Secretory Overexpression of PET Hydrolases for Scale Up Applications
Author
Rickert, Maren
Term
4. term
Education
Publication year
2024
Abstract
Denne afhandling adresserer behovet for skalerbar, ekstracellulær produktion af enzymer, der kan nedbryde polyethylenterephthalat (PET), et udbredt plastmateriale med betydelige miljømæssige konsekvenser. To ingeniørdesignede PET-hydrolaser, LCCICCG (en mutant af LCC-cutinase) og FastPETase (en mutant af IsPETase), blev udvalgt som kandidater. Projektets forskningsspørgsmål var, hvilken sekretionsstrategi og værtsorganisme der bedst muliggør effektiv ekstracellulær overekspression til industrielle anvendelser. To tilgange blev undersøgt: (1) i E. coli BL21 (DE3) blev type I α-hemolysin (HlyA)-sekretionssystemet, som kræver C-terminal HlyA-signalpeptid og co-ekspression af HlyB/HlyD, sammenlignet med den SecB-afhængige PelB-signalpeptidvej; (2) i Bacillus subtilis RIK 1285 blev et bibliotek af Sec-afhængige signalpeptider screenet for at udnytte den endogene sekretionsmaskine. Metoder omfattede plasmidkonstruktion og -biblioteker, ekspressionsforsøg, analyse af lokalisation (SDS-PAGE/Western blot), samt screening for ekstracellulær aktivitet. FastPETase gav udtryksmæssige udfordringer under projektet. For LCCICCG i E. coli blev der observeret ekstracellulær lokalisering selv uden signalpeptid, mens brug af signalpeptid førte til delvis intra- eller periplasmatisk ophobning og dermed lavere udbytte. I B. subtilis identificeredes signalpeptidet yqgA som mest effektivt for LCCICCG og opnåede 3,7 mg/L i kulturoverfladevæsken; dette niveau er dog endnu utilstrækkeligt til at matche ekspressionsrater i E. coli og industrielle krav. Samlet peger resultaterne på, at ekstracellulær overekspression af PET-hydrolaser er mulig, men kræver yderligere optimering af værtsvalg, signalpeptider og produktionsbetingelser for industriel relevans.
This thesis addresses the need for scalable, extracellular production of enzymes that depolymerize polyethylene terephthalate (PET), a widely used plastic with significant environmental impacts. Two engineered PET hydrolases, LCCICCG (a mutant of LCC cutinase) and FastPETase (a mutant of IsPETase), were selected as candidates. The research question asked which secretion strategy and host would best enable efficient extracellular overexpression for industrial applications. Two approaches were evaluated: (1) in Escherichia coli BL21 (DE3), the type I α-hemolysin (HlyA) secretion system, which requires a C-terminal HlyA signal peptide and co-expression of HlyB/HlyD, was compared to the SecB-dependent PelB signal peptide pathway; (2) in Bacillus subtilis RIK 1285, a library of Sec-dependent signal peptides was screened to leverage the endogenous secretion machinery. Methods included plasmid construction and libraries, expression trials, localization analysis (SDS-PAGE/Western blot), and screening for extracellular activity. FastPETase posed expression challenges during the project. For LCCICCG in E. coli, extracellular localization was observed even without a signal peptide, whereas using a signal peptide led to partial intra- or periplasmic accumulation and reduced overall yield. In B. subtilis, the yqgA signal peptide was most effective for LCCICCG, reaching 3.7 mg/L in the culture supernatant; however, this remains below expression levels in E. coli and industrial needs. Overall, the results indicate that extracellular overexpression of PET hydrolases is feasible but requires further optimization of host, signal peptides, and process conditions to become industrially competitive.
[This summary has been generated with the help of AI directly from the project (PDF)]
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