Development of <i>Escherichia coli</i> Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production

Gialama, Dimitra; Kostelidou, Kalliopi; Michou, Myrsini; Delivoria, Dafni Chrysanthi; Kolisis, Fragiskos N.; Σκρέτας, Γεώργιος

doi:10.1021/acssynbio.6b00174

Cited by 38 publications

(81 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1 b) indicates that directly transferring TtgABC to other strains may be challenging. Recently identified mutations in E. coli leading to a reduced burden of expressing membrane proteins may help to reduce the toxicity of the efflux pump [ 36 ], as it may chaperone overexpression [ 37 ]. Strategies for regulating membrane protein production via genetic circuits are also promising [ 6 ].…”

Section: Discussionmentioning

confidence: 99%

A Pseudomonas putida efflux pump acts on short-chain alcohols

Basler

Thompson

Tullman‐Ercek

et al. 2018

Biotechnol Biofuels

View full text Add to dashboard Cite

BackgroundThe microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists.ResultsWe report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n-butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n-butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n-butanol and isopentanol.ConclusionsTtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1133-9) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 99%

A Pseudomonas putida efflux pump acts on short-chain alcohols

Basler

Thompson

Tullman‐Ercek

et al. 2018

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…The complexity of transmembrane structures probably poses a major challenge for the overexpression of transporters, which has been reported to be detrimental to the host cell growth. [ 39,45 ] Previous hypotheses for this growth inhibition are mainly related to the excess translocation burden of the membrane protein complex, transmembrane protein overloading on limited membrane size, and impact on the central metabolism. [ 46–48 ] However, transporter overexpression is vital for a crystal structure analysis and functional enhancement of the transport systems.…”

Section: Transporter Engineering In Microbial Cellsmentioning

confidence: 99%

“…Transporter for the target compound was mined from candidates via strategies including an omics‐guided knockout assay, [ 23,24 ] gain‐of‐function strategy for heterologous transporters, [ 30 ] and biosensor‐based high‐throughput screening. [ 32,34 ] The next step utilized promoter optimization [ 37,38 ] and host engineering [ 39 ] for regulation of transporter expression, and the transporter function was enhanced by evolution [ 40 ] and fusion approaches. [ 41 ] Finally, by comprehensive metabolic analysis of the target pathway, intensified strains were obtained through a transport system redesign [ 42 ] and used in industrial biomanufacturing.…”

Section: Introductionmentioning

confidence: 99%

Transporter Engineering for Microbial Manufacturing

Zhu

Zhou

Wang

et al. 2020

Biotechnology Journal

View full text Add to dashboard Cite

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.

show abstract

“…In general, it seems that the enhanced effect is not due to a direct interaction of these genes with the target proteins, but instead by indirect effects, namely, induction of stress responses or changes in the composition of the bacterial periplasm (Skretas et al 2012). Foreseeing the identification of genes whose co-expression can supress MPinduced toxicity, a genome wide screen identified two potent suppressors, namely, djlA (encoding the membrane-bound DNAk cochaperone DjlA) and rraA (encoding RRaA), an inhibitor of the mRNA-degrading activity of the E. coli RNase E (Gialama et al 2017). E. coli strains co-expressing djlA or rrA, referred as SuptoxD and SuptoxR, respectively, strains were found to have a consistent behavior regarding an enhancement production of distinct MP, namely, from mammalian and bacterial origin and with different topologies, and perform better than other commercially available strains (Gialama et al 2017).…”

Section: Strain Engineeringmentioning

confidence: 99%

Smoothing membrane protein structure determination by initial upstream stage improvements

Pedro

Queiroz

Passarinha

2019

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

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.

show abstract

Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production

Cited by 38 publications

References 46 publications

A Pseudomonas putida efflux pump acts on short-chain alcohols

A Pseudomonas putida efflux pump acts on short-chain alcohols

Transporter Engineering for Microbial Manufacturing

Smoothing membrane protein structure determination by initial upstream stage improvements

Contact Info

Product

Resources

About