Ki, D. | bioRxiv Science

bioRxiv2022

Balance-directed protein engineering of IsPETase enhances both PET hydrolysis activity and thermostability

A mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low thermostability limits its industrial applications. We herein developed an engineering strategy for IsPETase to enhance PET hydrolysis activity, thermostability, and protein folding of the enzyme. Balance-directed Z1-PETase variant outperforms the stability-directed Z2-PETase variant under both mesophilic and thermophilic conditions, although Z2-PETase exhibits higher thermostability than Z1-PETase. The Z1-PETase is also superior to Fast-PETase, Dura-PETase, and LC-CICCG in terms of depolymerization rate regardless of temperature conditions we tested. Thus, maintaining a balance between PET hydrolysis activity and thermostability is essential for the development of high-performance PET hydrolases. In a pH-stat bioreactor, Z1-PETase depolymerized >90% of both transparent and colored post-consumer PET powders within 24 and 8 hours at 40{degrees}C and 55{degrees}C, respectively, demonstrating that the balance-directed IsPETase variant produced herein may be applicable in the bio-recycling of PET.

bioengineering↗

bioRxiv2022

Geobacter sulfurreducens' unique metabolism results in cells with a high iron and lipid content

Geobacter sulfurreducens is a ubiquitous iron reducing bacterium in soils, and in engineered systems it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C,H,O,N, ash content) showed a high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with a high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 {+/-} 0.2 g/gdw) and lipids (32 {+/-} 0.5% dw/dw) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. ImportanceGeobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively breathing metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal, and as a ubiquitous iron reducer in soils around the world, G. sulfurreducens and its relatives impact the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organisms composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria -- these observations are important for future microbiologists and biotechnologists working with the organism.

microbiology↗