Part:BBa_K3772013
ZWF1p-VS-SAG1t expression cassette
Express valencene synthase under the control of promoter ZWF1p (BBa_K3772009). We will use different promoters to initiate transcription of valencene synthase gene. Target promoters were screened by yield analysis of valencene. Then, target promoters will be changed the upstream regulatory sequence to achieve the maximum yield of valencene.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 785
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 785
Illegal NheI site found at 304
Illegal NotI site found at 140 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 785
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 785
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 785
Illegal AgeI site found at 1805 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This Composite Part is composed of basic parts ZWF1p (BBa_K3772009), valencene synthase coding sequence (BBa_K3772005) and terminator SAG1t (BBa_K3772014). ) and the terminator SAG1t (BBa_K3772014).14 yeast promoters with potential in different metabolic pathways (glucose degradation, gluconeogenesis and pentose phosphate pathways) were found through literature review [1]. The Promoter-VS-SAG1t expression cassette was used to allow the transcriptional expression of valencene synthases in the presence of their respective promoters. Since valencene synthase is a key enzyme in the rate limiting reaction of naringenone synthesis, we expect that increased expression of valencene synthase will more likely lead to a smoother transformation of our project. Therefore, we demonstrated the difference in transcriptional intensity of valencene synthase coding sequence by verifying the yield of valencene for these 14 promoters. The promoters with higher intensity will be analyzed more closely and will be the subject of our next transformation or auxiliary depending on their properties.
Because of the instability of regulatory element substitution across subspecies, the promoters in the Promoter-VS-SAG1t expression cassette are derived from Saccharomyces cerevisiae itself. Different types of carbon sources have different activation effects on cells, making them express different transcription activators. Certain transcription activators bind to UASs of promoters, recruit RNA polymerase, and regulate genes globally.
Because of the instability of substitution of regulatory elements across subspecies, the promoters in the Promoter-VS-SAG1t expression cassette are all derived from brewer's yeast itself. These promoters possess different characteristics due to their different carbon source responsive properties. Different types of carbon sources have different activation effects on cells, making them express different transcription Activators. Certain transcription Activators bind to UASs of promoters, recruit RNA polymerase, and regulate genes globally.
Fig.1:Search for natural promoters.
Characterization
Expression intensity of promoters
The product valenece was tested after incubation for 64 h in a 10 ml YPD system at 30 ℃ and 220 rpm inoculated with 0.05 OD of the engineered S. cerevisiae BJ5464-N strain. The results are shown in Fig.2.
Fig.2 Fermentation results-genome level LEU2::P-VS-T. In which it was observed that although the individual Promoter-VS-SAG1t had little effect on the growth of the strain, the intensity of the promoters varied widely. If we set the intensity of PCD1 was 100%, the intensity distribution of different promoters ranged from 3% to 74%. Among them, we selected PDC1p from Glycolysis pathway Promoters, ALD4p from Ethanol metabolism Promoters, SED1Lp from Other pathway Promoters and ZWF1 from Pentose Phosphate. These four Promoter-VS-SAG1t expression cassettes were subsequently analyzed for time curve and the ability to respond to different carbon sources.
Characteristics of promoters
Time curve:
Fig.3 Time curve of valencene production. We found that different promoters were expressed with different intensities at different fermentation times. The basic expression level of PDC1p was high, and it was mainly expressed in the prophase (0-24h); ALD4p was not expressed during 0-24h, and was mainly expressed during the later period (24-56h); The basic expression level of SED1Lp was not high and tended to increase in the later period (32-56h).
Different UASs on each promoter give it the ability to respond to different carbon sources [2].
Fig.4 Glucose, Ethanol concentration and Valencene production. From the figures above, we have learned key information and further understood the reasons behind: PPDC1 is mainly expressed in the glucose consumption stage (0-16h), and valencene production reaches a stable level at 24h, indicating that PPDC1 is a promoter that regulates the synthesis of valencene in the early stage. PALD4 is strictly limited in controlling valencene synthesis and is not expressed during glucose utilization by cells and is only effective when ethanol is consumed. The strength of SED1Lp is weak relatively during glucose consumption and increases significantly at the late fermentation stage (32-56 h). The overall expression level of PZWF1 is low, so it is not considered to be used for our subsequent promoter engineering.
Production Rate:
Fig.5 Specific production rate of product. The production rate of valencene can more directly reflect the expression characteristics of PDC1p, SED1Lp, ALD4p, ZWF1p.
Reference
[1] Nambu-Nishida, Y., et al., Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation. Journal of Bioscience and Bioengineering, 2018. 125(1): p. 76-86.
[2] Cao, L., et al., Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metabolic Engineering, 2014. 24: p. 150-159.
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