Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Nishizawa, Masafumi; Ozawa, Fumiko; Hishinuma, Fumio

doi:10.1007/bf00184982

Cited by 7 publications

(2 citation statements)

References 27 publications

(22 reference statements)

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“…Invertase is a naturally secreted enzyme that catalyzes conversion of sucrose to fructose and glucose. In Sc , the wild-type signal and its mutants have been used to secrete complex recombinant proteins such as human interferon and α-amylase [ 16 , 78 , 85 ]. In Sb , the SUC2 signal peptide enabled an NPA titer of 121 mg/L, the lowest od the 6 signals we tested.…”

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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show abstract

“…Invertase is a naturally secreted enzyme that catalyzes conversion of sucrose to fructose and glucose. In Sc , the wild-type signal and its mutants have been used to secrete complex recombinant proteins such as human interferon and α-amylase (Chang et al, 1986; Nishizawa et al, 1989; Rothe & Lehle, 1998). In Sb , the SUC2 signal peptide enabled an NPA titer of 121 mg/L, the lowest od the 6 signals we tested.…”

Section: Resultsmentioning

confidence: 99%

Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach

Durmusoglu

Al'Abri

Williams

et al. 2022

Preprint

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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 6-fold (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 - 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 >10-fold 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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Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nishizawa

Ozawa

Higashizaki

et al. 1993

Appl Microbiol Biotechnol

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Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast alpha-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 microgram/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.

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Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Cited by 7 publications

References 27 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

Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

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