Biology subjects

Ranaivoarisoa, T. O.

Publications and source records attributed to Ranaivoarisoa, T. O..

6 recordsLinked to original sources

bioRxiv2023

Improving bioplastic production by Rhodopseudomonas palustris TIE-1 using synthetic biology and metabolic engineering

With the increasing demand for sustainably produced renewable resources, it is important to look towards microorganisms capable of producing bioproducts such as biofuels and bioplastics. Though many systems for bioproduct production are well documented and tested in model organisms, it is essential to look beyond to non-model organisms to expand the field and take advantage of metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple, non-sulfur autotrophic, and anaerobic bacterium capable of producing bioproducts that are comparable to their petroleum-based counterparts. To induce bioplastic overproduction, genes that might have a potential role in the PHB biosynthesis such as the regulator, phaR, and phaZ known for its ability to degrade PHB granules were deleted using markerless deletion. Mutants in pathways that might compete with polyhydroxybutyrate (PHB) production such as glycogen and nitrogen fixation previously created to increase n-butanol production by TIE-1 were also tested. In addition, a phage integration system was developed to insert RuBisCO (RuBisCO form I and II genes) driven by a constitutive promoter PaphII into TIE- 1 genome. Our results show that deletion of the phaR gene of the PHB pathway increases PHB productivity when TIE-1 was grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to make glycogen or fix dinitrogen gas show an increase in PHB productivity under photoautotrophic growth conditions with hydrogen. In addition, the engineered TIE-1 overexpressing RuBisCO form I and form II produces significantly more polyhydroxybutyrate than the wild type under photoheterotrophy with butyrate and photoautotrophy with hydrogen. Inserting RuBisCO genes into TIE-1 genome is a more effective strategy than deleting competitive pathways to increase PHB production in TIE-1. The phage integration system developed for TIE-1 thus creates numerous opportunities for synthetic biology in TIE-1.

microbiology↗

bioRxiv2023

Improved Ethanol Tolerance and Production in Pyruvate Dehydrogenase Mutant of Saccharomyces cerevisiae

Ethanol, a naturally synthesized compound by Saccharomyces cerevisiae yeast through alcoholic fermentation, has previously been studied as a renewable alternative to traditional fossil fuels. However, current challenges of engineering S. cerevisiae strains for ethanol production remain: low ethanol productivity, inefficient substrate catabolism, and a buildup of toxic products to inhibitory levels. In this study, we proposed a method of metabolic rewiring via the deletion of the pda1 gene, which leads to pyruvate dehydrogenase (PDH) deficiency. The {Delta}pda1 mutant strain was created by CRISPR Cas-9 knockout using the constructed pCRCT-PDA1 plasmid. Subsequently, mutant candidates were screened by PCR and Sanger sequencing, confirming a 17 bp deletion in the pda1 gene. The wild-type and mutant strains were analyzed for growth under aerobic and anaerobic conditions in glucose and glycerol, as well as ethanol production and tolerance. The {Delta}pda1 mutant displays a ~two-fold increase in anaerobic ethanol production and an aerobic growth defect with no observed increase in ethanol production. The mutant is also hyper-tolerant to ethanol, which allows a faster buildup of products in growth media with minimal reduction in growth. This new S. cerevisiae strain deficient in PDH may provide a solution to the efficient and abundant synthesis of biofuels such as ethanol by redirecting metabolic flux and altering stress response.

microbiology↗

bioRxiv2020

Sustainable Production of the Biofuel n-Butanol by Rhodopseudomonas palustris TIE-1

Anthropogenic carbon dioxide (CO2) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO2 to fuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO2. However, oxygen generation during oxygenic photosynthesis affects biofuel production efficiency. To produce n-butanol (biofuel) from CO2, here we introduced an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieved n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produced highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produced n-butanol using CO2, solar panel-generated electricity, and light, with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

bioengineering↗

bioRxiv2020

Genetic redundancy in iron and manganese transport in the metabolically versatile bacterium Rhodopseudomonas palustris TIE-1

