Formamide-based production of amines by metabolically engineering Corynebacterium glutamicum

Abstract

Formamide is rarely used as nitrogen source by microorganisms. Therefore, formamide and formamidase have been used as protection system to allow for growth under non-sterile conditions and for non-sterile production of acetoin, a product lacking nitrogen. Here, we equipped Corynebacterium glutamicum, a renowned workhorse for industrial amino acid production for 60 years, with formamidase from Helicobacter pylori 26695, enabling growth with formamide as sole nitrogen source. Thereupon, the formamide/formamidase system was exploited for efficient formamide-based production of the nitrogenous compounds L-glutamate, L-lysine, N-methylphenylalanine, and dipicolinic acid by transfer of the formamide/formamidase system to established producer strains. Stable isotope labeling verified the incorporation of nitrogen from formamide into biomass and the representative product L-lysine. Moreover, we showed ammonium leakage during formamidase-based access of formamide to be exploitable to support growth of formamidase-deficient C. glutamicum in co-cultivation and demonstrated that efficient utilization of formamide as sole nitrogen source benefitted from overexpression of formate dehydrogenase. Key points • C. glutamicum was engineered to access formamide. • Formamide-based production of nitrogenous compounds was established. • Nitrogen cross-feeding supported growth of a formamidase-negative strain.

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

“…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. Since formamide is also a carbon source accessible to some methylotrophs, formamide may be relevant to fine‐tune and impose nitrogen and/or carbon dependencies in synthetic interspecies microbial consortia that grow with one‐carbon C sources such as methanol or formate (Schwardmann, Benninghaus, et al., 2023).…”

“…Formamidase‐dependent formamide hydrolysis to enable the use of formamide as the sole source of nitrogen was established by plasmid‐based expression of amiF in P. putida KT2440 Δ argH , yielding strain P. putida Δ argH FORM. Based on our previous work on formamidase‐positive C. glutamicum strains, which released sufficient ammonium to allow growth of co‐cultivated formamidase‐deficient C. glutamicum strains (Schwardmann, Wu, et al., 2023 ), it was tested if strain P. putida Δ argH FORM can grow in media with formamide as the sole nitrogen source and if it releases enough ammonium to support growth of co‐cultivated formamidase‐negative C. glutamicum strain ARG2. Since the crimson gene was replaced by the amiF gene in strain P. putida KT2440 Δ argH FORM, Gfp UV fluorescence was used to identify C. glutamicum cells and all non‐fluorescent cells were assigned to P. putida .…”

“…Formamide is a reduced C1 nitrogen compound, such as methylamine, ammonium carbamate and carbamoyl phosphate. The latter two decompose to yield ammonia, while the first two need enzyme‐catalysed reactions for nitrogen liberation (Pols et al., 2021 ; Schwardmann, Wu, et al., 2023 ). 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 +/+).…”

“…The latter two decompose to yield ammonia, while the first two need enzyme‐catalysed reactions for nitrogen liberation (Pols et al., 2021 ; Schwardmann, Wu, et al., 2023 ). 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 .…”

“…In the consortia described here, ammonium and formamide were used as nitrogen sources. Recently, nitrogen deprivation‐inducible promoters were used for dynamic growth‐decoupled gene expression or CRISPRi‐mediated gene knockdown in C. glutamicum for the production of the growth‐inhibitory sugar alcohol xylitol from xylose and were shown to be superior to standard overexpression (Schwardmann, Wu, et al., 2023 ). The use of arginine auxotrophy and overproduction in the consortia described here may also be developed further by using arginine‐dependent gene expression, for example based on the promoter P arg and the transcriptional repressor ArgR from C. glutamicum (Yim et al., 2011 ).…”