Title

Engineering yeast for the high-level synthesis of polyketide biobased chemicals

Conference Dates

July 14-18, 2019

Abstract

Polyketides are an important class of biobased chemicals, both as products and as precursors for subsequent conversion to a wide range of compounds. They are synthesized via complex polyketide synthases, and many using acetyl-CoA and malonyl-CoA as starter and extender units. We have combined engineering of the pathway and synthase enzymes, metabolic pathway engineering, and improved cultivation strategies to substantially increase titers and yields in two important yeast species, Saccharomyces cerevisiae and the thermotolerant Kluyveromyces marxianus. Our work has focused on the polyketide triacetic acid lactone (TAL) as it is can be converted into a wide range of high-value and commodity products. TAL is also a simple polyketide that requires expression of only one synthase enzyme and is easily assayed; it can thus be used as an effective and rapid indicator of strains with the high acetyl-CoA and malonyl-CoA pools needed for polyketide production. We have then extended our successful strategies to the synthesis of 6-methylsalicylic acid and a novel biosurfactant. For high-level TAL production in S. cerevisiae, our work has focused on overexpression of native and variant Gerbera hybrida 2-pyrone synthases, extensive engineering of the yeast metabolic pathways for increased cofactor and precursor pools, and implementation of fed-batch cultivation strategies. These interventions increased TAL titer from 0.07 g/L to 10.5 g/L and yield from <1% to 44% of theoretical. Recent work has included engineering of native regulatory systems to increase synthesis of polyketides and implementation of CRISPR-based combinatorial methods. In the presentation, we will highlight the critical pathways engineered, and examine the synergy between successful strategies for the polyketide products. We have also demonstrated the promise of the alternate yeast species K. marxianus for the high-level synthesis of polyketides from low cost substrates. K. marxianus has excellent growth characteristics for biobased chemical production, including 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 that in glucose. We have evaluated the effects of temperature, carbon source, 2-PS enzyme variant, and expression method on the synthesis of TAL. 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. Utilization of a multi-copy pKD1- based plasmid resulted in TAL titers of >1 g/L in testtube culture at 37oC prior to any metabolic engineering of the yeast. Yield on carbon is also the highest reported to date 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 modifying native and heterologous pathways in K. marxianus to increase levels of the acetyl-CoA and malonyl-CoA precursors. Continuing work also includes the synthesis of alternate products, and cultivation in controlled bioreactors to increase titer and yield. We will present our successful strategies to increase the synthesis of this class of biobased chemicals from lower-cost feedstocks in this rapidly growing thermotolerant yeast.

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