Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its favorable growth characteristics; these include rapid growth rate, ability to metabolize an array of substrates, high TCA cycle flux, and tolerance to low pH and high temperatures (in excess of 45oC). Unlike S. cerevisiae, K. marxianus is able to grow on low-cost substrates to high cell densities that equal or surpass cell densities in glucose. Metabolism of these carbon sources can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus as a platform for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The required 2-pyrone synthase (2-PS) was expressed using a K. marxianus CEN/ARS plasmid, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. Xylose and glycerol were the best carbon sources for TAL production. While the highest TAL titers were observed at 37oC, significant levels were also obtained at 41oC and 43oC. Expression system improvements including the introduction of high-stability 2-PS mutants and promoter substitution resulted in 10-fold higher titer in defined xylose medium, with only a 20% drop in specific titer in minimal medium. Utilization of a multi-copy pKD1-based plasmid (from Kluyveromyces lactis) improved TAL titers 5-fold over low-copy the CEN/ARS plasmid, reaching &gt;1 g/L for the wildtype strain in testtube culture at 37oC. This is an excellent titer and yield (from xylose) relative to other yeast hosts (from glucose) prior to metabolic engineering, and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production from a variety of carbon sources. Using our new CRISPR system, we are now optimizing the number of 2-PS genes, and modifying native and heterologous pathways for higher levels of the acetyl-CoA and malonyl-CoA precursors. Continuing work also includes the synthesis of alternate products, and analysis in controlled bioreactors. This should further improve the production of value-added biobased chemicals from lower-cost feedstocks.

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

2019 Spring National Meeting

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