Dataset Information

Biosensor-guided improvements in salicylate production by recombinant Escherichia coli.

ABSTRACT: BACKGROUND:Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict. Here, a salicylate-responsive transcription factor was used to optimize the expression levels of shikimate/salicylate pathway genes in recombinant E. coli, and to screen a chromosomal transposon insertion library for improved salicylate production. RESULTS:A high-throughput colony screen was first developed based on a previously designed salicylate-responsive variant of the E. coli AraC regulatory protein ("AraC-SA"). Next, a combinatorial library was constructed comprising a series of ribosome binding site sequences corresponding to a range of predicted protein translation initiation rates, for each of six pathway genes (>?38,000 strain candidates). Screening for improved salicylate production allowed for the rapid identification of optimal gene expression patterns, conferring up to 123% improved production of salicylate in shake-flask culture. Finally, transposon mutagenesis and screening revealed that deletion of rnd (encoding RNase D) from the host chromosome further improved salicylate production by 27%. CONCLUSIONS:These results demonstrate the effectiveness of the salicylate sensor-based screening platform to rapidly identify beneficial gene expression patterns and gene knockout targets for improving production. Such customized high-throughput tools complement other cell factory engineering strategies. This approach can be generalized for the production of other shikimate-derived compounds.

SUBMITTER: Qian S

PROVIDER: S-EPMC6350385 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Biosensor-guided improvements in salicylate production by recombinant Escherichia coli.

Qian Shuai S Li Ye Y Cirino Patrick C PC

Microbial cell factories 20190129 1

<h4>Background</h4>Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate pr ...[more]

PMID: 30696431

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Project description:The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24 °C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24 °C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Biosensor-guided improvements in salicylate production by recombinant Escherichia coli.

Publications

Biosensor-guided improvements in salicylate production by recombinant Escherichia coli.

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