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Stabilized biological production by product addiction

ABSTRACT: Stabilized biological production by product addiction

PROVIDER: PRJEB24416 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR2247250_1.fastq.gz	Fastqsanger.gz
	ERR2247250_2.fastq.gz	Fastqsanger.gz
	ERR2247260_1.fastq.gz	Fastqsanger.gz
	ERR2247260_2.fastq.gz	Fastqsanger.gz
	ERR2247251_1.fastq.gz	Fastqsanger.gz

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Project description:Photosynthetic microbes can produce the clean-burning fuel hydrogen using one of natureâ??s most plentiful resources, sunlight 1,2. Anoxygenic photosynthetic bacteria generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and consumes nitrogen gas, ATP and electrons 3. One bacterium, Rhodopseudomonas palustris, has a remarkable ability to obtain electrons from green plant-derived material 4,5 and to efficiently absorb both high and low intensity light energy to form ATP 6. Manipulating R. palustris or a similar organism to produce hydrogen commercially will require us to identify all its genes that contribute to hydrogen production and to understand how this process is regulated in cells. Here we describe mutant strains in which metabolism is redirected such that hydrogen production is uncoupled from nitrogen fixation. Our data indicate that three different single amino acid changes in the transcriptional regulator NifA each yielded strains that produced hydrogen even in the presence of the repressing nitrogen source ammonium and in the absence of specific inducing metabolic signals. We used the mutants to show that, in addition to nitrogenase genes, 18 genes outside of the nitrogenase gene cluster may contribute to hydrogen production. Some of these genes are likely involved in efficient ATP acquisition and in channeling electrons to nitrogenase for reduction of protons to molecular hydrogen. Our results demonstrate that photosynthetic bacteria can be genetically manipulated for sustained production of pure hydrogen in a variety of cultivation conditions in the absence of oxygen, nitrogen or other gases as long as light and an electron donor are supplied. Transcriptome profile of wild type (CGA009) growing photosynthetically in the presence of amonium an acetate was compare with that of 4 different mutants (CGA570, CGA571, CGA572 and CGA574). We did 2 biological replicates per strain.

Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work. Fed batch cultures were carried out in Sartorius BIOSTAT B plus 5liter bioreactor. The data acquisition was done with the MFCS Shell software (version) provided with the bioreactor. The Sartorius BIOSTAT B plus had proportional integral derivative-based control loops for temperature, pH, antifoam, and dissolved-oxygen (DO) regulation. The culture was grown in a batch mode till 10-12 OD (µ=0.5) and then the feed was attached. In the next hour the culture was induced for product formation with 1M IPTG. In case of Q culture quiescence was induced with 0.5M Indole just 5min after induction with IPTG. Post induction every hour samples were collected and frozen with liquid nitrogen and stored at -80ºC for further analytical use. Total RNA extraction, RNA quality control (Agilent BioAnalyser), total RNA labeling, microarray hybridization and scanning were performed according to the Affymetrix GeneChip Expression analysis .

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Stabilized biological production by product addiction

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