The purple non-sulfur bacterium Rhodopseudomonas palustris TIE-1 can produce useful biochemicals such as bioplastics and biobutanol. Production of such biochemicals requires intracellular electron availability, which is governed by the availability and the transport of essential metals such as iron (Fe). Because of the distinct chemical properties of ferrous [Fe(II)] and ferric iron [Fe(III)], different transport systems are required for their transport and storage in bacteria. Although Fe(III) transport systems are well characterized, we know much less about Fe(II) transport systems except for the FeoAB system. Iron transporters can also import manganese (Mn). Here, we study Fe and Mn transport by five putative Fe transporters in TIE-1 under metal-replete, -deplete, oxic and anoxic conditions. We observe that by overexpressing feoAB, efeU, and nramp1AB, the intracellular concentration of Fe and Mn can be enhanced in TIE-1, under oxic and anoxic conditions, respectively. The deletion of a single gene/operon does not attenuate Fe or Mn uptake in TIE-1 regardless of the growth conditions used. This indicates that genetically dissimilar yet functionally redundant Fe transporters in TIE-1 can complement each other. Relative gene expression analysis shows that feoAB and efeU are expressed during Fe and Mn depletion under both oxic and anoxic conditions. The promoters of these transporter genes contain a combination of Fur and Fnr boxes suggesting that their expression is regulated by both Fe and oxygen availability. The findings from this study will help us modulate intracellular Fe and Mn concentration, ultimately improving TIE-1s ability to produce desirable biomolecules. IMPORTANCERhodopseudomonas palustris TIE-1 is a metabolically versatile bacterium that can use various electron donors including Fe(II) and poised electrodes for photoautotrophic growth. TIE-1 can produce useful biomolecules such as biofuels and bioplastics during various growth conditions. Production of such reduced biomolecules is controlled by intracellular electron availability, which in turn is mediated by various iron-containing proteins in the cell. Several putative Fe transporters exist in TIE-1s genome. Some of these transporters can also transport Mn, part of several important cellular enzymes. Therefore, understanding the ability to transport and respond to varying levels of Fe and Mn under different conditions is important to improve TIE-1s ability to produce useful biomolecules. Our data suggest that by overexpressing Fe transporter genes via plasmid-based expression, we can increase the import of Fe and Mn in TIE-1. Future work will leverage these data to improve TIE-1 as an attractive microbial chassis and future biotechnological workhorse.

microbiology↗

bioRxiv2017

Improving microbial electrosynthesis of polyhydroxybutyrate (PHB) from CO2 by Rhodopseudomonas palustris TIE-1 using an immobilized iron complex modified cathode

Microbial electrosynthesis (MES) is a promising bioelectrochemical approach to produce biochemicals. A previous study showed that Rhodopseudomonas palustris TIE-1 can directly use poised electrodes as electron donors for photoautotrophic growth at cathodic potentials that avoid electrolytic H2 production (photoelectroautotrophy). To make TIE-1 an effective biocatalyst for MES, we need to improve its electron uptake ability and growth under photoelectroautotrophic conditions. Because TIE-1 interacts with various forms of iron while using it as a source of electrons for photoautotrophy (photoferrotrophy), we tested the ability of iron-based redox mediators to enhance direct electron uptake. Our data show that soluble iron cannot act as a redox mediator for electron uptake by TIE-1 from a cathode poised at +100mV vs. Standard Hydrogen electrode. We then tested whether an immobilized iron-based redox mediator Prussian Blue (PB) can enhance electron uptake by TIE-1. Chronoamperometry indicates that cathodic current uptake by TIE-1 increased from 1.47 {+/-} 0.04 to 5.6 {+/-} 0.09 {micro}A/cm2 (3.8 times) and the production of the bioplastic, polyhydroxybutyrate (PHB) improved from 13.5 {+/-} 0.2 g/L to 18.8 {+/-} 0.5 g/L (1.4 times) on electrodes coated with PB. Overall, our data show that immobilized PB can increase direct electron uptake by TIE-1 and enhances PHB production.

microbiology↗

bioRxiv2017

Towards sustainable bioplastic production in resource limited environments using the photoferroautotrophic and photoelectroautotrophic bacterium Rhodopseudomonas palustris TIE-1

Bioplastics are an attractive alternative to petroleum-derived plastics because of the harmful environmental effects of conventional plastics and the impending fossil fuel crisis. Polyhydroxybutyrate (PHB) is a well-known bioplastic that is produced by several microbes using organic carbon sources. Autotrophic (using carbon dioxide or CO2) PHB production is reported for only a few organisms. Sustainable PHB bioproduction using other autotrophic microbes needs to be explored. Rhodopseudomonas palustris, a metabolically versatile purple non-sulfur bacterium (PNSB) is known to produce PHBs under photoheterotrophic conditions. Rhodopseudomonas palustris strain TIE-1 demonstrates extended metabolic versatility by using electron sources such as ferrous iron and poised electrodes for photoautotrophy. Here we report the ability of TIE-1 to produce PHB under photoferroautotrophic (light - energy source, ferrous iron - electron source and CO2 - carbon source) and photoelectroautotrophic (light - energy source, poised electrodes - electron source and CO2 - carbon source) growth conditions. PHB accumulation was observed both under nitrogen (N2) fixing and non-N2 fixing conditions. For comparison, we determined PHB production under chemoheterotrophic, photoheterotrophic and photoautotrophic conditions using hydrogen as the electron donor. Photoferroautotrophic and photoelectroautotrophic PHB production was on par with that observed from organic carbon substrates such as butyrate. PHB production increased during N2 fixation under photoheterotrophic conditions but not during photoautotrophic growth. Electron microscopy confirmed that TIE-1 cells accumulate PHBs internally under the conditions that showed highest production. However, gene expression analysis suggests that PHB cycle genes are not differentially regulated despite observable changes in biopolymer production.

microbiology↗