Understanding and Engineering the Secretome of Biotechnology Workhorse may Simplify and Reduce the Cost of Downstream Product Purification and Recovery

Abstract

Maintaining cellular homeostasis is central to cell survival. To do so, cells need to secrete excess metabolites to avoid accumulation of extragenous compounds that may trigger unnecessary reactions that result in a detrimental phenotype. At the same time, cells do not live in isolation, but rather, they need to communicate with their neighbouring microbes, and if necessary conduct chemical warfare to protect fellow brethren of the same species. This then sow the seeds for the evolution of metabolite and protein secretion systems in cells. Efforts to understand the secretome of biotechnology workhorse has been ongoing for some time. Perhaps, the quintessential approach is in using metabolomics methodology to profile the ensemble set of metabolites secreted by cells. But perhaps due to the complexity of the mixture, concomitant separation and detections of ensemble of small molecular metabolites and proteins remain difficult. On the other hand, reducing the complexity of the secretome of a biotechnology workhorse such as Escherichia coli or Saccharomyces cerevisiae may offer a path to streamlining downstream processing workflow which reduces cost. Specifically, one approach could specifically target secreted proteins which pose the most serious challenge in downstream purification. This line of thinking posits that extragenous small molecule metabolites could be removed through using appropriate column matrix in industrial chromatography separation systems. Hence, the goal of engineering the secretome of biotechnology workhorse could be reduced to reducing the secretion of extragenous proteins which are not part of the product portfolio from the fermentation. One approach for reducing the production or secretion of secreted proteins would be to knockout genes encoding such proteins in the genome. Bioinformatically, the first task is to identify secreted proteins through their signal peptide. Such peptides and their sequence have been well characterised in many of the biotechnology workhorses in use such as E. coli, Bacillus subtilis and S. cerevisiae, which afford their ready use in identifying secreted proteins targeted for gene knockout. Once the extragenous secreted proteins have been knockout, the purification task for the target protein product in the fermentation broth would be much simplified. While knocking out important secreted proteins may hamper a species ability to communicate with other cells or defend the population against another adversarial microbial species, the controlled environment of a bioreactor with reduced chances of microbial contamination provides a conducive habitat for cell growth. More importantly, knocking down of regulatory proteins such as those pertaining to quorum sensing that induce the secretion of additional metabolites may afford a simpler culture broth designated for downstream processing. Collectively, simplifying downstream processing of fermentation product would bring important benefits to reducing cost of medicinal products. Such an approach is particularly important as pharmaceutical companies turn their attention to biologics. To this end, knocking out genes that encode secreted proteins 2 is one viable approach for reducing amount and types of secreted products. Simplicity of the approach may afford its ready use in the biotech industry with as-yet underappreciated benefits to industrial bioprocessing.

Keywords: downstream processing, secreted protein, signal peptide, small molecule metabolites, chromatography, biotechnology, biochemistry, microbiology, bioinformatics, cell biology