Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

Abstract

Background: Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.

Results: S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.

Conclusions: Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.

Figure 1

The Mevalonate pathway of GPP and FPP biosynthesis in yeast. The non-boxed rectangles show the points of integration of the introduce plant enzymes producing monoterpenes and sesquiterpenes. The encircled enzymatic steps represent targets of modification. Solid lines represent introduction of extra gene copies (HMG2, IDI1), broken lines show the introduction of a strong inducible promoter in one of two alleles, and in dotted line is the deletion of one allele to generate a haploinsufficient strain.

Figure 2

Production of cineole in yeast. (A) EG60-01 yeast cells expressing SfCinS1, under the control of the galactose inducible promoter, were sampled by SPME and analyzed by gas chromatography. (B) EG60-06 cells harboring empty vector do not produce cineole. (C) Western blot showing expression of SfCinS1, lane 1 protein extracts from EG60 cells, lane 2 protein extracts from EG60-01 cells harboring the SfCinS1 grown in glucose, and in lane 3 grown in galactose, which induces protein expression. (D) Quantification of cineole production, in BY4741-02 cells (reference strain) versus EG60-02 cells.

Figure 3

Integration of HMG2(K6R) variant leads to cineole production increase. (A) Map of the homologous recombination cassette designed to integrate a stabilized copy of HMG2(K6R) into the HO locus under the control of the galactose promoter. The hisG-URA3-hisG part of the cassette is subsequently excised from the genome by plating in FOA plates; (B) Cineole production in AM63 and AM65 cells; (C) Cineole yields in AM63 cells co-expressing the P. abies geranyl pyrophosphate synthase (GDS). Exogenous GPP was also added to assess the level of saturation of the system.

Figure 4

Improved yeast strains generated by strong promoter integration and single allele deletion for ERG9. (A) Map of the COD2 cassette, designed for integration of the galactose inducible promoter into promoter regions of endogenous genes. Subsequent to integration, the loxP-URA3-loxP part of the cassette is removed by excision from the enzyme Cre recombinase; (B) Map of the COD3 cassette, designed for the integration of the constitutive ADH1 promoter; C) Cineole production increase in AM68-01 cells, which overexpress ERG20, under the galactose promoter. AM70-01 cells, which are additionally haploinsufficient for ERG9, present substantially reduced levels of cineole production; (D) Expression of a sesquiterpene synthase P330 from S. pomifera in AM68-02 cells; peak 1, α-cubebene; peak 2, α-copaene; peak 3, trans-β-caryophyllene; peak 4, δ-cadinene; (E) Trans-β-caryophyllene standard used for quantification (peak 1);. (F) Copaene standard used for quantification (peak 1); (G) Sesquiterpene yields in the newly developed yeast strains. AM70-02 cells exhibit a substantial yield increase compared to wild type EG60-03 cells;

Figure 5

High levels of IDI1 lead to high monoterpene production. (A) Co-expression from a plasmid of the yeast IDI1 gene in EG60-04, AM65-02, AM68-03, AM70-03, AM76-01 cells (dark bars) leads to substantial increase in cineole yields in all strains tested vs. their corresponding non expressing strains (light grey bars); (B) Map of COD4 cassette which can accept cDNAs under the control of the galactose promoter; (C) Map of COD40 cassette which integrates an extra copy of the IDI1 gene into the chromosome, under the control of the galactose promoter. Subsequent to integration, the loxP-his5-loxP part of the cassette is removed by excision from the Cre recombinase; (D) Cineole production in AM78-01 and AM78-02 cells, which have a copy of the P_Gal1-IDI1-ts in the leu2 locus.

Figure 6

The new strains are robust and allow further improvements through plasmid-driven gene expression. (A) The robustness of the newly developed AM66, AM68, AM70, AM78 strains were tested in a growth assay in galactose based media. No difference with wild type EG60 cells is observed; (B) AM78-01 cells (triangle), AM65-01 cells (square), and parental EG60-02 (rhomboid) cells induced for protein expression were sampled by SPME daily over a 19 day period and monitored for cineole production; (C) Co-expression in AM78-05 cells of IDI1 from two high copy number plasmids did not cause higher cineole production, whereas the co-expression of IDI1 with HMG2 (K6R) in AM78-07 doubled the yield.

Figure 7

Terpenoid yields from Hexane/ethyl acetate LL extraction in 3 days old cultures. (A) A 3.3-fold increase and 11-fold increase of terpenes extracted is observed between AM78-01, AM78-08 respectively compared to control cells. (B) A 2.6-fold increase and 4-fold increase of terpenes extracted is observed between AM68-05, AM78-10 respectively compared to control cells. (C) A 4.2-fold increase in terpenes extracted is observed between AM70-02 cells and BY4741-04 cells expressing the SpP330 sesquiterpene synthase.

Figure 8

The putative interacting HSP90 synergizes with SfCinS1 to increase cineole production. (A) EGY48 yeast reporter cells carrying the SfCinS1-LexA (top row) or the LexA plasmid (bottom row) with the five (C2, C4, D1, D4, D6) library cDNAs were replica plated on glucose/CM-his, trp, leu and galactose-raffinose/CM-his, trp, leu agar media. Growth is observed only for the SfCinS1-LexA fusion in the galactose based media, where proteins are expressed, confirming the specificity of interactions. (B) EG60-05 cells and AM78-08 cells carrying SfCinS1 with empty vector (light grey) and EG60-05 and AM78-09 cells carrying SfCinS1 with HSP90 (dark grey), were tested for cineole production. The presence of HSP90 enhances cineole yield for both strains.