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

Abstract

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 ​°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, P _SSA3 and P _ADH1 , increased expression above 40 ​°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 ​°C. Cultures at 41 ​°C exhibited a similar TAL biosynthesis capability, while at 30 ​°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis.

Keywords: Chemical production; K. marxianus; Non-conventional microbe; Promoter; Thermo-tolerance.

Fig. 1

Screening and characterization of K. marxianus promoters for heterologous protein expression. (A) Twenty-five EGFP expression cassettes were constructed by identifying and cloning the 700 base pairs upstream of a start codon of a given K. marxianus gene. (B) Growth and growth rate of K. marxianus CBS6556 ΔHIS3 ΔURA3 in 2% glucose harboring a low copy number plasmid with EGFP expression driven by P_TEF3. Shake flask cultures were inoculated with an initial OD₆₀₀ of 0.05. Triplicate cultures were grown at 30, 37, and 45 ​°C. Arrows indicate the early stationary phase time points used for expression characterization across the promoter library. (C) Cell density normalized EGFP fluorescence (Relative Fluorescence Unit, RFU) at 30 ​°C for the promoter set. The P_TEF3 result is shown in black for ease of reference to part (B). The effect of temperature (37 and 45 ​°C) is indicated above as the log₂ fold change from fluorescence observed at 30 ​°C. In the lower panel, the strength of S. cerevisiae TEF1 promoter is indicated by the horizontal blue line as a control. Vertical dashed lines show how promoters were grouped into low, medium and high strength sub-groups based on the EGFP RFU/OD at 30 ​°C. All data represent biological triplicates. Data points and bars indicate the mean; the error bars indicate the standard deviation. All RFU values are background substrate with CBS6556 ΔHIS3 ΔURA3 harboring empty plasmid pIW578 as the background. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

K. marxianus promoter dynamics using glucose as the carbon source. Time course data were collected from inoculation to stationary phase for 6 different K. marxianus promoters. Shake flask cultures were grown at 30 ​°C (blue), 37 ​°C (orange), and 45 ​°C (red) in synthetic defined media without histidine (SD-His) with 2% glucose. All data is normalized to the highest fluorescence signal intensity for each combination of promoter and temperature. The promoter strength upon reaching stationary phase is shown in Fig. 1. Data points represent the mean of biological triplicates, while the error bars represent the standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Characterization of six selected promoters at late exponential phase when grown on xylose. (A) Growth curves and growth rate of K. marxianus CBS6556 ΔHIS3 ΔURA3 harboring a blank expression vector (pIW578) in synthetic defined media with 2% xylose as a carbon source at 30, 37, 41 and 45 ​°C. Shake flask cultures with an initial OD₆₀₀ of 0.05 were inoculated with starter cultures grown on xylose. (B) Comparison of relative promoter strength with growth on xylose and glucose at 30 ​°C. EGFP fluorescence was normalized to P_TEF3 for xylose and glucose separately. The absolute value of RFU/OD for each promoter is 25 ​± ​18 (ADH1), 47 ​± ​7 (HHF1), 95 ​± ​14 (NC1), 10 ​± ​1 (PGK), 15 ​± ​11 (SSA3) and 86 ​± ​3 (TEF3) at 30 ​°C. (C) The temperature effect on promoter strength with xylose as a carbon source. The effect of temperature (37 and 41 ​°C) is indicated as the log₂-fold change from fluorescence observed at 30 ​°C. Time-points for late exponential phase in xylose are 22 ​h ​at 30 ​°C and 18 ​h ​at 37 ​°C and 41 ​°C. All data represent biological triplicates. Data points and bars indicate the mean and error bars indicate the standard deviation.

Fig. 4

Triacetic acid lactone (TAL; 4-hydroxy-6-methyl-2-pyrone) biosynthesis in xylose medium with varying levels of 2-pyrone synthase (2-PS) expression. (A) Simplified illustration of proposed TAL biosynthesis pathway in K. marxianus. (B) Specific TAL production at late exponential phase at 30, 37 and 41 ​°C. 2-PS was overexpressed with PGK, SSA3, HHF1, ADH1, TEF3, and NC1 promoters on a low copy number plasmid. All data represent biological triplicates and the error bars indicate the standard deviation.