Optimization of a Recombinant Lectin Production in Pichia pastoris Using Crude Glycerol in a Fed-Batch System

Abstract

The production of heterologous proteins for medical use is an important area of interest. The optimization of the bioprocesses includes the improvement of time, costs, and unit operations. Our study shows that a lectin fraction from Tepary bean (Phaseolus acutifolius) (TBLF) has cytotoxic effects on colon cancer cells and in vivo antitumorigenic activity. However, the low-yield, time-consuming, and expensive process made us focus on the development of a strategy to obtain a recombinant lectin using engineered Pichia pastoris yeast. Pure glycerol is one of the most expensive supplies; therefore, we worked on process optimization using crude glycerol from biodiesel production. Recombinant lectin (rTBL-1) production and purification were evaluated for the first time by an experimental design where crude glycerol (G65) was used and compared against pure glycerol (G99) in a controlled stirred-tank bioreactor with a fed-batch system. The recombinant lectin was detected and identified by SDS-PAGE, Western blot, and UHPLC–ESI–QTOF/MS analysis. The results show that the recombinant lectin can be produced from G65 with no significant differences with respect to G99: the reaction rates were 2.04 and 1.43 mg L−1 h−1, and the yields were 264.95 and 274.67 mgL−1, respectively. The current low cost of crude glycerol and our results show the possibility of producing heterologous proteins using this substrate with high productivity.

EGFR and p38MAPK Contribute to the Apoptotic Effect of the Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) in Colon Cancer Cells

“…In previous studies, it was possible to determine that the LC 50 for rTBL-1 on HT-29 cells was 0.056 mg protein/mL (0.228 mg lyophilized/mL) [ 32 ]. Using this concentration, the apoptotic effect of rTBL-1 after 8 h of incubation was determined and compared against the effect of TBLF (LC 50 0.055 mg lectin/mL or 0.402 mg of lyophilized TBLF/mL) previously determined in the same cell line [ 28 ].…”

“…TBLF contains at least two bioactive lectins and some other minority proteins [ 47 ], and its production involves several purification steps, resulting in a high-cost, low-yield procedure. Therefore, it was necessary to develop a method to obtain a recombinant lectin [ 29 , 30 , 31 ] rTBL-1 with high homology to one native bioactive lectin [ 32 ]. In this sense, we compared the apoptotic effects of both LC 50 for rTBL-1 versus TBLF on HT-29 colon cancer cells.…”

“…The recombinant Tepary bean lectin (rTBL-1) showed affinity to β 1-6 branched N-glycans, which are overexpressed in fundamental transmembrane receptors in some types of cancer, such as colon cancer. A high amino acid sequence homology between one native lectin and rTBL-1 was observed, and preliminary results suggested similar cytotoxic effects [ 31 , 32 ].…”

Construction of an l-Tyrosine Chassis in Pichia pastoris Enhances Aromatic Secondary Metabolite Production from Glycerol

“…pastoris is widely used as a chassis to produce heterologous proteins for research and industrial purposes, , which does not produce fermentative byproducts under aerobic conditions because its glycolytic flux does not exceed the respiratory capacity. , Glycerol is often used as a carbon source during P. pastoris fermentation because it encodes four H + /glycerol symporters in its genome, allowing for efficient metabolism and high biomass productivity. − Glycerol requires fewer enzymatic reactions for the biosynthesis of phosphoenolpyruvate (PEP) from glycolysis and erythrose-4-phosphate (E4P) from the pentose phosphate pathway (PPP) compared to glucose (Figure ). PEP and E4P are essential precursors for l -Tyr biosynthesis.…”

Second generation Pichia pastoris strain and bioprocess designs

“…In addition, 15,977 U.mg −1 lipase activity that is higher than that of pure glycerol was obtained and the key components responsible for the enhanced activity were specified as Na + , Ca 2+ , and grease in crude glycerol [ 111 ]. Likewise, crude glycerol was used to produce recombinant lectin, and the maximum yield was attained as 265 ± 13 mg.L −1 through optimization of bioreactor operation parameters, e.g., pH, aeration, agitation, and temperature [ 110 ]. Eleven different glycerol samples were obtained with methanolysis of soybean oil using different catalysts and purification steps, and they were used for the production of recombinant α-amylase [ 116 ].…”