Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach

Abstract

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb’s innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb’s ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76–458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae’s secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.

Thermally activated antibiotic production by probiotic bacteria for pathogen elimination

“…Earlier reports on engineered probiotics have highlighted the importance of maintaining the genetic stability of the heterologous protein encoding genes during bacterial growth (Landry and Tabor, 2018). Although, the most preferred route to achieve genetic stability is to integrate the heterologous genes in the chassis genome, studies have shown that this could significantly reduce the overall production (Aggarwal et al, 2020) and secretion (Durmusoglu et al, 2023) levels of recombinant proteins. As in our case, we were unable to observe darobactin production in the liquid cultures, using a medium-copy vector backbone (p15A ori), we decided not to proceed with genomic integration approaches for our genetic circuits.…”

Programming Probiotics: Diet-Responsive Gene Expression and Colonization Control in Engineered S. boulardii

“…Saccharomyces boulardii (S. boulardii or Sb ) is a probiotic yeast that was first isolated from lychee and mangosteen by Henri Boulard in 1923. , Compared to Saccharomyces cerevisiae (with which it shares 99% genomic relatedness), Sb can better tolerate low pH and human body temperature, enabling improved survival in the human gut. − Sb is currently used to treat ulcerative colitis, diarrhea, and recurrent Clostridium difficile infection. − Wild-type Sb is generally recognized as safe (GRAS) and persists in conventionally raised mice for up to 24 h, antibiotic-treated mice for up to 10 days, and in humans for approximately one week. − Recently, a suite of genetic engineering tools for Sb have been developed, including transformation methods, constitutive promoters, genome editing protocols, genomic integration sites, G protein-coupled receptors, and secretion-enhancing gene knockouts. ,− …”

Omics driven onboarding of the carotenoid producing red yeastXanthophyllomyces dendrorhousCBS 6938

“…This is because a high-quality genome, gene regulatory data, species-specific DNA parts for genetic manipulation, and efficient transformation procedures are often missing. The process of developing these elements, termed "onboarding," can be accelerated for nonconventional ascomycete yeasts closely related to S. cerevisiae because the genomics and genetics are similar, permitting transfer of similar promoter and plasmid designs (Durmusoglu et al 2023).…”