LuxS Coexpression Enhances Yields of Recombinant Proteins in
            <i>Escherichia coli</i>
            in Part through Posttranscriptional Control of GroEL

Tsao, Chen Yu; Wang, Liang; Hashimoto, Yoshifumi; Yi, Hyunmin; March, John C.; DeLisa, Matthew P.; Wood, Thomas K.; Valdés, James J.; Bentley, William E.

doi:10.1128/aem.02347-10

Cited by 19 publications

(13 citation statements)

References 49 publications

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“…It is worthy to note that the use of sorbitol is acceptable as a proof-of-concept but may not be routinely used in microbial fermentation due simply to the cost involved [29]. An alternative is to modify the host microbes (such as manipulation of cellular protein folding system [27,35]) to render these proteins more soluble for industrial applications.…”

Section: Discussionmentioning

confidence: 99%

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

et al. 2012

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BackgroundRecombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes.ResultsFour enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway.ConclusionThis study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites.

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Section: Discussionmentioning

confidence: 99%

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

et al. 2012

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“…This study demonstrated one cell population was able to guide protein synthesis process of another by guiding intraspecies communication. Moreover, it was reported in [55] that by simply adding conditioned medium (containing a high amount of AI-2) during recombinant protein induction, one can double the yield of active product. Also, by altering the coincident luxS expression to control the AI-2 concentration while also inducing heterologous protein expression, they found an optimal condition where protein yield is dramatically increased.…”

Section: Applicationsmentioning

confidence: 99%

Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

Wang¹,

Payne²,

Bentley³

2019

Gene Expression and Control

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Because cells have the extraordinary ability to sense and respond to even subtle environmental changes by intricately regulating their gene expression patterns, their behaviors can be intentionally "tuned" by altering the state of their environments in a prescribed or rational manner. Rational control of both external and internal molecular stimuli provides a basis for many biotechnological applications including the expression of foreign protein products. This is done by coordinately controlling product synthesis while retaining the cell in a productive state. Quorum sensing (QS), a molecular signaling modality that mediates cell-cell communication, autonomously facilitates both inter-and intra-species gene regulation. This process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. Recently, even electrical signals, which have long been used to control the most sophisticated of man-made devices, are now employed to alter cell signaling processes enabling computer programmed behavior, particularly in cells suitably engineered to accommodate electrical signals. By minimally engineering these genetic circuits, new applications have emerged for the repurposing of Escherichia coli, from creating innovative sensor concepts to stimulating the emerging field of electrogenetics.Gene Expression and Control 2 as well as the extracellular microenvironmental state in the vicinity of 'designer' production strains in order to program gene expression and behavior. These techniques incorporate the understanding of cell metabolism and the transcriptome, cell-cell communication (previously reviewed by [2,3]), and biological redox reactions (previously reviewed by [4]). This chapter will mainly focus on recent advances in how actuation of genes is accomplished in Escherichia coli through methods that require only minimal genetic rewiring and the technologies developed on such platforms, for instance those of biosensors and bioelectric devices.

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“…The assay for synthesizing autoinducer-2 in vitro was based upon the report of Chen-Yu Tsao with some modification (Tsao et al, 2011). The cultured cells of B. thailandensis (wild type and recombinants) were harvested by centrifugation, washed twice with ice-cold PBS (pH 7.4) and finally resuspended in 50 mM Tris-HCl (pH 7.8).…”

Section: In Vitro Autoinducer-2 Synthesis and Assaymentioning

confidence: 99%

Substrate preference of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase inBurkholderia thailandensis

Gao

Zheng

Yuan

2013

FEMS Microbiol Lett

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5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and AdoHcyase from Burkholderia thailandensis were cloned and characterized. The K(m) values of MTAN for 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) were 19 and 58 μM, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B. thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, AdoHcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the K(m) value of AdoHcyase for SAH was determined to be 40 μM. Sequence analysis revealed that MTAN had much higher diversity than AdoHcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS(+) bacteria could be discriminated from LuxS(-) bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle.

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LuxS Coexpression Enhances Yields of Recombinant Proteins in Escherichia coli in Part through Posttranscriptional Control of GroEL

Cited by 19 publications

References 49 publications

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

Substrate preference of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase inBurkholderia thailandensis

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