Sustainable production of a biotechnologically relevant ß-galactosidase in Escherichia coli cells using crude glycerol and cheese whey permeate.

Responsible use of natural resources and waste reduction are key concepts in bioeconomy. This study demonstrates that agro-food derived-biomasses from the Italian food industry, such as crude glycerol and cheese whey permeate (CWP), can be combined in a high-density fed-batch culture to produce a recombinant ß-galactosidase from Marinomonas sp. ef1 (M-ßGal). In a small-scale process (1.5 L) using 250 mL of crude glycerol and 300 mL of lactose-rich CWP, approximately 2000 kU of recombinant M-ßGal were successfully produced along with 30 g of galactose accumulated in the culture medium. The purified M-ßGal exhibited high hydrolysis efficiency in lactose-rich matrices, with hydrolysis yields of 82 % in skimmed milk at 4 °C and 94 % in CWP at 50 °C, highlighting its biotechnological potential. This approach demonstrates the effective use of crude glycerol and CWP in sustainable and cost-effective high-density Escherichia coli cultures, potentially applicable to recombinant production of various proteins.

PGM3 inhibition shows cooperative effects with erastin inducing pancreatic cancer cell death via activation of the unfolded protein response.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Methods: Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP's Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Results: Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Conclusion: Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

Diverse Repertoire and Relationship of Exopolysaccharide Genes in Cold-Adapted Acinetobacter sp. CUI-P1 Revealed by Comparative Genome Analysis.

This study focused on the exploration of microbial communities inhabiting extreme cold environments, such as the Passu and Pisan glaciers of Pakistan, and their potential utilization in industrial applications. Among the 25 initially screened strains, five were found to be suitable candidates for exopolysaccharide (EPS) production, with strain CUI-P1 displaying the highest yield of 7230.5 mg/L compared to the other four strains. The purified EPS from CUI-P1 was tested for its ability to protect probiotic bacteria and E. coli expressing green fluorescence protein (HriGFP) against extreme cold temperatures, and it exhibited excellent cryoprotectant and emulsification activity, highlighting its potential use in the biotechnological industry. Furthermore, the genome of Acinetobacter sp., CUI-P1 comprised 199 contigs, with a genome size of 10,493,143bp and a G + C content of 42%, and showed 98.197% nucleotide identity to the type genome of Acinetobacter baumannii ATCC 17978. These findings offer promising avenues for the application of EPS as a cryoprotectant, an essential tool in modern biotechnology.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.