Hexanoic acid production in Kluyveromyces marxianus using simultaneous multiple gene integration system

Many yeast strains are widely employed to produce numerous bioproducts, including fuels and various compounds used in bioenergy and biorefinery. Among yeast strains, Kluyveromyces marxianus is now receiving considerable attention because of its thermotolerance, high growth rate, ability to assimilate various sugars, and high secretory capacity. To better utilize these advantages, we used infamously high 'Non-Homologous End Joining (NHEJ)' activity of K. Marxianus for developing simultaneous integration system which can integrate multiple genes to chromosome of K. marxianus in a single step. Using simultaneous integration system, we integrated six genes (bktB, phbA, atoB, hbd, crt and ter) to chromosome of KM for producing hexanoic acid. The constructed strain (H6) produced 214 mg/L hexanoic acid in 16h by using galactose as a substrate. However, the H6 strain showed inexplicable sudden decrease of hexanoic acid concentration in the middle of fermentation regardless of sugars and conditions tested. H6m strain, which was constructed by replacing atoB with MCT1 from the synthetic pathway, did not show such a sudden drop of hexanoic acid concentration due to efficient condensation of acetyl-CoA into acetoacetyl-CoA. Our finding not only reduces the labor and time required for such procedures, but also removes a number of preconditions, such as pre-made vectors, selection markers and knockout mutants, which are needed to introduce many genes into K. marxianus. Furthermore, this system enables K. marxianus to produce hexanoic acid. hexanoic acid could be produced from K. marxianus for the first time through this simultaneous integration system.