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Analysis of the Involvement of the Isoleucine Biosynthesis Pathway in Photoheterotrophic Metabolism of Rhodospirillum rubrum.

ABSTRACT: Purple non-sulfur bacteria (PNSB) are recognized as a highly versatile group of bacteria that assimilate a broad range of carbon sources. Growing heterotrophically, PNSB such as Rhodospirillum rubrum (Rs. rubrum) generate reduced equivalents that are used for biomass production. However, under photoheterotrophic conditions, more reduced electron carriers than required to produce biomass are generated. The excess of reduced equivalents still needs to be oxidized for the metabolism to optimally operate. These metabolic reactions are known as electron sinks. Most PNSB rely on the CO₂-fixing Calvin cycle and H₂ production to oxidize these reduced equivalents. In addition to these well-described electron sinks, the involvement of some pathways, such as polyhydroxyalkanoate (PHA) biosynthesis, in redox poise is still controversial and requires further studies. Among them, isoleucine biosynthesis has been recently highlighted as one of these potential pathways. Here, we explore the role of isoleucine biosynthesis in Rs. rubrum. Our results demonstrate that the isoleucine content is higher under illuminated conditions and that submitting Rs. rubrum to light stress further increases this phenomenon. Moreover, we explore the production of (p)ppGpp in Rs. rubrum and its potential link with light stress. We further demonstrate that a fully functional isoleucine biosynthesis pathway could be an important feature for the onset of Rs. rubrum growth under photoheterotrophic conditions even in the presence of an exogenous isoleucine source. Altogether, our data suggest that isoleucine biosynthesis could play a key role in redox homeostasis.

SUBMITTER: Bayon-Vicente G

PROVIDER: S-EPMC8490811 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. Rhodospirillum rubrum has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in Rs. rubrum, the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of Rs. rubrum in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance.IMPORTANCE Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. Rhodospirillum rubrum S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In Rs. rubrum, purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.

Project description:Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by Rhodospirillum rubrum has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of R. rubrum in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB-co-HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process.IMPORTANCE The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by Rhodospirillum rubrum, a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of R. rubrum in industrial processes.

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Analysis of the Involvement of the Isoleucine Biosynthesis Pathway in Photoheterotrophic Metabolism of Rhodospirillum rubrum.

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