Quantum modeling simulates nutrient effect of bioplastic polyhydroxyalkanoate (PHA) production in Pseudomonas putida.

Polyhydroxyalkanoates (PHAs) could be used to make sustainable, biodegradable plastics. However, the precise and accurate mechanistic modeling of PHA biosynthesis, especially medium-chain-length PHA (mcl-PHA), for yield improvement remains a challenge to biology. PHA biosynthesis is typically triggered by nitrogen limitation and tends to peak at an optimal carbon-to-nitrogen (C/N) ratio. Specifically, simulation of the underlying dynamic regulation mechanisms for PHA bioprocess is a bottleneck owing to surfeit model complexity and current modeling philosophies for uncertainty. To address this issue, we proposed a quantum-like decision-making model to encode gene expression and regulation events as hidden layers by the general transformation of a density matrix, which uses the interference of probability amplitudes to provide an empirical-level description for PHA biosynthesis. We implemented our framework modeling the biosynthesis of mcl-PHA in Pseudomonas putida with respect to external C/N ratios, showing its optimization production at maximum PHA production of 13.81% cell dry mass (CDM) at the C/N ratio of 40:1. The results also suggest the degree of P. putida's preference in channeling carbon towards PHA production as part of the bacterium's adaptative behavior to nutrient stress using quantum formalism. Generic parameters (kD, kN and theta θ) obtained based on such quantum formulation, representing P. putida's PHA biosynthesis with respect to external C/N ratios, was discussed. This work offers a new perspective on the use of quantum theory for PHA production, demonstrating its application potential for other bioprocesses.

Effect of mutation of phaC on carbon supply, extracellular polysaccharide production, and pathogenicity of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice. Polyhydroxyalkanoates (PHAs) consitute a diverse group of biopolyesters synthesized by bacteria under nutrient-limited conditions. The phaC gene is important for PHA polymerization. We investigated the effects of phaC gene mutagensis in Xoo strain PXO99A. The phaC gene knock-out mutant exhibited reduced swarming ability relative to that of the wild-type. Under conditions where glucose was the sole sugar source, extracellular polysaccharide (EPS) production by ΔphaC declined by 44.8%. ΔphaC showed weak hypersensitive response (HR) induction in the leaves of non-host Nicotiana tabacum, concomitant with downregulation of hpa1 gene expression. When inoculated in rice leaves by the leaf-clipping method, ΔphaC displayed reduced virulence in terms of lesion length compared with the wild-type strain. The complemented strain showed no significant difference from the wild-type strain, suggesting that the deletion of phaC in Xoo induces significant alterations in various physiological and biological processes. These include bacterial swarming ability, EPS production, transcription of hrp genes, and glucose metabolism. These changes are intricately linked to the energy utilization and virulence of Xoo during plant infection. These findings revealed involvement of phaC in Xoo is in the maintaining carbon metabolism by functioning in the PHA metabolic pathway.

The phototrophic purple non-sulfur bacteria Rhodomicrobium spp. are novel chassis for bioplastic production.

Petroleum-based plastics levy significant environmental and economic costs that can be alleviated with sustainably sourced, biodegradable, and bio-based polymers such as polyhydroxyalkanoates (PHAs). However, industrial-scale production of PHAs faces barriers stemming from insufficient product yields and high costs. To address these challenges, we must look beyond the current suite of microbes for PHA production and investigate non-model organisms with versatile metabolisms. In that vein, we assessed PHA production by the photosynthetic purple non-sulfur bacteria (PNSB) Rhodomicrobium vannielii and Rhodomicrobium udaipurense. We show that both species accumulate PHA across photo-heterotrophic, photo-hydrogenotrophic, photo-ferrotrophic, and photo-electrotrophic growth conditions, with either ammonium chloride (NH4Cl) or dinitrogen gas (N2) as nitrogen sources. Our data indicate that nitrogen source plays a significant role in dictating PHA synthesis, with N2 fixation promoting PHA production during photoheterotrophy and photoelectrotrophy but inhibiting production during photohydrogenotrophy and photoferrotrophy. We observed the highest PHA titres (up to 44.08 mg/L, or 43.61% cell dry weight) when cells were grown photoheterotrophically on sodium butyrate with N2, while production was at its lowest during photoelectrotrophy (as low as 0.04 mg/L, or 0.16% cell dry weight). We also find that photohydrogenotrophically grown cells supplemented with NH4Cl exhibit the highest electron yields - up to 58.89% - while photoheterotrophy demonstrated the lowest (0.27%-1.39%). Finally, we highlight superior electron conversion and PHA production compared to a related PNSB, Rhodopseudomonas palustris TIE-1. This study illustrates the value of studying non-model organisms like Rhodomicrobium for sustainable PHA production and indicates future directions for exploring PNSB metabolisms.

