 19420 (MSM), Streptomyces coelicolor A3(2) (SCO), Halomonas elongata ATCC 33173 (HEL) and Virgibacillus salexigens ATCC 700290 with amino acid exchanges A163C and S244C (VSA) [32], in the pClik 5a (pClik) and pCES-PLPV (pCES) vector. Cultures were grown on minimal glucose medium in a microbioreactor and analyzed for growth (on-line measurement of OD620) and the conversion of ectoine into hydroxyectoine (final titers after depletion of glucose). Screening for different initial ectoine concentrations at 30 °C (a). Screening at different temperatures with 1 mM and 5 mM initial ectoine, respectively (b, c). n = 2. Figure S3. Impact of ectoine and 5-hydroxyectoine on intracellular metabolite levels in C. glutamicum. For 5-hydroxyectoine production C. glutamicum ATCC 13032, episomally expressed a codon-optimized ectD gene from Pseudomonas stutzeri A1501 (PST), Mycobacterium smegmatis ATCC 19420 (MSM), or Virgibacillus salexigens ATCC 700290 (VSA), respectively. The gene from V. salexigens encoded for an enzyme variant that carried the amino acid exchanges A163C and S244C [32]. All strains were cultivated at 37 °C on minimal glucose medium, supplemented with 5 mM ectoine. Cells were analyzed for intracellular metabolite levels during the mid-exponential phase (10 h). As a control, C. glutamicum, harboring the empty vector, investigated during growth on glucose and ectoine (Control) and during growth on glucose alone (Control -Ect), respectively. n = 3. Figure S4. Production of 5-hydroxyectoine in a fed-batch process in stirred tank bioreactors using metabolically engineered C. glutamicum. The C. glutamicum type strain ATCC 13032 episomally expressed the codon optimized ectD genes from Mycobacterium smegmatis ATCC 19420 (a, b) and Pseudomonas stutzeri A1501 (c, d). The process was operated in fed-batch-mode at 37 °C with initial sucrose and ectoine concentrations of 100 g L−1 and 60 g L−1, and the addition of feed (600 g L−1 sucrose, 15 g L−1 yeast extract) at constant rate, respectively. n = 2. Figure S5. Impact of process conditions on fed-batch production of 5-hydroxyectoine using strain C. glutamicum Ptuf ectDMSM in a stirred tank bioreactor at 37 °C. In each setup one process parameter was varied as compared to the standard layout (Fig. 7) to study its impact (from left to right): DO maintained at 60%, twice as high as in the control; supplementation of batch and feed medium with l-glutamate; limited nitrogen supply by elimination of small amounts of yeast extract from the batch medium and the use of a nitrogen-free feed; addition of ectoine at a reduced initial amount (10 g L−1) plus separate feeding later on. n = 1. Figure S6. Production of 5-hydroxyectoine in a batch process in stirred tank bioreactors using C. glutamicum Ptuf ectDPST which episomally expressed codon optimized ectD from Pseudomonas stutzeri A1501. The strain was cultivated in batch-mode with initial sucrose and ectoine concentrations of 100 g L−1 and 50 g L−1 (a, b), 150 g L−1 and 75 g L−1 (c, d) and 200 g L−1 and 100 g L−1 (e, f) at 37 °C. n = 2. Figure S7. Selectivity of 5-hydroxyectoine production in a batch process using C. glutamicum Ptuf ectDPST. The C. glutamicum type strain ATCC 13032 episomally expressed codon-optimized ectD from Pseudomonas stutzeri A1501. It was cultivated with initial sucrose and ectoine concentrations of 100 g L−1 and 50 g L−1 (a), 150 g L−1 and 75 g L−1 (b), and 200 g L−1 and 100 g L−1 (c) at 37 °C. n = 2.