Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

Pasini, Martina; Fernández-Castañé, Alfred; Jaramillo, Alfonso; Mas, Carles de; Caminal, Glòria; Ferrer, Pau

doi:10.1016/j.nbt.2015.08.003

Cited by 38 publications

(38 citation statements)

References 27 publications

(21 reference statements)

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“…The high level of expression of multiple LHR-encoded proteins also supports the conclusion that LHR-mediated heat resistance is a multifactorial phenotype Wang et al, 2020). High level protein expression in E. coli imposes a substantial metabolic burden on the cell and may lead to metabolic alterations and a reduced growth rate (Pasini et al, 2016). The maintenance of the LHR in a substantial proportion of E. coli (Mercer et al, 2015;Lee et al, 2018) demonstrates that some of the environmental niches After 60 • C for 5 min 1 or more 62.9 ± 5.0 29.8 ± 6.6…”

Section: Abundance Of Lhr-encoded Proteinsmentioning

confidence: 52%

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

Mercer

Behr

et al. 2020

Front. Microbiol.

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The locus of heat resistance (LHR) confers extreme heat resistance in Escherichia coli. This study explored the role of the LHR in heat and pressure resistance of E. coli, as well as its relationship with protein folding and aggregation in vivo. The role of LHR was investigated in E. coli MG1655 and the pressure resistant E. coli LMM1010 expressing an ibpA-yfp fusion protein to visualize inclusion bodies by fluorescence microscopy. The expression of proteins by the LHR was determined by proteomic analysis; inclusion bodies of untreated and treated cells were also analyzed by proteomics, and by fluorescent microscopy. In total, 11 proteins of LHR were expressed: sHSP20, ClpK GI , sHSP, YdfX1 and YdfX2, HdeD, KefB, Trx, PsiE, DegP, and a hypothetical protein. The proteomic analysis of inclusion bodies revealed a differential abundance of proteins related to oxidative stress in strains carrying the LHR. The LHR reduced the presence of inclusion bodies after heat or pressure treatment, indicating that proteins expressed by the LHR prevent protein aggregation, or disaggregate proteins. This phenotype of the LHR was also conferred by expression of a fragment containing only sHSP20, ClpK GI , and sHSP. The LHR and the fragment encoding only sHSP20, ClpK GI , and sHSP also enhanced pressure resistance in E. coli MG1655 but had no effect on pressure resistance of E. coli LMM1010. In conclusion, the LHR confers pressure resistance to some strains of E. coli, and reduces protein aggregation. Pressure and heat resistance are also dependent on additional LHR-encoded functions.

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Section: Abundance Of Lhr-encoded Proteinsmentioning

confidence: 52%

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

Mercer

Behr

et al. 2020

Front. Microbiol.

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“…Efforts to increase control and predictability of engineered biological systems has led to tools to measure and reduce burden which have come about through improvements in our understanding of host-construct interactions 9 10 . This has led, among other things, to the development of new orthogonal expression systems 11 , a transcriptional resource allocator able to tune the transcriptional capacity available for a synthetic system 12 , and libraries of promoters able to tune the expression of burdensome proteins and decrease cellular stress 13 . These are all strategies that bypass some of the resource-sharing required for gene expression but do not fully-eliminate it.…”

Section: Introductionmentioning

confidence: 99%

Burden-driven feedback control of gene expression

et al. 2018

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Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

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“…Generally, heterologous expression could represent a substantial metabolic burden for the host (37), and several measures, such as antibiotic-free plasmid maintenance and tuning of expression levels, have been proposed to mitigate this effect (38). However, as both engineered strains possess identical expression vectors with similar insert lengths (3,141 and 3,084 bp for the RCM Bmas and RCM Ktus variants, respectively) and were incubated under the same conditions, the remarkably low growth and product yields of the RCM Ktus culture are somewhat surprising.…”

Section: Discussionmentioning

confidence: 99%

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

Rohde

Tischer

Harms

et al. 2017

Appl Environ Microbiol

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The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B 12 -dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564 -4572, 2015, https://doi.org/10.1128/ AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter Ϫ1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g Ϫ1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1.IMPORTANCE In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C 4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C 1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.KEYWORDS acyl-CoA mutase, bulk chemicals, fed-batch bioreactor, overflow metabolism, polyhydroxybutyrate

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Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

Cited by 38 publications

References 27 publications

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

Burden-driven feedback control of gene expression

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

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