Metabolic engineering and adaptive laboratory evolution of Kluyveromyces Marxianus for lactic acid production.

Background: Poly lactic acid (PLA) is one of the most promising bioplastics due to its interesting mechanical and physical properties, low carbon footprint, and biodegradability. PLA is produced from lactic acid (LA) that is either sourced from petrochemical industries or obtained through microbial fermentation using lactic acid bacteria, with the latter accounting for 90% of total LA production. While the bio-based production is more sustainable, it requires complex and expensive feedstocks and large amounts of neutralization agents for pH control during fermentation.

Results: We explored the potential of a non-conventional, acid-tolerant yeast Kluyveromyces marxianus for LA production. First, we analyzed 168 genetically diverse K. marxianus strains to identify the best candidate chassis strains and each of the 10 selected strains was genetically engineered to produce LA. The best candidate strain, Km3, was subjected to adaptive laboratory evolution, yielding a further 18% increase in LA production, reaching titers of 120 g L^- 1 LA and a yield of 0.81 g g^- 1, while requiring less neutralization agent and showing capacity to efficiently ferment xylose-containing feedstocks. Genome sequencing identified a mutation in the general transcription factor gene SUA7 that proved causal for the increased performance of the evolved clone.

Conclusions: Our results highlight the potential of integrating state-of-the-art techniques with the genetic diversity of non-standard microbes to obtain superior microbial cell factories that can ferment xylose-containing media and can be harnessed for sustainable commercial production of fine chemicals through precision fermentation.