Growth of Corynebacterium glutamicum in minimal medium relies on the supplementation of essential biotin and the siderophore protocatechuate (PCA) to compensate for transient iron limitation [1,2]. Previously, biotin-prototroph C. glutamicum was engineered [1] and iron-responsive, enforced PCA synthesis was implemented in strain C. glutamicum IRON+ to improve intracellular iron availability [2].

Here, a prototrophic C. glutamicum strain was engineered that is independent of both, biotin and PCA. Therefore, C. glutamicum IRON+ was genomically equipped with heterologous bioF and bioI genes catalyzing the missing steps of biotin synthesis [1] which yielded the designated strain C. glutamicum IRON+BioFI. Growth properties were characterized in shake flask cultivations in biotin- and PCA-free CgXII minimal medium (CgXII_MM) with 20 g glucose L^-1­.

In the shake flask, C. glutamicum IRON+BioFI grew to a final biomass of 11.3 ± 0.8 g CDW L^-1 in CgXII_MM, however, showed biphasic growth with reduced growth rates of 0.27 ± 0.00 h^-1 and 0.34 ± 0.02 h^-1 in each phase, respectively. In contrast, the wild type (WT) exhibited continuous exponential growth with a µ of 0.40 ± 0.01 h^-1 reaching 10.2 ± 0.5 g CDW L^-1 in fully supplemented CgXII medium. The biphasic growth was observed before for the WT grown in PCA-depleted CgXII medium which was traced back to a temporary iron limitation [2,3]. Thus, we assume that intracellular iron availability of C. glutamicum IRON+BioFI is impaired again.

Upon bioFI integration, the regulator of the biotin metabolism bioQ was deleted which previously led to decreased transcription levels of genes involved in the iron metabolism, such as cg0924 [4] which encodes a component of a putative siderophore ABC transporter [5]. To investigate the underlying physiological effects of bioQ deletion on the iron metabolism, we deleted cg0924, integrated the bioFI genes into the neutral landing pad CgLP5 [6] and characterized growth of the engineered strains on different carbon sources.

[1] Peters-Wendisch et al., J. Biotechnol. (2014)

[2] Thoma et al., Microb. Biotechnol. (2023)

[3] Müller et al., mBio (2020)

[4] Brune et al., J. Biotechnol. (2012)

[5] Küberl et al., Appl. Environ. Microbiol. (2020)

[6] Lange et al., Microb. Biotechnol. (2018)