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A proteomic study of bacterial growth arrest in a synthetic biology application

ABSTRACT: Scaling up the functioning of synthetic circuits from microplates to bioreactors is far from trivial to achieve. We here test the scalability performance of a previously developed growth switch for increasing product yields in bacteria, based on external control of RNA polymerase expression. We show that, in liter-scale bioreactors operating in fed-batch mode, growth-arrested Escherichia coli cells are able to convert glucose to glycerol at an increased yield. A multi-omics quantification of the physiology of the cells shows that apart from acetate production, few metabolic side-effects occur, while a number of specific responses to growth slow-down and growth arrest are launched on the transcriptional level. These responses include the downregulation of genes involved in growth-associated processes, such as amino acid and nucleotide metabolism and translation, and the upregulation of heat shock genes. Interestingly, these transcriptional responses are buffered on the proteomic level, probably due to the strong decrease of the total mRNA concentration after the diminution of transcriptional activity and the absence of growth dilution of proteins. This transforms the growth-arrested cells in “bags of proteins“ with a functioning metabolism. More generally, the analysis shows that physiological characterization of bacterial cells hosting a synthetic circuit may reveal complex patterns of adaptation on different time-scales, dynamically interacting with the bioreactor environment.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Yohann Couté

LAB HEAD: Virginie Brun

PROVIDER: PXD024231 | Pride | 2021-11-25

REPOSITORIES: Pride

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Project description:Saccharomyces cerevisiae is currently widely used as a model to study chronological aging of metazoan cells. Chronological aging is typically studied in aerobic stationary phase (SP) cultures, i.e. the final stage of batch cultures in which growth is arrested due to exogenous carbon source exhaustion. Survival of yeast cells in SP defines their chronological lifespan (CLS). S. cerevisiae SP cultures have strongly contributed to the understanding of cellular mechanisms involved in aging and indicated a key role for oxygen. Oxygen is the natural starting point for reactive oxygen species (ROS) that may both have malignant and beneficial effects on aging. In addition, oxygen allows yeast to grow on ethanol and organic acids formed during the initial respiro-fermentative growth phase on glucose. This post-diauxic phase is hallmarked by reduced growth rates, increased expression of genes involved in SP survival, and increased stress resistance. To date, the role of oxygen and respiration in aging has mostly been studied using respiratory deficient mutants, and respiration repressing agents. However, genetic or chemical interventions may result in unwanted side effects that influence survival in SP. We therefore followed a different approach to evaluate the impact of oxygen availability on yeast robustness in SP, i.e. its CLS and stress resistance, by using the capability of S. cerevisiae to grow under anaerobic conditions. A thorough physiological comparison of strictly anaerobic and aerobic SP cultures revealed that the presence of oxygen during growth and aging of S. cerevisiae strongly affects its energetic status, longevity and stress tolerance in a positive way. Combining the physiological data with genome-wide expression analysis revealed that the oxygen-dependent diauxic growth phase enabled the full induction of robustness in S. cerevisiae, and points to appropriate pre-conditioning of cells as a crucial factor to survive starvation. These findings highlight the importance of exogenous energy availability in the conditions leading to growth arrest, and bring new insight on the role of oxygen in the aging of eukaryotes. The goal of the present study is to evaluate the impact of oxygen availability on yeast longevity. More specifically, the questions addressed are whether the presence of a â??conditioningâ?? post-diauxic phase and the ability to utilize efficiently reserves, characteristics of aerobicity, affects yeast survival during stationary phase. To this end, S. cerevisiae was cultivated in well controlled bioreactors under the presence or absence of oxygen. S. cerevisiaeâ??s response to oxygen availability was monitored by an in-depth physiological analysis combined with genome wide expression analysis.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. Fermentations were carried out in 0.5L bioreactors (Sartorius) containing 0.3L of SynH, SynH lacking osmoprotectants, SynH+LT, or SynH lacking osmoprotectants but containing lignotoxins and cultures were diluted into SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), early stationary (Stat1), and late stationary (Stat2) growth phases.

			Action	DRS
	Proline_export.mzid.gz	Mzid
	R1-Replicate1.mgf	Mgf
	R1-Replicate1.raw	Raw
	R1-Replicate2.mgf	Mgf
	R1-Replicate2.raw	Raw

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A proteomic study of bacterial growth arrest in a synthetic biology application

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