Dataset Information

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System.

ABSTRACT: In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 ?g/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7314905 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Establishing a Eukaryotic <i>Pichia pastoris</i> Cell-Free Protein Synthesis System.

Zhang Lingkai L Liu Wan-Qiu WQ Li Jian J

Frontiers in bioengineering and biotechnology 20200618

In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic <i>Pichia pastoris</i> is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report he ...[more]

PMID: 32626695

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Project description:BackgroundLipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility.ResultInspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts.ConclusionWe have proposed a new concept of integrated biodiesel production. This integrated system couples lipase production to lipase-catalyzed biodiesel synthesis in one pot. The proof-of-concept was established through construction of a recombinant P. pastoris yeast strain that was able to grow, overexpress T. lanuginosus lipase, and efficiently catalyze biodiesel production from fed waste cooking oils and methanol simultaneously. This simplified single-step process represents a significant advance toward achieving economical production of biodiesel at industrial scale via a 'green' biocatalytic route.

Dataset Information

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System.

Publications

Establishing a Eukaryotic <i>Pichia pastoris</i> Cell-Free Protein Synthesis System.

Similar Datasets

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