Nitrogen-controlled Valorization of Xylose-derived Compounds by Metabolically Engineered Corynebacterium glutamicum

Abstract

The implementation of bioprocesses in an economically feasible and industrial competitive manner requires the optimal allocation of resources for a balanced distribution between biomass formation and product synthesis. The decoupling of growth and production in two-stage bioprocesses, aiming to ensure sufficient growth before the onset of production, is particularly relevant when target products inhibit growth. In order to avoid expensive inducer molecules, continuing process monitoring, elaborate individual process optimization, and strain engineering, we developed and applied nitrogen deprivation-induced expression of genes for product biosynthesis. Two native nitrogen deprivation-inducible promoters were identified and shown to function for dynamic growth-decoupled gene expression or CRISPRi-mediated gene knockdown in C. glutamicum with superior induction factors than the standard IPTG-inducible Ptrc promoter. Valorization of xylose to produce either the sugar acid xylonic acid or the sugar alcohol xylitol from xylose as sole source of carbon and energy was demonstrated. Competitive titers of up to 34 g•L −1 xylonate and 13 g•L −1 xylitol were achieved in two-stage processes. We discussed that the transfer to bioprocesses with C. glutamicum using carbon sources other than xylose appears straightforward in particular regarding production of growth-inhibitory compounds by their growthdecoupled fermentative production.

Establishment of synthetic microbial consortia with Corynebacterium glutamicum and Pseudomonas putida: Design, construction, and application to production of γ‐glutamylisopropylamide and l‐theanine

“…Formamide is a rare nitrogen source in biotechnology (Schwardmann, Wu, et al., 2023) and was used in the here developed consortia with ammonium dependency (+/0 and +/+). Cross‐feeding of ammonium was shown between formamidase‐positive and formamide‐negative C. glutamicum strains (Schwardmann, Rieks, et al., 2023) and here for interspecies consortia with P. putida and C. glutamicum . Production of glutamate with formamide as a nitrogen source was also established by engineering C. glutamicum for expression of amiF from H. pylori .…”

“…A more balanced ratio between the strains could only be reached within 24 h in the mutualistic (+/+) consortium, in which P. putida releases ammonium as a nitrogen source for C. glutamicum , which in turn secretes arginine to supplement growth by the arginine‐auxotrophic P. putida . In a consortium with formamidase‐positive and formamidase‐negative strains of C. glutamicum , only lower ratios of formamidase‐positive strains were necessary to support growth of formamidase‐negative C. glutamicum strain (Schwardmann, Rieks, et al., 2023).…”

“…Production of glutamate with formamide as a nitrogen source was also established by engineering C. glutamicum for expression of amiF from H. pylori . Comparing the product titre, there was a 2.5‐fold increase in glutamate production with formamide instead of ammonium sulphate and urea (Schwardmann, Rieks, et al., 2023). This is in line with our observations for the highest production of theanine with strain Thea1 FORM (Figure 4C) with formamide as a nitrogen source.…”

Prospects of formamide as nitrogen source in biotechnological production processes

“…Similarly, it could be elaborated for formamide to completely decouple biomass and biosynthesis of nitrogen-free products. For the bioconversion of xylose to produce xylitol and xylonate in C. glutamicum (Schwardmann et al 2023a ) , nitrogen starvation–inducible promoters combined with balanced provision of ammonia allowed for growth-decoupled production. What these approaches have in common is that they only work for non-nitrogenous target compounds such as acetoin or xylitol.…”