Food grade microbial synthesis of the butter aroma compound butanedione using engineered and non-engineered...

The design-build-test-learn (DBTL) cycle has been implemented in metabolic engineering processes for optimizing the production of valuable compounds, including food ingredients. However, the use of recombinant microorganisms for producing food ingredients is associated with different challenges, e.g., in the EU, a content of more than 0.9% of such ingredients requires to be labeled. Therefore, we propose to expand the DBTL cycle and use the "learn" module to guide the development of non-engineered strains for clean label production. Here, we demonstrate how this approach can be used to generate engineered and natural cell factories able to produce the valuable food flavor compound - butanedione (diacetyl). Through comprehensive rerouting of the metabolism of Lactococcus lactis MG1363 and re-installment of the capacity to metabolize lactose and dairy protein, we managed to achieve a high titer of diacetyl (6.7 g/L) in pure dairy waste. Based on learnings from the engineering efforts, we successfully achieved the production of diacetyl without using recombinant DNA technology. We accomplish the latter by process optimization and by relying on high-throughput screening using a microfluidic system. Our results demonstrate the great potential that lies in combining metabolic engineering and natural approaches for achieving efficient production of food ingredients.