Saccharomyces cerevisiae as a Host for Chondroitin Production
Abstract
:1. Introduction
2. Results
2.1. Heterologous Production of Chondroitin in S. cerevisae
2.2. Bioinformatics Tool for Identification of Gene Targets
3. Materials and Methods
3.1. Strains and Plasmids
3.2. Biosynthetic Pathway Construction
3.3. Flask Fermentation Conditions
3.4. Analytical Methods
3.5. Model Construction
3.6. Conditions for In Silico Simulations and Optimization
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound ID Code | Compound Name | Template Model | Corrected Model 1% Chitin | ||
---|---|---|---|---|---|
Stoichiometry | Percentage (%) | Stoichiometry | Percentage (%) | ||
s_0001_ce | (1→3)-β-D-glucan [cell envelope] | 0.748514964 | 33.88 | 0.748514964 | 33.54 |
s_0004_ce | (1→6)-β-D-glucan [cell envelope] | 0.250091654 | 11.32 | 0.250091654 | 11.21 |
s_0773_c | glycogen [cytoplasm] | 0.361414528 | 16.36 | 0.361414528 | 16.20 |
s_1107_c | mannan [cytoplasm] | 0.710939625 | 32.18 | 0.710939625 | 31.86 |
s_1520_c | trehalose [cytoplasm] | 0.138275712 | 6.26 | 0.138275712 | 6.20 |
s_0509_c | chitin [cytoplasm] | 0 | 0.00 | 0.022313288 | 1.00 |
Solution | BPCY | Genes Modified | Predicted Phenotype (pFBA) | FVA | |||
---|---|---|---|---|---|---|---|
Under Expression | Over Expression | Biomass (h−1) | Chondroitin Flux (mmol/gDW/h) | Minimum Chondroitin Flux (mmol/gDW/h) | Maximum Chondroitin Flux (mmol/gDW/h) | ||
- | - | - | - | 0.8612 | 0.0000 | - | - |
1 | 0.04375 | - | QRI1 | 0.7317 | 0.5980 | 0.5980 | 0.9358 |
2 | 0.04375 | - | GNA1 | 0.7317 | 0.5980 | 0.5980 | 0.9358 |
3 | 0.04375 | - | PCM1 | 0.7317 | 0.5980 | 0.5980 | 0.9358 |
Solution | BPCY | WYIELD | Genes Modified | Predicted Phenotype (pFBA) | FVA | ||||
---|---|---|---|---|---|---|---|---|---|
Knock-Out | Under Expression | Over Expression | Biomass (h−1) | Chondroitin Flux (mmol/gDW/h) | Minimum Chondroitin Flux (mmol/gDW/h) | Maximum Chondroitin Flux (mmol/gDW/h) | |||
1 | 0.04375 | 0.60872 | DNF2, CTS1 | CRD1, LYP1, FAA1 | YAH1, QRI1, PCM1, CAB5 | 0.7317 | 0.5980 | 0.5980 | 0.9358 |
2 | 0.04374 | 0.60872 | FAA1, POT1, CTS1 | MAL31, ARG8 | YAH1, PCM1, ALD2 | 0.7316 | 0.5980 | 0.5980 | 0.9358 |
3 | 0.04335 | 0.60876 | ENO1, CTS1 | ERG10 | FAA1, YAH1, AAT1, POF1, CTA1, PCM1, MAL31 | 0.7248 | 0.5981 | 0.5981 | 0.9365 |
Solution | BPCY | WYIELD | Genes Modified | Predicted Phenotype (pFBA) | FVA | ||||
---|---|---|---|---|---|---|---|---|---|
Knock-Out | Under Expression | Over Expression | Biomass (h−1) | Chondroitin Flux (mmol/gDW/h) | Minimum Chondroitin Flux (mmol/gDW/h) | Maximum Chondroitin Flux (mmol/gDW/h) | |||
1 | 0.04375 | 0.83445 | - | - | QRI1 | 0.7317 | 0.5980 | 0.5980 | 0.9358 |
2 | 0.01488 | 2.80306 | CTM1 | CDS1 | QRI1 | 0.0541 | 0.6131 | 0.6131 | 3.7416 |
3 | 0.04359 | 0.83489 | HAM1 | SDH2 | QRI1 | 0.7288 | 0.5980 | 0.5980 | 0.9364 |
Strains | Relevant Genotype | Source |
---|---|---|
Escherichia coli NZY5α | fhuA2 Δ(argF-lacZ)U169 phoA glnV44Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 | NZYTech (MB00401) |
Saccharomyces cerevisiae CEN.PK2-1C | MATa ura3-52 his3Δ1 leu2-3,112 trp1-289 MAL2-8c SUC2 | Euroscarf 30000A [61] |
S. cerevisiae BY4741 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Euroscarf Y00000 [62] |
Plasmids | Description | Source |
pSP-GM1 | pUC ori, AmpR, 2 µ ori, URA3 PTEF1 PPGK1 | Addgene #64739 [63] |
pBEVY-L | pUC ori, AmpR, 2 µ ori, LEU2 PGPD PADH1 | ATCC 51226 |
pUC57_Giuae | pMB1 ori, AmpR; pUC57 carrying Giardia intestinalis uridine diphosphate-glucosamine-4-epimerase gene (GiUAE) codon-optimized for S. cerevisiae | NZYTech |
pUC57_Btchsy1 | pMB1 ori, AmpR; pUC57 carrying Bos taurus chondroitin synthase 1 gene (BtCHSY) codon-optimized for S. cerevisiae | NZYTech |
pETM6_kfoCA | pETM6 carrying chondroitin synthase, kfoC, and, uridine diphosphate-glucosamine-4-epimerase, kfoA, genes from E. coli O5:K4:H4 | [5] |
pSP-GM1_Zmugd | pSP-GM1 carrying Zymomonas mobilis uridine diphosphate glucose 6-dehydrogenase gene (Zmugd) | [46] |
pSP-GM1_Giuae_Zmugd | pSP-GM1 carrying GiUAE and Zmugd | This study |
pBEVY_Btchsy | pBEVY-L carrying BtCHSY | This study |
pSP-GM1_kfoA_Zmugd | pSP-GM1 carrying kfoA and Zmugd | This study |
pBEVY_kfoC | pBEVY-L carrying kfoC | This study |
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Couto, M.R.; Rodrigues, J.L.; Dias, O.; Rodrigues, L.R. Saccharomyces cerevisiae as a Host for Chondroitin Production. SynBio 2024, 2, 125-141. https://doi.org/10.3390/synbio2020008
Couto MR, Rodrigues JL, Dias O, Rodrigues LR. Saccharomyces cerevisiae as a Host for Chondroitin Production. SynBio. 2024; 2(2):125-141. https://doi.org/10.3390/synbio2020008
Chicago/Turabian StyleCouto, Márcia R., Joana L. Rodrigues, Oscar Dias, and Lígia R. Rodrigues. 2024. "Saccharomyces cerevisiae as a Host for Chondroitin Production" SynBio 2, no. 2: 125-141. https://doi.org/10.3390/synbio2020008
APA StyleCouto, M. R., Rodrigues, J. L., Dias, O., & Rodrigues, L. R. (2024). Saccharomyces cerevisiae as a Host for Chondroitin Production. SynBio, 2(2), 125-141. https://doi.org/10.3390/synbio2020008