Search bioRxiv⌕ Search

Biology subjects

Tee, K. L.

Publications and source records attributed to Tee, K. L..

5 recordsLinked to original sources

Rapid and Cost-Effective Preparation of a RuBisCO-Rich Protein Fraction from Dried Leafy Biomass

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant protein on Earth, is an attractive and sustainable food ingredient owing to its favourable nutritional and techno-functional properties. Leafy vegetables are particularly rich sources of RuBisCO; however, large-scale vegetable production generates substantial quantities of residual biomass throughout agri-food supply chains. Drying is widely used to stabilise this biomass and facilitate storage, transport, and handling, yet most reported RuBisCO extraction methods have been developed for fresh material and are poorly suited to dried feedstocks. Here, we present a simple, scalable, and cost-effective process for the recovery of food-grade RuBisCO from dried leafy biomass. Using spinach, rocket, and kale as model systems, efficient protein extraction was achieved from both freshly dried leaves and commercially available leaf powders without the need for resource-intensive processing. Application of the method to spinach yielded approximately 75 mg of high-purity RuBisCO per 100 g fresh-leaf equivalent, corresponding to an extraction efficiency of [~]70%, which increased to [~]90% following supplementation with 20 mM CaCl2. The recovered protein fraction also exhibited favourable foaming capacity and foam stability, demonstrating its potential as a functional food ingredient. This work provides a practical route for the valorisation of dried vegetable residues and supports the development of circular, waste-to-value supply chains for sustainable plant protein production.

biochemistry↗

Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. Here, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving C. necator H16 for enhanced tolerance towards elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. Our method offers several advantages over traditional ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially hazardous DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

synthetic biology↗

Sweetwater: an underrated crude glycerol for sustainable lipid production in non-conventional yeasts

Sweetwater, a methanol-free by-product of the fat splitting process, is an emerging alternative feedstock for microbial utilization of crude glycerol. Yeasts are promising candidates in this context due to their versatility in delivering a wide range of value-added products including lipids. To enhance sweetwater utilization, we investigated the growth and lipid production of 21 oleaginous yeast strains in sweetwater and found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. To decipher the underlying mechanism, the transcriptomic changes of R. toluroides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock and suggested potential sweetwater type and strain engineering targets for increasing microbial lipid production. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/558176v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1ad5738org.highwire.dtl.DTLVardef@139227eorg.highwire.dtl.DTLVardef@7a8e1org.highwire.dtl.DTLVardef@e072c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Directed evolution for improved total secretory protein production in Escherichia coli

Production of secretory protein in Gram-negative bacteria simplifies downstream processing in recombinant protein production, accelerates protein engineering, and advances synthetic biology. Signal peptides and secretory carrier proteins are commonly used to effect the secretion of heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have successfully applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate the directed evolution workflow. In this study, we applied directed evolution on OsmY to enhance its total secretory protein production. After just one round of directed evolution followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 {+/-} 0.3 fold and 2.9 {+/-} 0.8 fold more secretory Tfu0937 {beta}-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 {degrees}C, 30 {degrees}C and 25 {degrees}C). Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 {+/-} 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. This is the first report of applying directed evolution on a carrier protein to enhance total secretory protein production. The methodology can be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli and other bacteria. In this study, OsmY(M3) improved the production of three proteins, originating from diverse organisms and with diverse properties, in secreted form, clearly demonstrating its wide-ranging applications.

synthetic biology↗

Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay

Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its receptor binding domain (RBD)] is an important step in the manufacturing process. Simple and high-capacity protein purification schemes for spike, RBD, and CR3022 mAb, recombinantly expressed in CHO and HEK293 cells, are reported in this article. The schemes consist of an affinity chromatography step and a desalting step. Purified proteins were validated in ELISA-based serological tests. Interestingly, extracellular matrix proteins [most notably heparan sulfate proteoglycan (HSPG)] were co-purified from spike-expressing CHO culture with a long cultivation time. HSPG-spike interaction could play a functional role in the pathology and the pathogenesis of SARS-CoV-2 and other coronaviruses.

biochemistry↗