Biology subjects

Tiong, E.

Publications and source records attributed to Tiong, E..

3 recordsLinked to original sources

bioRxiv2024

Further characterization and engineering of a 11-amino acid motif for enhancing recombinant protein expression

BackgroundRecombinant protein production in Escherichia coli (E. coli) is a widely used system in both academic and industrial research owing to its low cost and wide availability of genetic tools. Despite its advantages, this system still struggles with soluble expression of recombinant proteins. To address this, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. However, traditional tags such as small ubiquitin-related modifier (SUMO) and Glutathione S-transferase (GST) can interfere with protein folding or require removal post-translation, which adds complexity and cost to production. To circumvent these issues, smaller solubility tags (<10 kDa) are preferred. This study specifically focuses on an 11-amino acid solubility-enhancing tag (NT11) derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. ResultsA comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we increased its activity and identified key residues for further development. Screening with the alanine mutant library consistently led to at least a two-fold improvement in protein yield for three different proteins. We also discovered that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing protein expression constructs. With these new insights, we have successfully doubled the recombinant protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and an originally low-yielding human epidermal growth factor (hEGF), in E. coli. ConclusionThe further characterisation and development of the NT11 tag have provided valuable insights into the optimization process for such small tags and expanded our understanding of its potential applications. The ability of NT11 tag to be positioned at different locations within the protein construct without compromising its effectiveness to enhance recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings have the potential to simplify and enhance the efficiency of recombinant protein production.

synthetic biology↗

bioRxiv2024

Hyperporous encapsulation of microbes for whole cell biocatalysis and biomanufacturing

Compared to traditional synthetic chemical processes, biocatalysts offer a more sustainable and eco-friendly approach to producing complex molecules. In particular, whole-cell biocatalysts boast numerous advantages, including scalable, self-containing co-factor recycling systems, the use of cost-effective raw materials, and reduced purification costs. However, challenges arise when working with microbial consortia for biotransformation cascades. Our encapsulation strategy addresses these challenges by controlling microbial cell populations through physical constraints, offering a promising approach in biomanufacturing. In this work, we describe the immobilization of cells in a hyper-porous hydrogel block, which provides ample nutrient access while simplifying media changes. We encapsulated E. coli cells in a hydrogel matrix with suitable mechanical properties, effectively limiting their proliferation while sustaining recombinant GFP production. Furthermore, we successfully maintained different microbial strains spatially in a single porous hydrogel block for at least 10 days, demonstrating the potential of this method for achieving stable co-culture. Finally, we demonstrated the application of immobilized E. coli for co-culture fermentation. The immobilization of E. coli heterologously expressing RadH halogenase significantly improved the efficiency of genistein halogenation in a co-culture with genistein-producing Streptomyces compared to its non-immobilized counterpart.

bioengineering↗

bioRxiv2023

Expression and engineering of unexplored PET degrading enzymes from Microbispora, Nonomuraea, Micromonospora genus

Low recycling rates have resulted in the alarming rate of accumulation of a widely used plastic material, polyethylene terephthalate (PET). With the build-up of plastics in our environment, there is an urgent need to source for more sustainable solutions to process them. Biological methods such as enzyme-catalyzed PET recycling or bioprocessing are seen as a potential solution to this problem. Actinobacteria, known for producing enzymes involved in the degradation of complex organic molecules, are of particular interest due to their potential to produce PET degrading enzymes. The highly thermostable enzyme, leaf-branch compost cutinase (LCC) found in Actinobacteria is one such example. This work expands on the discovery and characterization of new PET degrading enzymes from Microbispora, Nonomuraea, and Micromonospora genus. Within this genus, we analyzed enzymes from the polyesterase-lipase-cutinase family, which have [~]60% similarity to LCC, where one of the enzymes was found to be capable of breaking down PET and BHET at 45-50 {degrees}C. Moreover, we were able to enhance the enzymes depolymerization rate through further engineering, resulting in a two-fold increase in activity. IMPORTANCEThe proliferation of PET plastic waste poses a significant threat to human and environmental health, making it an issue of increasing concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Despite the existence of enzymes capable of degrading PET, their scarcity in nature limits their applicability. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel ones that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation.

microbiology↗