Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

Abstract

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, ) can overcome such production limitations owing to a natural propensity for high flux through acetyl-CoA. By exploring three distinct metabolic engineering strategies for acetyl-CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression () can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.

Expanding the Chemical Palette of Industrial Microbes: Metabolic Engineering for Type III PKS‐Derived Polyketides

“…The most successful strategy for triacetic acid lactone production deviated from traditional hosts and employed a precursor overexpression strategy in the non‐conventional yeast, Y. lipolytica . By overexpression of the pyruvate bypass pathway combined with ACC, this strain was able to generate 35.9 g L −1 of triacetic acid lactone in bioreactor fermentation . This result suggests that the oleaginous property of this yeast provides a more optimal starting point for acetyl‐CoA‐ and malonyl‐CoA‐derived polyketides.…”

“…It was recently shown that this high flux through acyl‐CoA precursors could be diverted away from lipid production and into production of the polyketide, triacetic acid lactone . While no other type III PKS‐derived polyketides have yet been produced by Y. lipolytica , the extraordinarily high titer achieved in this rewired strain holds promise as an exciting starting point for expanding the reach of this organism as a type III PKS‐derived polyketide host …”

“…A recently proposed host for polyketide production is the oleaginous yeast Yarrowia lipolytica owing to the high acetyl‐CoA and malonyl‐CoA pools implicit in this organism as an industrial workhorse for lipid and organic acid production . It was recently shown that this high flux through acyl‐CoA precursors could be diverted away from lipid production and into production of the polyketide, triacetic acid lactone . While no other type III PKS‐derived polyketides have yet been produced by Y. lipolytica , the extraordinarily high titer achieved in this rewired strain holds promise as an exciting starting point for expanding the reach of this organism as a type III PKS‐derived polyketide host …”

“…This compound has been identified as a potential biorenewable platform chemical as it can be chemically converted into a variety of different valuable products traditionally sourced from petroleum, such as sorbic acid . Additionally, triacetic acid lactone can be converted into antibiotics, such as pogostone, as well as into novel polymers . Biochemically, triacetic acid lactone is a common derailment product formed during the synthesis of other, longer polyketides.…”

“…However, a type III PKS from Gerbera hybrida was identified that natively produces triacetic acid lactone (Figure a). As a result, the 2‐pyrone synthase (encoded by g2ps1 ) has been imported into E. coli , S. cerevisiae , and Y. lipolytica hosts and coupled with strain engineering . Expression of this synthase in E. coli led to initial production of 0.5 g L −1 .…”