Coupling bacterial nitrogen fixation to yeast whole-cell biocatalysis enables amine production using air as the sole nitrogen source.

Abstract

Background: Alkylamine production depends on ammonium produced via the energy-intensive, fossil-based Haber-Bosch process. Diazotrophic bacteria could offer a biological alternative by supplying nitrogen directly from air to heterotrophic whole-cell biocatalysts for alkylamine production. However, their integration with yeast production systems remains largely unexplored.

Results: Here, we evaluated the use of an ammonium-excreting strain of Azotobacter vinelandii (AZBB664) to supply nitrogen to an engineered Saccharomyces cerevisiae biocatalyst for vanillylamine production using air as the sole nitrogen source. A modified Burk's medium was developed to support S. cerevisiae growth and enable evaluation of biologically supplied nitrogen. Co-cultivation of both organisms was feasible under diazotrophic conditions, however, vanillin oxidation by A. vinelandii prevented effective vanillylamine production. To address this, a sequential process was implemented in which A. vinelandii was first used to generate ammonium in a modified Burk's medium, followed by cell removal and use of the resulting medium (AZM1) for yeast cultivation. In this medium, the yeast strain CEN.PK 113-7d exhibited a growth rate of 0.27 h^- 1 and reached higher final cell density compared to the control medium supplemented with synthetic ammonium. Cultivation in AZM1 was associated with a more stable pH profile, which may have contributed to the improved growth of the yeast. S. cerevisiae strain TMBNM033 expressing recombinant transaminase and alanine dehydrogenase was able to convert vanillin to vanillylamine, although a significant amount of vanillic alcohol was also observed due to competing endogenous reductase activity.

Conclusions: A. vinelandii-derived medium enables yeast growth and whole-cell reductive amination using air as the sole nitrogen source. This demonstrates that biologically fixed nitrogen can directly sustain heterotrophic whole-cell biotransformations, establishing a strategy for coupling diazotrophy with biocatalytic production systems.