Project description:Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.
Project description:Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on minimal media with one strain, named “LAbrini”, exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of mutations in potentially sporulation-related genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain “LAbrini” to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism.
Project description:Gas fermentation of CO₂ and H₂ is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO-to-ethanol and presents an opportunity for CO₂-to-ethanol processes. As we have previously characterized its CO₂/H₂ chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO₂/H₂. Seven ALE lineages were generated, all with improved specific growth rates. Developed with 2% CO supplementation of CO₂/H₂, Evolved lineage D has the highest ethanol/acetate of ALE lineages when fermenting CO₂/H₂. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Complete multi-omics analyses at steady-state revealed that although Evolved D has widespread proteome changes, intracellular metabolites prevent phenotype shifts. Yet, we observe numerous insights to CO₂/H₂ metabolism via these multi-omics results and link these to mutations, suggesting novel targets for metabolic engineering.
Project description:Gas fermentation of CO₂ and H₂ is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO-to-ethanol and presents an opportunity for CO₂-to-ethanol processes. As we have previously characterized its CO₂/H₂ chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO₂/H₂. Seven ALE lineages were generated, all with improved specific growth rates. Developed with 2% CO supplementation of CO₂/H₂, Evolved lineage D has the highest ethanol/acetate of ALE lineages when fermenting CO₂/H₂. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Complete multi-omics analyses at steady-state revealed that although Evolved D has widespread proteome changes, intracellular metabolites prevent phenotype shifts. Yet, we observe numerous insights to CO₂/H₂ metabolism via these multi-omics results and link these to mutations, suggesting novel targets for metabolic engineering.
Project description:This agent-based model is based on an adaptive laboratory evolution (ALE) experiment scenario of two mutually cross feeding strains of bacteria and yeast. The bacterial strain secretes vitamins for which the yeast strain is auxotrophic and the yeast strain secrets amino acids for which the bacterial strain is auxotrophic. In particular, the model simulates a situation where a mutation arises in the bacterial strain that results in the emergence of individuals (mutant bacteria) with a higher secretion of vitamins as compared to the wild type (WT). This increase in secretion comes with a cost in terms of fitness (growth rate) of the mutant bacteria. The model can be used to assess if this mutant is able to persist and increase in frequency in the cross-feeding community.
Project description:To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of docosahexaenoic acid (DHA)-producing Crypthecodinium cohnii, fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87% and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87% and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE.
Project description:A recent study reported that daptomycin-resistant MRSA (DAPR) strain biofilm is more resistant to daptomycin and vancomycin, as compared to the WT strain biofilm. This pose a great danger since DAPR MRSA is prevalent in clinics and they often form biofilms in medical devices. In this study, we investigate the anti-biofilm activity of elasnin against DAPR MRSA biofilms, and we observed that elasnin is not only effective to eradicate the biofilm of the DAPR strain, but it shows a superior activity compared to the susceptible WT strain. Using proteomics, we compared the proteome profile of the DAPR and the WT strain biofilm cells under elasnin treatment to reveal why elasin is superior against the DAPR strain. Besides, we also employed adaptive laboratory evolution (ALE) experiment by repetitively treating MRSA culture with high dose of elasnin, and generated evolved MRSA strain with increased elasnin tolerance. Using quantitative proteomics, we compared the proteome differences in the elasnin-susceptible WT MRSA and elasnin-tolerant evolved strain.
Project description:The growth of E. coli is inhibited by millimolar concentrations of L-homoserine, which may be problematic for the industrial production of this compound or for products derived from it. In this work, an adapted laboratory evolution (ALE) was applied, which resulted in the isolation of an E. coli strain at least 10-fold more tolerant to L-homoserine than the original (MG1655) strain. Only four genomic modifications were identified after genome sequencing of this evolved strain (designed 4E), including a 49 bp truncation starting from the codon stop of thrL and leading to a modified thrL locus carrying a thrL* allele encoding a 30 amino acids polypeptide, which is 9 amino acids more than the leader peptide encoded by thrL. Replacement of thrL with thrL* enabled the initial strain to be as or slightly more tolerant to L-homoserine than the evolved 4E strain, which is explained by the rapid metabolization of L-homoserine to threonine resulting from ThrL*-dependent transcriptional activation of the threonine thrABC operon. Interestingly, in 4E strain, L-homoserine degradation went beyond threonine degradation, as tdh and kbl encoding threonine degradation pathway II and genes of the glycine cleavage system were strongly upregulated. To infer about the toxicity of L-homoserine, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which identified a potent repression of locomotion-motility-chemotaxis process. Since the magnitude of this repression was reduced in a ΔthrL mutant concomitantly with a twofold lower sensitivity of this mutant to L-homoserine, one could argue that the repression of this biological process contributed to growth inhibition by L-homoserine. Furthermore, in both wild type and thrl mutant, a strong repression of the branched-chain amino acids synthesis and transport, as well as activation of the sulphate assimilation pathway process to cysteine synthesis were observed in the presence of L-homoserine, which may also contribute to toxic effect of this compound. How this non-canonical amino acid triggers these transcriptomic changes is discussed.
Project description:Whole genome sequencing was performed on E. coli BL21 (DE3) evolved at 25°C in pH 9 terrific broth media buffered with Tris-HCl (pH 9). The evolved E. coli was characterized and compared to the parent strain.