Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

Abstract

Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h-1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.

Keywords: disaccharide; isomaltase; laboratory evolution; multiple gene deletion; real-time PCR; reverse engineering.

Figure 1.

Strategy for deletion of the IMA genes using a shared Cas9 target site. (A) Plasmid pUDR127, that contains two gRNA-cassettes: ‘gRNA-IMAs’ (in red) with a target sequence to cleave all IMA genes except IMA5 and ‘gRNA-IMA5’, in purple. The SNR52 promoter and SUP4 terminator are shown in green and yellow, respectively. Sequences encoding structural RNAs are shown in grey. 2 μm: origin of replication in yeast; pMB1: origin of replication in bacteria; amp^R: cassette for ampicillin resistance in E. coli; amdSYM: dominant marker that allows use of acetamide as nitrogen source. (B) Illustration of the shared Cas9 target sequences among the IMA genes. The coding sequence of each IMA is represented by black lines. The red boxes correspond to the shared target sequence. The dark- and light-grey fragments are the homology regions (60 bp) up- and downstream of the target site where the repair fragment (120 bp) can recombine. In view of its sequence divergence from the other IMA genes, an exclusive target sequence, in purple, and repair sites, in dark and light green, were chosen for inactivation of IMA5.

Figure 2.

Non-biological hydrolysis of sucrose caused by medium acidity. (A) Growth of S. cerevisiae strain IMU051 (malΔ mphΔ suc2Δ) in SM with 20 g L⁻¹ sucrose as sole carbon source with initial pH set at different values: pH 2, filled squares; pH 3, filled triangles; pH 4, inverted filled triangles; pH 5, open squares; pH 6, ‘crosses’ and pH 7, ‘plus’. The symbols filled square, open square, cross and plus are overlapping. (B) Sugar concentrations in flasks with SM at an initial pH of 3. Closed symbols: glucose (filled diamonds) and sucrose (filled circles) concentrations in flasks inoculated with IMU051. Open symbols; glucose (open diamonds) and sucrose (open circles) concentrations in flasks without inoculum. At day 0.00 and 1.06: filled circles overlapping with open circles and filled diamonds overlapping with open diamonds. Data were not corrected for evaporation to keep the experimental setup identical to that used for growth studies. The experiment was conducted with two independent replicates, of which one representative replicate is shown. 500 mL shake flasks containing 100 mL SM with sucrose 20 g L⁻¹ were incubated at 30°C and at 200 rpm.

Figure 3.

Relative expression of IMA1-5 in S. cerevisiae strains evolved for faster growth on SM with sucrose as the sole carbon source. Relative expression of IMA5(A); IMA1(B); and IMA2, IMA3 and IMA4(C). Expression of IMA2, IMA3 and IMA4 was assayed by a single primer pair since these genes are highly similar in nucleotide sequence. CEN.PK113-7D: reference strain (IMAx MALxx MPHx SUC2); IMU048: unevolved parental strain (genotype: malΔ mphΔ suc2Δ MAL11); and three independently evolved strains (IMS422, IMA423 and IMS424). CEN.PK113-7D was used as calibrator sample (expression set to 1). Averages and standard errors were obtained from independent duplicate biological experiments and three technical replicates. Cells were harvested at mid-exponential phase (OD_660nm = 3) from shake-flask cultures on SM with sucrose as sole carbon source.

Figure 4.

Depth of coverage analysis of chromosome III from unevolved strain IMU048 (A) and the evolved strain IMS422 (B). Sequencing reads from the IMU048 and IMS422 have been mapped onto the CEN.PK113-7D genome sequence (Nijkamp et al.2012). Read count data represent the average coverage of non-overlapping 500 bp window. These data are representative of the other two evolved strains (IMS423 and IMS424).