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

Abstract

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined.The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

Developing a broad-range promoter set for metabolic engineering in the thermotolerant yeast Kluyveromyces marxianus

“…CEN/ARS, P NC1 –2PS-T CYC1Sc , HIS3 marker This study P Sc ADH2 -2PS K.m. CEN/ARS, P Sc ADH2 -2PS-T CYC1Sc , HIS3 marker This study pKD-A2PS K. lactis pKD1, P Sc ADH2 .c -2PS-T CYC1Sc , URA3 marker McTaggart et al. (2019) pKD-N2PS K. lactis pKD1, P NC1 –2PS-T CYC1Sc , URA3 marker This study …”

“…This was necessary due to the presence of an internal Spe I restriction site in the selected TEF3 promoter region. Finally to create pKD-N2PS, the ADH2 promoter from S. cerevisiae was substituted by the K. marxianus NC1 promoter in the high-copy plasmid pKD-A2PS ( McTaggart et al., 2019 ) by introducing the PCR amplification product from primer pair FW-pKD1-NC1-2PS ​+ ​RV-pKD1-NC1-2PS using P NC1 -2PS as a template and the restriction sites Zra I and Spe I.…”

“…This culture was used as the inoculum for the experimental cultures in 25 ​mL of SX-His 250 ​mL baffled flasks (initial OD 600 ​= ​0.05), which were grown until late exponential or late stationary phase. KM1 ΔURA3 strains were cultivated as described by ( McTaggart et al., 2019 ). Briefly, cells were inoculated from fresh plates into 3 ​mL of liquid 1% SXCA or 0.95% SLCA and grown overnight at 37 ​°C.…”

“…Like other non-conventional microbes, there has been considerably less research effort put towards developing well-characterized genetic parts for manipulating gene expression in comparison to the common laboratory host and ethanol producer Saccharomyces cerevisiae ( Löbs et al., 2017 ). Despite this general lack of metabolic engineering tools, simple genetic manipulations and media optimization have been used to demonstrate the potential of K. marxianus for a variety of bioprocesses including protein production, high temperature ethanol fermentation, the biosynthesis of ethyl acetate (a native metabolite produced in grams per liter quantities in many K. marxianus strains) and heterologous products such as the polyketide triacetic acid lactone (TAL) ( Cernak et al., 2018 ; Raimondi et al., 2013 ; Kushi et al., 2000 ; Lertwattanasakul et al., 2011 ; Yarimizu et al., 2015 ; Madeira and Gombert, 2018 ; McTaggart et al., 2019 ). The development of new metabolic engineering tools ( e.g.…”

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

“…The past decade has seen development of tools for its metabolic engineering such as CRISPR/Cas9 systems, libraries of regulatory elements, secretory peptides, and in vivo assembly protocols (Nurcholis et al, 2020). In parallel, K. marxianus has been successfully used as a platform to produce heterologous compounds such as short-chain alcohols, carotenoids and polyketides (Cheon et al, 2014;Lin et al, 2017;McTaggart et al, 2019). However, all the latter studies focus on the overexpression of heterologous enzymes or pathways and only a few studies attempt engineer metabolism to improve production (Cernak et al, 2018;Kong et al, 2019).…”