Isolation and characterization of a Halomonas species for non-axenic growth-associated production of bio-polyesters from sustainable feedstocks.

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01." Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain's exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE: The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

Copolymers as a turning point for large scale polyhydroxyalkanoates applications.

Traditional plastics reshaped the society thanks to their brilliant properties and cut-price manufacturing costs. However, their protracted durability and limited recycling threaten the environment. Worthy alternatives seem to be polyhydroxyalkanoates, compostable biopolymers produced by several microbes. The most common 3-hydroxybutyrate homopolymer has limited applications calling for copolymers biosynthesis to enhance material properties. As a growing number of researches assess the discovery of novel comonomers, great endeavors are dedicated as well to copolymers production scale-up, where the choice of the microbial carbon source significantly affects the overall economic feasibility. Diving into novel metabolic pathways, engineered strains, and cutting-edge bioprocess strategies, this review aims to survey up-to-date publications about copolymers production, focusing primarily on precursors origins. Specifically, in the core of the review, copolymers precursors have been divided into three categories based on their economic value: the costliest structurally related ones, the structurally unrelated ones, and finally various low-cost waste streams. The combination of cheap biomasses, efficient pretreatment strategies, and robust microorganisms paths the way towards the development of versatile and circular polymers. Conceived to researchers and industries interested in tackling polyhydroxyalkanoates production, this review explores an angle often underestimated yet of prime importance: if PHAs copolymers offer advanced properties and sustainable end-of-life, the feedstock choice for their upstream becomes a major factor in the development of plastic substitutes.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

The metabolic pathways of carbon assimilation and polyhydroxyalkanoate production by Rhodospirillum rubrum in response to different atmospheric fermentation.

The purple nonsulfur bacteria, Rhodospirillum rubrum, is recognized as a potential strain for PHAs bioindustrial processes since they can assimilate a broad range of carbon sources, such as syngas, to allow reduction of the production costs. In this study, we comparatively analyzed the biomass and PHA formation behaviors of R. rubrum under 100% CO and 50% CO gas atmosphere and found that pure CO promoted the PHA synthesis (PHA content up to 23.3% of the CDW). Hydrogen addition facilitated the uptake and utilization rates of CO and elevated 3-HV monomers content (molar proportion of 3-HV up to 9.2% in the presence of 50% H2). To elucidate the genetic events culminating in the CO assimilation process, we performed whole transcriptome analysis of R. rubrum grown under 100% CO or 50% CO using RNA sequencing. Transcriptomic analysis indicated different CO2 assimilation strategy was triggered by the presence of H2, where the CBB played a minor role. An increase in BCAA biosynthesis related gene abundance was observed under 50% CO condition. Furthermore, we detected the α-ketoglutarate (αKG) synthase, converting fumarate to αKG linked to the αKG-derived amino acids synthesis, and series of threonine-dependent isoleucine synthesis enzymes were significantly induced. Collectively, our results suggested that those amino acid synthesis pathways represented a key way for carbon assimilation and redox potential maintenance by R. rubrum growth under syngas condition, which could partly replace the PHA production and affect its monomer composition in copolymers. Finally, a fed-batch fermentation of the R. rubrum in a 3-l bioreactor was carried out and proved H2 addition indeed increased the PHA accumulation rate, yielding 20% ww-1 PHA production within six days.

Enhancing Pseudomonas cell growth for the production of medium-chain-length polyhydroxyalkanoates from Antarctic krill shell waste.

Antarctic krill shell waste (AKSW), a byproduct of Antarctic krill processing, has substantial quantity but low utilization. Utilizing microbial-based cell factories, with Pseudomonas putida as a promising candidate, offers an ecofriendly and sustainable approach to producing valuable bioproducts from renewable sources. However, the high fluoride content in AKSW impedes the cell growth of P. putida. This study aims to investigate the transcriptional response of P. putida to fluoride stress from AKSW and subsequently conduct genetic modification of the strain based on insights gained from transcriptomic analysis. Notably, the engineered strain KT+16840+03100 exhibited a remarkable 33.7-fold increase in cell growth, capable of fermenting AKSW for medium-chain-length-polyhydroxyalkanoates (mcl-PHA) biosynthesis, achieving a 40.3-fold increase in mcl-PHA yield compared to the control strain. This research advances our understanding of how P. putida responds to fluoride stress from AKSW and provides engineered strains that serve as excellent platforms for producing mcl-PHA through AKSW.

ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction.

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

Boosting hydrogen production in Rhodospirillum rubrum by syngas-driven photoheterotrophic adaptive evolution.

