Dataset Information

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

ABSTRACT: Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA(-) endA(-)), we synthesized 550±40 ?g?mL(-1) of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine. This yield was increased to ?1300 ?g?mL(-1) when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

SUBMITTER: Hong SH

PROVIDER: S-EPMC4418657 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Hong Seok Hoon SH Kwon Yong-Chan YC Martin Rey W RW Des Soye Benjamin J BJ de Paz Alexandra M AM Swonger Kirsten N KN Ntai Ioanna I Kelleher Neil L NL Jewett Michael C MC

Chembiochem : a European journal of chemical biology 20150303 5

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five ...[more]

PMID: 25737329

Similar Datasets

Project description:UNLABELLED:Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. IMPORTANCE:The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.

Dataset Information

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Publications

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets