A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions

Microbial autotrophic biorefineries: Perspectives for biopolymer production

The use of autotrophic microorganisms to fabricate biochemical products has attracted much attention in both academia and industry. Unlike heterotrophic microorganisms that require carbohydrates and amino acids for growth, autotrophic microorganisms have evolved to utilize either light (photoautotrophs) or chemical compounds (chemolithotrophs) to fix carbon dioxide (CO₂) and drive metabolic processes. Several biotechnological approaches, including synthetic biology and metabolic engineering, have been proposed to harness autotrophic microorganisms as a sustainable/green production platform for commercially essential products such as biofuels, commodity chemicals, and biopolymers. Here, we review the recent advances in natural autotrophic microorganisms (photoautotrophic and chemoautotrophic), focusing on the biopolymer production. We present current state-of-the-art technologies to engineer autotrophic microbial cell factories for efficient biopolymer production

Acetate-Inducing Metabolic States Enhance Polyhydroxyalkanoate Production in Marine Purple Non-sulfur Bacteria Under Aerobic Conditions

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that a variety of microorganisms accumulate as carbon and energy storage molecules under starvation conditions in the presence of excess carbon. Anoxygenic photosynthetic bacteria exhibit a variety of growth styles and high PHA production activity. Here, we characterized PHA production by four marine purple non-sulfur bacteria strains (Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakhapatnamense) under different growth conditions. Unlike the well-studied PHA-producing bacteria, nutrient limitation is not appropriate for PHA production in marine purple non-sulfur bacteria. We found that marine purple non-sulfur bacteria did not accumulate PHA under aerobic conditions in the presence of malate and pyruvate. Interestingly, PHA accumulation was observed upon the addition of acetate under aerobic conditions but was not observed upon the addition of reductants, suggesting that an acetate-dependent pathway is involved in PHA accumulation. Gene expression analysis revealed that the expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions and increased with the addition of acetate, indicating that TCA cycle activity is involved in PHA production under aerobic conditions. We also found that expression of PdhRrs, which belongs to the GntR family of transcription regulators, in Rhodovulum sulfidophilum was upregulated upon the addition of acetate. Taken together, the results show that the changes in the metabolic state upon the addition of acetate, possibly regulated by PdhR, are important for PHA production under aerobic conditions in marine purple non-sulfur bacteria

Engineered Nanogel Particles Enhance the Photoautotrophic Biosynthesis of Polyhydroxyalkanoate in Marine Photosynthetic Bacteria

Improving polyhydroxyalkanoate (PHA, a biodegradable plastic) production under photoautotrophic cultivation is challenging for sustainable bioproduction. In this study, we demonstrated the use of engineered nanogel particles to enhance PHA accumulation in the marine photosynthetic bacterium Rhodovulum sulfidophilum under photoautotrophic culture. We screened the effect of 13 engineered nanogel particles on the cell growth and PHA accumulation of R. sulfidophilum. The addition of anionic nanogel particles significantly enhanced PHA accumulation in R. sulfidophilum up to 157-fold compared to that without nanogel particles. By performing ¹³C tracer experiments and gas chromatography–mass spectrometry analysis, we confirmed that HCO₃⁻ was assimilated throughout the central carbon metabolism and that the accumulated PHA was indeed incorporated from HCO₃⁻. Our results indicate successful PHA production with the supplementation of engineered nanogel particles under photoautotrophic cultivation in R. sulfidophilum. Furthermore, the strategy of using engineered nanoparticles demonstrated in this study may be applicable to other microbial cell factories to produce other commodity metabolites

Engineered Mutants of a Marine Photosynthetic Purple Nonsulfur Bacterium with Increased Volumetric Productivity of Polyhydroxyalkanoate Bioplastics

Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer

Optimal iron concentrations for growth-associated polyhydroxyalkanoate biosynthesis in the marine photosynthetic purple bacterium Rhodovulum sulfidophilum under photoheterotrophic condition.

Polyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study, Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production in R. sulfidophilum was growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 μM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA volumetric productivity during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth in R. sulfidophilum and also enhances PHA utilization

Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites

A marine photosynthetic microbial cell factory as a platform for spider silk production

光合成細菌を用いてクモ糸を作ることに成功 --天然資源を利用した物質生産のモデル微生物--. 京都大学プレスリリース. 2020-07-13.Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks