Fig 1 Schematic Representation of our Experimental Design
We introduced muscone gas molecule receptors derived from mouse olfactory epithelial cells into chassis bioengineered bacteria. The muscone gas molecule receptor is a G protein coupled receptor in eukaryotic cells, and we chose Saccharomyces cerevisiae as the chassis bioengineering bacterium. By modifying the mating pathway of Saccharomyces cerevisiae, the muscone gas molecule receptor is integrated into the signaling pathway of Saccharomyces cerevisiae. And downstream of the modified mating pathway, lactate dehydrogenase was introduced to alter the anaerobic metabolism pathway of Saccharomyces cerevisiae, synthesizing lactate for the treatment of IBD disease.
Our experimental design consists of two parts: the verification of the mating pathway in Saccharomyces cerevisiae containing muscone gas molecule switches and the modification of the Saccharomyces cerevisiae genome. We independently verified each component of the mating pathway in the modified Saccharomyces cerevisiae and ultimately integrated them. Additionally, we knocked out the original receptor of the Saccharomyces cerevisiae mating signal to eliminate signal interference caused by the yeast’s own growth.
Our designed Therapy system primarily consists of three parts:
Input signal - Muscone molecular switch
Signaling pathway - Saccharomyces cerevisiae mating pathway
Output signal - Lactic acid secretion
Fig 2 Schematic Diagram of the Therapy System
We selected muscone gas molecules as the upstream control signal for our therapy system. We used the muscone receptor sequence from mouse olfactory epithelial cells, as employed by the Ye Haifeng team [1]; for details, please refer to the description, and introduced it into the plasmid system expressed in Saccharomyces cerevisiae. We chose the mating pathway in Saccharomyces cerevisiae as the transmission pathway for the muscone signal within Saccharomyces cerevisiae. Based on the Benjamin M Scott team's optimization[2], we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway, allowing the muscone receptor to be integrated into the Saccharomyces cerevisiae mating pathway.
We used the galactose promoter to induce the expression of the muscone signal receptor and the optimized Gα protein, and screened the successfully transformed Saccharomyces cerevisiae with a His nutritional deficiency. By controlling the induction conditions of galactose and muscone, we tested the effectiveness of the muscone gas molecule switch. For details, please refer to the protocol.
Aim:
To validate the effectiveness of the muscone gas molecule switch in Saccharomyces cerevisiae.
Constructs:
MOR215&Ga-pESC
Fig 3 MOR215&Ga-pESC plasmid
We chose the mating pathway in Saccharomyces cerevisiae as the conduit for muscone signaling in yeast. Using the mating pathway's pFUS1 promoter, we expressed the downstream lactate dehydrogenase to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae, secreting lactic acid for the treatment of IBD[3]. Initially, we designed a plasmid with the pFUS1 promoter expressing the GFP reporter gene and screened the successfully transformed yeast using Ura nutritional deficiency. We then tested the effectiveness of the muscone molecular switch using confocal microscopy; for details, please refer to the protocol. Subsequently, we designed the pFUS1 promoter to express lactate dehydrogenase from E. coli. By co-transforming it with Muscone Receptor & Gα (pESC) into Saccharomyces cerevisiae, we achieved the construction of the complete pathway.
Aim:
To check the reporter signals downstream of the muscone molecular switch.
To check the synthesis of the secretion system downstream of the muscone molecular switch.
Constructs:
pFUS1 promoter-GFP-pYES、pFUS1 promoter-ldhA-pYES2
Fig 4 pFUS1 promoter-GFP-pYES plasmid
Fig 5 pFUS1 promoter-ldhA-pYES2 plasmid
Lactic acid is the small molecule we selected to target the abnormally activated autoimmune cells in IBD diseases. For details, please refer to the description. We chose lactate dehydrogenase from E. coli to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize and secrete D-lactic acid. We used the galactose promoter to induce the expression of lactate dehydrogenase and screened the successfully transformed yeast with a Ura nutritional deficiency. By controlling the induction with galactose and glucose and establishing gradients of induction time and post-induction culture time, we tested the synthesis and secretion of lactic acid and searched for the optimal induction conditions for lactic acid secretion. For details, please refer to the protocol.
Aim:
To test the effectiveness of the lactate secretion system in Saccharomyces cerevisiae.
To explore the optimal induction conditions for the lactate secretion system.
Constructs:
GAL1 promoter-ldhA-pYES2
Fig 6 GAL1 promoter-ldhA-pYES2 plasmid
To avoid interference from the mating signals of Saccharomyces cerevisiae's own growth on the signal transduction controlled by the muscone molecule and to ensure biological safety, we have made modifications to the genome of Saccharomyces cerevisiae. This is reflected in our knockout of the original receptor STE2 in the mating pathway of Saccharomyces cerevisiae. Our knockout system includes a gRNA targeting the STE2 gene and the Cas9 protein.
We expressed the gRNA targeting the STE2 receptor gene and the Cas9 protein through a constitutive promoter and screened the successfully transformed yeast using leu nutritional deficiency. The genome of the successfully transformed Saccharomyces cerevisiae strains was sequenced to screen for strains with a successful knockout of STE2, followed by the transformation of the muscone molecular switch signaling pathway.
Aim:
To remove the interference of Saccharomyces cerevisiae's own growth and mating signals on the secretory system.
Constructs:
STE2 gRNA&Cas9-pML107
Fig 7 STE2 gRNA&Cas9-pML107 plasmid
Through our efforts, we have obtained some experimental results to support our system design. We divided the therapy system into an upstream muscone molecular signal switch and a downstream lactate secretion system, and we have tested their operational effectiveness through a series of wet lab experiments. Finally, we integrated them to test the effectiveness of the complete therapy system. You can see our experimental results in the following content:
In our system, the muscone molecule serves as the signal controlling the secretion system. Introducing the muscone molecular signal switch into the signaling pathway of Saccharomyces cerevisiae is one of our core tasks. You can view our experimental design through the design.
To verify the effectiveness of the muscone molecular switch in Saccharomyces cerevisiae, we used the GFP reporter gene to reflect the signal intensity downstream of the muscone molecular switch. By using the galactose promoter to induce the expression of the muscone molecular switch, we established a glucose-induced control group during the induction process. Additionally, we set up control groups with and without muscone under two different carbon source induction conditions. Details of the induction experiment can be found in the protocol. We captured fluorescence signal images of different groups of Saccharomyces cerevisiae under a confocal microscope and conducted quantitative analysis of relative fluorescence intensity and fluorescence proportion.
In the galactose-induced experimental group, the fluorescence intensity and proportion of the GFP reporter gene under muscone induction were significantly higher than those in the control group without muscone. In the glucose control group, there was no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone-induced experimental group and the control group. The experiment preliminarily proves the effectiveness of the introduced muscone molecular switch in Saccharomyces cerevisiae.
At the same time, we found that compared to the galactose-induced experimental group, the glucose control group showed a higher background noise of mating signals in Saccharomyces cerevisiae. In our subsequent experimental design, we knocked out the original receptor of the Saccharomyces cerevisiae mating signal pathway, which reduced the background signal intensity of the Saccharomyces cerevisiae mating signal pathway and improved the reliability of the system.
Fig 8 Muscone molecular switch fluorescence signal test, A. Galactose-induced, add muscone organic solution. B. Galactose-induced, without muscone. C. Glucose control group, add muscone organic solution. D. Glucose control group, without muscone.
Fig 9 Quantitative analysis of muscone molecular switch fluorescence signal.
In our system, the lactic acid molecule is the small molecule that ultimately treats IBD diseases and alleviates the abnormal activation of autoimmune cells. We have altered the anaerobic metabolic pathway of Saccharomyces cerevisiae by inducing the expression of lactate dehydrogenase from E. coli with galactose, and tested the effectiveness of the secretion system. You can view our experimental design through the design.
We used the galactose promoter to control the expression of lactate dehydrogenase within Saccharomyces cerevisiae. We established a glucose-induced control group during the induction process. And we also conducted an induction experiment with untransformed wild-type yeast as a control to exclude the background signal noise of Saccharomyces cerevisiae. Details of the induction experiment can be found in the protocol. After induction, the bacterial solution was centrifuged, and the supernatant was collected. The WST colorimetric method was used to measure the lactic acid content in the supernatant of different groups. The absorbance of the colorimetric reaction was recorded at 455 nm, which reflects the concentration of D-lactic acid in the supernatant.
The experimental results preliminarily demonstrate that in the transformed groups, galactose induction led to the expression of lactate dehydrogenase, which successfully altered the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize D-lactic acid. Meanwhile, after the synthesis of D-lactic acid, it can be secreted into the surrounding environment of the Saccharomyces cerevisiae.
Fig 10 Galactose-induced D-lactic acid secretion system
Taking into account that different carbon sources for induction and cultivation systems may affect the growth of Saccharomyces cerevisiae and the working rate of lactate dehydrogenase. We further established different combinations of induction and cultivation with glucose and galactose groups based on previous experiments, and set up a time gradient to sample the supernatant of Saccharomyces cerevisiae cultures for the measurement of D-lactic acid concentration.
The experimental results show that, compared to the galactose group, the glucose group has a non-specific promotional effect on the secretion of D-lactic acid by Saccharomyces cerevisiae. In the short term of induction and cultivation, glucose will synthesize more D-lactic acid by promoting yeast growth more strongly and accelerating the reaction rate of lactate dehydrogenase. However, in longer-term induction experiments, the galactose group will ultimately synthesize more D-lactic acid due to the specific induction of lactate dehydrogenase expression. This experimental result confirms the impact of different carbon source cultivation systems on the secretion system. To eliminate unnecessary influences as much as possible, we adopted a muscone molecular switch expressed by a constitutive promoter in subsequent experiments, removing the galactose-induced experimental step.
Fig 11 Changes in lactic acid secretion with different carbon source combinations in short-term culture.
Fig 12 Changes in lactic acid secretion with different carbon source combinations in long-term culture.
Based on the previous experimental results of the muscone molecular switch signal, we designed a knockout system targeting the original receptor STE2 of the mating pathway in Saccharomyces cerevisiae, including the gRNA and Cas9 protein targeting STE2. We performed sequencing on the successfully transformed strains and confirmed that base deletions and frameshift mutations occurred in the original receptor STE2 sequence. We retransformed the strains with successful STE2 knockout with MOR215&Ga-pESC and pFUS1 promoter-GFP-pYES and repeated the previous induction experimental protocol. The results of the muscone analysis switch signal intensity in the knockout Saccharomyces cerevisiae strain were obtained under a confocal microscope.
The experimental results show that under the muscone induction condition in the galactose group, the fluorescence intensity and proportion of the GFP reporter gene remained significantly higher than that of the control group without muscone. In the glucose group, there was still no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone induction group and the control group without muscone. Compared to the strain with the original receptor STE2 not knocked out, the background signal noise in the glucose group significantly decreased after the original receptor STE2 was knocked out. The experiment confirmed that knocking out the original receptor STE2 of the Saccharomyces cerevisiae mating pathway reduced the noise of the background mating signal, thus improving the reliability of the muscone molecular switch signal.
Fig 13 Quantitative analysis of muscone molecular switch fluorescence signal of knocking out STE2 strain
After separately verifying the effectiveness of the muscone molecular switch and the lactic acid secretion system in Saccharomyces cerevisiae, we constructed a complete therapeutic system within the yeast. We linked the lactate dehydrogenase behind the pFUS1 promoter, which is downstream of the mating pathway in Saccharomyces cerevisiae. For details, refer to design. The muscone receptor, the modified Gα protein, and the lactate dehydrogenase were simultaneously introduced into Saccharomyces cerevisiae. We tested the efficacy of the complete therapeutic system by using the same induction scheme as the muscone molecular switch and the WST colorimetric method to measure the supernatant content of D-lactic acid. For details, refer to protocol.
In the galactose-induced experimental group, the content of D-lactic acid in the supernatant of the muscone-induced group was significantly higher than that in the control group without muscone. However, in the glucose control group, there was no significant difference in the content of D-lactic acid in the supernatant between the muscone-induced group and the control group without muscone. This preliminarily verified that the complete therapeutic system constructed in Saccharomyces cerevisiae is effective. At the same time, we also found that, similar to the previous results of the lactic acid secretion system alone, the carbon source in the culture medium used during the culture and induction process has a significant impact on the lactic acid secretion results of Saccharomyces cerevisiae. Glucose promotes the background rate of lactic acid synthesis compared to galactose. In addition, we found that there were significant differences in lactic acid secretion values among different strains under the same induction conditions. We speculate that this may be related to the growth status of different strains and the copy number of the transformed plasmid.
Fig 14 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)
Subsequently, we plan to replace the muscone receptor and the modified Gα protein expression promoter with a constitutive promoter, thereby eliminating the interference caused by differences in culture medium carbon source components during the experimental process. Later, we will transfer the related genes of the system into the genome of Saccharomyces cerevisiae to avoid the inter-group differences caused by plasmid copy number. At the same time, we will screen dominant strains for subsequent experiments to minimize the interference of strain differences on the results.
Fig 15 Muscone-induced lactate measurement results of the treatment system after induced operation optimization. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)
In order to reduce the interference of carbon source differences in the induction operation (based on the previous experimental results on lactate secretion), we further extended the joint induction time of galactose and muskone, extending the induction time from 24h to 48h. In the experimental protocol for optimized induction time, we obtained more significant group difference results.
[1] Wu X, Yu Y, Wang M, Dai D, Yin J, Liu W, Kong D, Tang S, Meng M, Gao T, Zhang Y, Zhou Y, Guan N, Zhao S, Ye H. AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation. Nat Commun. 2024 Feb 6;15(1):1122. doi: 10.1038/s41467-024-45383-z. PMID: 38321056; PMCID: PMC10847102.
[2] Scott BM, Gutiérrez-Vázquez C, Sanmarco LM, da Silva Pereira JA, Li Z, Plasencia A, Hewson P, Cox LM, O'Brien M, Chen SK, Moraes-Vieira PM, Chang BSW, Peisajovich SG, Quintana FJ. Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nat Med. 2021 Jul;27(7):1212-1222. doi: 10.1038/s41591-021-01390-x. Epub 2021 Jun 28. PMID: 34183837.
[3] Sanmarco LM, Rone JM, Polonio CM, Fernandez Lahore G, Giovannoni F, Ferrara K, Gutierrez-Vazquez C, Li N, Sokolovska A, Plasencia A, Faust Akl C, Nanda P, Heck ES, Li Z, Lee HG, Chao CC, Rejano-Gordillo CM, Fonseca-Castro PH, Illouz T, Linnerbauer M, Kenison JE, Barilla RM, Farrenkopf D, Stevens NA, Piester G, Chung EN, Dailey L, Kuchroo VK, Hava D, Wheeler MA, Clish C, Nowarski R, Balsa E, Lora JM, Quintana FJ. Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells. Nature. 2023 Aug;620(7975):881-889. doi: 10.1038/s41586-023-06409-6. Epub 2023 Aug 9. PMID: 37558878; PMCID: PMC10725186.