1 Introduction
Fig.1 Model for a dual temperature control system constructed by heat-responsive expression of a cs Gal4p mutant. The cs Gal4p mutant activity was lower at 30 °C than at 37 °C. For the cs Gal4p mutant under the control of a heat-shock promoter, the activity at 37 °C was amplified, while that at 30 °C was further reduced and even fell below the threshold (detection limit). |
2 Experimental
2.1 Construction of plasmids and strains
2.2 Media and culture conditions in sealed vials
2.3 GC analysis of isoprene
2.4 High-performance liquid chromatography (HPLC) analysis of glucose
2.5 Fluorescence intensity measurement
2.6 Real-time quantitative PCR (qPCR) analysis
2.7 Batch fermentation in a 5 L bioreactor
3 Results and discussion
3.1 Directed evolution for cs Gal4p
Fig.2 Strategy and flowchart of Gal4p-directed evolution. (a) Principle of the 5-FOA/URA3 cytotoxicity-based screening method. 5-FOA could be catalyzed into cytotoxic 5-fluorouridine by Ura5p and Ura3p, resulting in cell death (Ura5p: orotate phosphoribosyltransferase; Ura3p: orotidine-5′-phosphate decarboxylase; 5-FUMP: 5-fluorouridine monophosphate; 5-FdUMP: 5-fluorodeoxyuridine monophosphate; dTMP: deoxythymidine monophosphate). (b) Flowchart of Gal4p directed evolution. A mutant library was constructed by error-prone PCR. The transformants were first incubated on SD/5-FOA plates at 30 °C and then photoprinted onto two SD-URA plates: one was cultured at 30 °C, and the other was cultured at 37 °C. Colonies grown at 37 °C but not at 30 °C were selected and subjected to gradient dilution. Five microliters of diluted cells were spotted on SD plates lacking uracil. Positive variants were selected for further verification in the isoprene producer M08H-CS after propagation in E. coli. |
Fig.3 Rescreening of potential cs Gal4p mutants. YCS113-URA cells transformed with WT Gal4p and the corresponding mutant were cultured on SD-URA. Triangles represent serial 10-fold dilutions, and the starting concentration was 1 OD600. Colonies that grew well at 37 °C but not at 30 °C or grew badly at 30 °C were considered positive mutants (indicated by blue squares). |
Fig.4 Isoprene production and biomass of strains harboring WT and mutant Gal4p. (a) Comparison of the biomass at 30 °C and 37 °C among the cs Gal4p mutants and WT Gal4p; (b) comparison of isoprene production at 30 °C and 37 °C among the Gal4p cs mutants and WT Gal4p (Cells were cultured in sealed vials. The data presented are the means of three biological replicates. Error bars represent the standard deviations. Significant levels of the t-test: * P<0.05, ** P<0.01, *** P< 0.001). |
Tab.1 Mutation sites of the Gal4 mutants |
Mutants a) | DNA binding domain | DNA activation domain | DUF | ||||||
---|---|---|---|---|---|---|---|---|---|
ep5 | H53H | K82E | A152A | W174R | L187P | P423L | |||
ep16 | D12G | L81L | E176G | F211L | S219S | ||||
ep19 | – | – | T262A | M265T | N308D | R378G | |||
ep21 | K25E | L184S | K212K | Q270R | S350R | W383R |
a) Majuscule represents amino acid changes in the Gal4 mutants. |
3.2 Heat-shock expression of Gal4p
Fig.5 Structure and characterization of the SSA4 promoter: (a) Structure of the SSA4 promoter. HSEs are essential elements for the heat response in the SSA4 promoter. Under extreme conditions, HSFs are activated and bind tightly with HSE to initiate gene transcription. PSSA4-1, PSSA4-2 and PSSA4 contain 1, 2 or 3 HSE modules, respectively (ATF: activating transcription factor). (b) Fluorescence characterization of heat-shock promoters of different lengths. Y113EGFP-0 served as a control, where the EGFP gene was regulated by the constitutive promoter PERG9, whereas EGFP in Y113EGFP-1, Y113EGFP-2, and Y113EGFP-S was regulated by PSSA4-1, PSSA4-2, and PSSA4, respectively (The error bars represent standard deviations calculated from triplicate experiments). (c) Qualitative analysis of WT and mutant Gal4p under PSSA4 in yeast. PERG9-GAL4WT and PSSA4-GAL4WT cassettes were carried on the pESC-URA plasmid and transformed into the Yelacz strain. Phenotypes were analyzed by monitoring UASG-lacZ reporter gene expression driven by WT Gal4p. The hydrolysis of X-Gal by β-gal yielded a blue-colored product. The blue color was used as a measure of the expression level of β-gal, which in turn represented the activity of Gal4p (The data presented are the means of three biological replicates. Error bars represent the standard deviations. Significant levels of the t-test: * P<0.05, ** P<0.01, *** P<0.001). |
Fig.6 Temperature control systems and their effects on isoprene production and yeast growth: (a) Schematic diagram of the temperature control systems. Control (constitutive expression of WT Gal4p): PERG9-GAL4WT; single temperature control system constructed by heat-shock promoter-mediated expression regulation of WT Gal4p or by Gal4p directed evolution: PSSA4-GAL4WT and PERG9-GAL4ep19; dual temperature control system constructed by both directed evolution and expression regulation of Gal4p: PSSA4-GAL4ep19. (b) Effect of the temperature control systems on isoprene production. (c) Effect of the temperature control systems on yeast growth. (Cells were cultured in sealed vials. The data presented are the means of three biological replicates. Error bars represent the standard deviations. Significant levels of t-test: * P<0.05, ** P<0.01, *** P<0.001). |
3.3 Batch fermentation for isoprene production
Fig.7 Aerobic batch fermentation of isoprene by recombinant yeast strains and transcriptional levels of some genes in M08H-CS-MLN-SSA4-ep19 during fermentation: (a) Time course of the OD600; (b) time course of isoprene production. The control strain M08H-CS-MLN-WT was cultured constantly at 30 °C, whereas the temperature-controlled strains M08H-CS-MLN-SSA4-WT, M08H-CS-MLN-ep19 and M08H-CS-MLN-SSA4-ep19 were cultured with a 30 °C /37 °C temperature shift at the late log-phase. The arrows indicate the time point of the temperature shift; (c) time course of isoprene productivity; (d) transcriptional levels of GAL4, tHMG1, and ISPS during the fermentation of M08H-CS-MLN-SSA4-ep19 (The error bars represent standard deviations calculated from triplicate experiments). |