Kluyveromyces marxianus as a robust synthetic biology platform host

Abstract

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traitsthermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

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

“…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.…”

Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii

“…CRISPR‐Cas9 genome editing is becoming a standard tool for metabolic engineering in yeast. It has been successfully used not only in S. cerevisiae but also in many nonconventional yeast strains, such as Yarrowia lipolytica, Rhodosporidium toluroides (Schultz et al, 2019), Kluyveromyces lactis (Juergens et al, 2018), and Kluyveromyces marxianus (Cai et al, 2019; Cernak et al, 2018). To test if an existing CRISPR‐Cas9 tool, which has been optimized for other yeast, can be functional in Z. bailii , we obtained a plasmid containing the PAN autonomously replicating sequence (ARS), a ribozyme based gRNA expression cassette, and Cas9 (pUDP004) (#101165; Addgene; Gorter de Vries et al, 2017).…”

“…The recent advent and maturation of CRISPR Cas9‐based genome editing has allowed applying it for precise and efficient genome editing of many nonconventional yeasts as well as S. cerevisiae (Cai et al, 2019; Cernak et al, 2018; Gorter de Vries et al, 2017). We extended the CRISPR Cas9 mediated genome editing portfolio to Z. bailii .…”

Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products

“…In parallel, K. marxianus has been successfully used as a platform to produce heterologous compounds such as short-chain alcohols, carotenoids and polyketides [18][19][20]. However, all the latter studies focus on the overexpression of heterologous enzymes or pathways and only a few studies attempt to engineer metabolism to improve production [21,22]. With regards to aromatic compounds, K. marxianus has been evolved to overproduce phenylalanine and by extension the aromatic alcohol 2-phenylethanol (2-PE) via an enhanced Ehrlich pathway [23], but this has shed little light on appropriate metabolic engineering strategies.…”