Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

Gast, Veronica; Sandegren, Anna; Dunås, Finn; Ekblad, Siri; Güler, Rezan; Thorén, Staffan; Mohedano, Marta Tous; Molin, Mikael; Engqvist, Martin K. M.; Siewers, Verena

doi:10.1186/s12934-022-01761-0

Cited by 10 publications

(2 citation statements)

References 57 publications

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“…Both are mainly present in the vacuole, but Prb1p is also present extracellularly. Deletion of these 3 proteases alone or in combination improved secretion of a human serum albumin (HSA)—human parathyroid hormone fusion protein in Pp and three cancer-targeting affibodies in Sc [ 29 , 102 ]. In Sb , deletion of YPS1 , PRB1 and PEP4 improved secretory NPA concentration by 124%, 402% and 390%, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Durmusoglu

Al'Abri

et al. 2023

Microb Cell Fact

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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.

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Section: Resultsmentioning

confidence: 99%

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

Durmusoglu

Al'Abri

et al. 2023

Microb Cell Fact

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“…For example, the S. cerevisiae was engineered to produce and secrete 1-alkenes by manipulation of the fatty acid metabolism, enzyme selection, engineering the electron transfer system and expressing a transporter [ 120 ]. Furthermore, a dynamic regulation strategy were implemented to control the expression of membrane enzyme and transporter, which improved 1-alkene production [ 121 – 123 ].…”

Section: Technical Approaches For Overcoming Low Efficiency In Carbon...mentioning

confidence: 99%

Biosynthesis pathways of expanding carbon chains for producing advanced biofuels

Lin

2023

Biotechnol Biofuels

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Because the thermodynamic property is closer to gasoline, advanced biofuels (C ≥ 6) are appealing for replacing non-renewable fossil fuels using biosynthesis method that has presented a promising approach. Synthesizing advanced biofuels (C ≥ 6), in general, requires the expansion of carbon chains from three carbon atoms to more than six carbon atoms. Despite some specific biosynthesis pathways that have been developed in recent years, adequate summary is still lacking on how to obtain an effective metabolic pathway. Review of biosynthesis pathways for expanding carbon chains will be conducive to selecting, optimizing and discovering novel synthetic route to obtain new advanced biofuels. Herein, we first highlighted challenges on expanding carbon chains, followed by presentation of two biosynthesis strategies and review of three different types of biosynthesis pathways of carbon chain expansion for synthesizing advanced biofuels. Finally, we provided an outlook for the introduction of gene-editing technology in the development of new biosynthesis pathways of carbon chain expansion.

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Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability

Diankristanti,

Lin,

et al. 2024

Bioresource Technology

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Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

Cited by 10 publications

References 57 publications

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

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

Biosynthesis pathways of expanding carbon chains for producing advanced biofuels

Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability

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