Nitrogen is an essential element for every organism on Earth. Modern agricultural activity depletes
soil in nitrogen much faster than it is naturally replenished. Therefore, fertilization is key for feeding a
fast-growing population. However, the use efficiency of fertilizer nitrogen is only about 60%. The rest
of reactive nitrogen leaches to the environment, and the pollution caused demands modern societies
tens of billions of euros annually as remediation costs. Rationalisation of current agricultural practices
is essential, including a more targeted application of fertilizers, to tackle the nitrogen crisis. One way
is the use of nitrogen-fixing organisms as biofertilizer in close association to agricultural crops. In this
thesis, a stoichiometric model was reconstructed for the heterocystous nitrogen-fixing
cyanobacterium Anabaena sp. PCC 7120 to understand the nature of metabolite exchange between
its photosynthetic and diazotrophic cell types, and design metabolic engineering strategies for
nitrogen excretion. Using flux balance analysis of diazotrophically grown filaments, excretion of
ammonia followed by urea achieved the highest molar nitrogen flux. To achieve a similar effect
experimentally, glutamine synthetase (GS) was inhibited using L-methionine sulfoximine. It was
possible to accumulate about 760 μM ammonia in a 7-day assay with stagnating growth. For stable
excretion, GS has been replaced for an active-site mutant exhibiting decreased specific activity for
ammonia. The metabolic changes have been implemented in both wild type and an ammonium uptake
transporter mutant (Δamt). The resulting strains displayed increased ammonia excretion up to about
8-fold compared to the wild type. Furthermore, IF7A, a small oligopeptide controlling the activity of
GS, has been overexpressed under four different promoters. The constructs driven by two of these
promoters, PnifHDK and PpetE enabled the growth of the non-diazotrophic alga Chlorella vulgaris at 68%
of that of the algal monoculture on combined nitrogen. Overall, the ammonia-excreting strains
provided an important proof-of-principle for the development of more efficient biofertilizers in future
agriculture.