Microbial expression systems for membrane proteins

Dilworth, Marvin V.; Piel, Mathilde S.; Bettaney, Kim E.; Ma, Pikyee; Luo, Ji; Sharples, David; Poyner, David R.; Gross, Stephane R.; Moncoq, Karine; Henderson, Peter J. F.; Miroux, Bruno; Bill, Roslyn M.

doi:10.1016/j.ymeth.2018.04.009

Cited by 61 publications

(55 citation statements)

References 91 publications

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“…Host strain engineering can optimize transporter expression by improving host robustness. [ 57,58 ] Efficient methods to obtain strains with improved robustness include random gene deletions, [ 59 ] point mutations, [ 60 ] and gene coexpression, followed by a high‐throughput screening. [ 39,61 ] In eukaryotic microorganisms, such as yeast cells, random genome‐wide mutants can be constructed using the CRISPR/Cas9 system, [ 62 ] the Cre/LoxP system, and the synthetic chromosome rearrangement and modification by loxP‐mediated evolution (SCRaMbLE).…”

Section: Transporter Engineering In Microbial Cellsmentioning

confidence: 99%

Transporter Engineering for Microbial Manufacturing

Zhu

Zhou

Wang

et al. 2020

Biotechnology Journal

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Microbes play an important role in biotransformation and biosynthesis of biofuels, natural products, and polymers. Therefore, microbial manufacturing has been widely used in medicine, industry, and agriculture. However, common strategies including enzyme engineering, pathway optimization, and host engineering are generally inadequate to obtain an efficient microbial production system. Transporter engineering provides an alternative strategy to promote the transmembrane transfer of substrates, intermediates, and final products in microbial cells and thus enhances production by alleviating feedback inhibition and cytotoxicity caused by final products. According to the current studies in transport engineering, native transporters usually have low expression and poor transportation ability, resulting in inefficient transport processes and microbial production. In this review, current approaches for transporter mining, characterization, and verification are comprehensively summarized. Practical approaches to enhance the transport system in engineered cells, such as balancing transporter overexpression and cell growth, and evolution of native transporters are discussed. Furthermore, the applications of transporter engineering in microbial manufacturing, including enhancement of substrate utilization, concentration of metabolic flux to the target pathway, and acceleration of efflux and recovery of products, demonstrate its outstanding advantages and promising prospects.

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Section: Transporter Engineering In Microbial Cellsmentioning

confidence: 99%

Transporter Engineering for Microbial Manufacturing

Zhu

Zhou

Wang

et al. 2020

Biotechnology Journal

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“…For structural biologists, membrane protein production still represents a major biochemical challenge (Zoonens and Miroux 2010). Despite the emergence of eukaryotic expression systems, microbial expression systems are the most popular vehicle for membrane protein production (Dilworth et al 2018). Historically, genetically modified strains have been developed to enhance membrane protein production (Miroux and Walker 1996;Baumgarten et al 2017;Angius et al 2018).…”

Section: Membrane Overproduction As a Platform For Biotechnological Amentioning

confidence: 99%

Intracellular Membranes: Conserved Mechanisms of Formation and Regulation Throughout Evolution

Mir¹,

Biou²,

Dautin³

et al. 2020

Preprint

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Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering membrane proliferation share two major common features: 1) they promote the formation of highly curved membrane domains and 2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, inspiring new biotechnological applications.

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“…Yeasts and particularly P. pastoris are highly attractive alternatives for MP expression as they represent low-cost cultivation and high-quantity production platforms, meeting the demand for criteria of safety and authentically process proteins (Emmerstorfer-Augustin et al 2019). Pichia pastoris systems usually rely on the use of integrative plasmids containing the gene of interest which are integrated into the yeast genome, generating stable production strains (Dilworth et al 2018). Moreover, protein production is usually accomplished resorting the alcohol oxidase promoter (AOX), which is inducible by methanol and depending on the functionality of 1 or both aox genes, recombinant strains may present a Mut S or Mut + phenotype exhibiting different growth behaviors (in methanol) and different methanol requirements for induction.…”

Section: Genetic-level Strategiesmentioning

confidence: 99%

“…In the last years, studies have shown that distinct recombinant gene dosages and codon usage optimizations greatly influence MP expression levels in P. pastoris. As mentioned above, P. pastoris expression systems usually rely on expression plasmids that are integrated into the yeast genome and multi-copy clones-the so-called Bjackpot clones^-can be selected experimentally by screening several colonies in increasing concentrations of antibiotic (Dilworth et al 2018). Nordén et al (2011) performed a two-step antibiotic selection, initially with 100 μg/mL zeocin and then with higher concentrations, from which they isolated multi-copy clones and observed that the expression of different aquaporins strongly respond to an increase in recombinant gene dosage, independently of the amount of protein expressed from a single gene copy clone.…”

Section: Genetic-level Strategiesmentioning

confidence: 99%

“…Nordén et al (2011) performed a two-step antibiotic selection, initially with 100 μg/mL zeocin and then with higher concentrations, from which they isolated multi-copy clones and observed that the expression of different aquaporins strongly respond to an increase in recombinant gene dosage, independently of the amount of protein expressed from a single gene copy clone. However, despite higher recombinant gene dosages can lead to higher titers of recombinant proteins, this correlation is not always linear and strains with low copy number may be preferred (Aw and Polizzi 2013;Dilworth et al 2018). Aiming to exclude possible false-positives while establishing accurate correlations, along with the levels of the target protein, the recombinant DNA levels must be evaluated, for which qPCR protocols have been reported using pPICZ vectors (Nordén et al 2011) and resorting to SYBR Green or TaqMan (Abad et al 2010).…”

Section: Genetic-level Strategiesmentioning

confidence: 99%

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Smoothing membrane protein structure determination by initial upstream stage improvements

Pedro

Queiroz

Passarinha

2019

Appl Microbiol Biotechnol

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Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

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Microbial expression systems for membrane proteins

Cited by 61 publications

References 91 publications

Transporter Engineering for Microbial Manufacturing

Transporter Engineering for Microbial Manufacturing

Intracellular Membranes: Conserved Mechanisms of Formation and Regulation Throughout Evolution

Smoothing membrane protein structure determination by initial upstream stage improvements

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