Genomic analysis of high-cell-density recombinantEscherichia coli fermentation and ?cell conditioning? for improved recombinant protein yield

Gill, Ryan T.; DeLisa, Matthew P.; Valdés, James J.; Bentley, William E.

doi:10.1002/1097-0290(20010105)72:1<85::aid-bit12>3.0.co;2-h

Cited by 61 publications

(45 citation statements)

References 54 publications

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“…Research labs, including ours, turned to the elicitation of proteases (Harcum and Bentley, 1993), creating deletion strains (Meerman and Georgiou, 1994) or fermentation operating policies (DeLisa et al, 1991;Gill et al, 2001;Ramirez and Bentley, 1995) that avoided protein degradation. Various protein foldases were later found altered in their level and activity in response to metabolic perturbations.…”

Section: Introductionmentioning

confidence: 78%

Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Bentley

Mirjalili

Andersen

et al. 2009

Biotech & Bioengineering

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Section: Introductionmentioning

confidence: 78%

Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Bentley

Mirjalili

Andersen

et al. 2009

Biotech & Bioengineering

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“…We previously hypothesized that an adaptation of this nature was likely occurring during the transition to high cell density and during recombinant protein overexpression (20), and while this hypothesis was not specifically tested here, the dissimilar responses observed following the initial and subsequent pulse addition of IPTG is likely indicative of culture adaptation to heterologous protein formation. Similar adaptation, consistent with this result, has been demonstrated for glucose uptake and protease up-regulation in response to two consecutive pulses inducing recombinant protein expression (27).…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, little is known about the attenuated induction of heterologous proteins commonly observed at high cell densities (11,19,50), where factors such as decreased growth rate, increased cell death, increased lysis, decreased metabolic activity, and segregation into viable but nonculturable cells can negatively impact reactor productivity (2,28,43). We have shown dramatically altered transcript levels of several stress-related genes at cell densities near 80 g (dry weight) liter Ϫ1 (20), and recent understanding of autoinduction suggests possible mechanisms that contribute to the altered metabolic and physiological state of cells grown to these cell densities. In this work, we have also explored the transcriptional response of 24 genes, including four known quorum-related genes as well as a known subset of genes up-regulated in response to human interleukin-2 (hIL-2) production, in order to begin mapping quorum-dependent transcriptional regulation with bacterial stress responses.…”

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confidence: 95%

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Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

2001

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Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-␤-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.Synthesis and perception of a self-produced, freely diffusible signal molecule, termed autoinducer AI-2, by the gram-negative bacterium Escherichia coli is thought to regulate the expression of a variety of genes in response to population density. This process, termed autoinduction or quorum sensing, was first described in Vibrio fischeri (39), and similar autoregulatory mechanisms have since been reported in a wide range of bacteria, including Pseudomonas aeruginosa (34), Erwinia caratovora (5), and Agrobacterium tumefaciens (40), as well as E. coli (18,44,57). Although the existence of such a mechanism in E. coli has been uncovered, the genetic, physiological, and environmental factors that contribute to and regulate the quorum circuitry remain poorly defined.Evidence of intercellular communication in E. coli came with the discovery of a quorum-regulated transcriptional transactivator (SdiA) of the cell division genes in the ftsQAZ locus homologous to LuxR of V. fischeri (18, 44, 57) and a synthase protein (LuxS E.c. ) responsible for AI-2 signal molecule production (49). Further elucidation of native E. coli quorum circuit architecture resulted from the Vibrio harveyi cross-species activity assay of Surette and Bassler (47), which has g...

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“…It is also recognized that the abundance of heat shock proteins (HSPs, including chaperones and proteases) is influenced by heterologous protein overexpression and, in turn, can affect the protein yield (4,14,21,23,32,42). We have previously demonstrated that avoiding (32) or intentionally downregulating (39) the heat shock response coincident with protein overexpression, as well as stimulation of HSPs prior to induction (14), can facilitate increased yield and activity of CAT (14) and OPH (39). To ascertain whether luxS coexpression leads to increased yield through the pleiotropic regulation of HSPs, we measured the levels of two important heat shock proteins, GroEL and DnaK, as well as the transcription of these and several other proteins in the presence and absence of varied LuxS expression (48).…”

Section: Resultsmentioning

confidence: 99%

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

Tsao

Wang

Hashimoto

et al. 2011

Appl Environ Microbiol

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We found conditions in which the protein yield was dramatically increased. Further studies demonstrated coincident upregulation of the chaperone GroEL, which may have facilitated higher yields and is shown for the first time to be positively regulated at the posttranscriptional level by AI-2. This report is the first to demonstrate that the protein synthesis capacity of E. coli can be altered by rewiring quorum sensing circuitry.

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Genomic analysis of high-cell-density recombinantEscherichia coli fermentation and ?cell conditioning? for improved recombinant protein yield

Cited by 61 publications

References 54 publications

Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

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

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