Rhodospirillum rubrum is a photosynthetic purple non-sulphur bacterium with great potential to be used for complex waste valorisation in biotechnological applications due to its metabolic versatility. This study investigates the production of hydrogen (H2) and polyhydroxyalkanoates (PHA) by R. rubrum from syngas under photoheterotrophic conditions. An adaptive laboratory evolution strategy (ALE) has been carried out to improve the yield of the process. After 200 generations, two evolved strains were selected that showed reduced lag phase and enhanced poly-3-hydroxybutyrate (PHB) and H2 synthesis compared to the parental strain. Genomic analysis of the photo-adapted (PA) variants showed four genes with single point mutations, including the photosynthesis gene expression regulator PpsR. The proteome of the variants suggested that the adapted variants overproduced H2 due to a more efficient CO oxidation through the CO-dehydrogenase enzyme complex and confirmed that energy acquisition was enhanced through overexpression of the photosynthetic system and metal cofactors essential for pigment biosynthesis.

Natural Polyhydroxyalkanoates-An Overview of Bacterial Production Methods.

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers that microorganisms use for energy and carbon storage. They are mechanically similar to petrochemical plastics when chemically extracted, but are completely biodegradable. While they have potential as a replacement for petrochemical plastics, their high production cost using traditional carbon sources remains a significant challenge. One potential solution is to modify heterotrophic PHA-producing strains to utilize alternative carbon sources. An alternative approach is to utilize methylotrophic or autotrophic strains. This article provides an overview of bacterial strains employed for PHA production, with a particular focus on those exhibiting the highest PHA content in dry cell mass. The strains are organized according to their carbon source utilization, encompassing autotrophy (utilizing CO2, CO) and methylotrophy (utilizing reduced single-carbon substrates) to heterotrophy (utilizing more traditional and alternative substrates).

Synergistic effects of peracetic acid and free ammonia pretreatment on anaerobic fermentation of waste activated sludge to promote short-chain fatty acid production for polyhydroxyalkanoate biosynthesis: Mechanisms and optimization.

Peracetic acid (PAA) combined with free ammonia (FA) pretreatment can be utilized to promote anaerobic fermentation (AF) of waste activated sludge (WAS) to produce short-chain fatty acids (SCFAs), and the resulting SCFAs are desirable carbon sources (C-sources) for polyhydroxyalkanoate (PHA) biosynthesis. This work aimed to determine the optimum conditions for PAA + FA pretreatment of sludge AF and the feasibility of using anaerobic fermentation liquor (AFL) for PHA production. To reveal the mechanisms of integrated pretreatment, the impacts of PAA + FA pretreatment on different stages of sludge AF and changes in the microbial community structure were explored. The experimental results showed that the maximum SCFA yield reached 491.35 ± 6.02 mg COD/g VSS on day 5 after pretreatment with 0.1 g PAA/g VSS +70 mg FA/L, which was significantly greater than that resulting from PAA or FA pretreatment alone. The mechanism analysis showed that PAA + FA pretreatment promoted sludge solubilization but strongly inhibited methanogenesis. According to the analysis of the microbial community, PAA + FA pretreatment changed the microbial community structure and promoted the enrichment of bacteria related to hydrolysis and acidification, and Proteiniclasticum, Macellibacteroides and Petrimonas became the dominant hydrolytic and acidifying bacteria. Finally, after alkali treatment, the AFL was utilized for batch-mode PHA production, and a maximum PHA yield of 55.05 wt% was achieved after five operation periods.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Corroborative studies on chain packing characteristics of biological medium-chain-length poly-3-hydroxyalkanoates with different monomeric composition.

Medium-chain-length poly-3-hydroxyalkanoates (mcl-PHAs) with varied monomeric compositions were biosynthesized by producer bacteria fed with different fatty acids as carbon source. Octanoic-, lauric-, stearic-, and oleic acids were used to produce four types of mcl-PHAs viz. PHA-OC, PHA-LA, PHA-ST, and PHA-OL, respectively. The mcl-PHAs as film-casted preparations exhibit distinct traits e.g., PHA-OC and PHA-ST films are less flexible than PHA-LA while PHA-OL is a sticky, glue-like material; PHA-ST is opaque whereas PHA-OC, PHA-LA, and PHA-OL displayed transparent layers. The observation is attributed to polymer chain packing and side chain crystallization. A structure-property investigation of these biopolymers was carried out employing different spectroscopic and microscopic analyses in addition to thermal analyses. Comparative analyses of the results were applied in the interpretation and discussion of structure-property relationship.

Food wastes for bioproduct production and potential strategies for high feedstock variability.

Unavoidable food wastes could be an important feedstock for industrial biotechnology, while their valorization could provide added value for the food processor. However, despite their abundance and low costs, the heterogeneous/mixed nature of these food wastes produced by food processors and consumers leads to a high degree of variability in carbon and nitrogen content, as well as specific substrates, in food waste hydrolysate. This has limited their use for bioproduct synthesis. These wastes are often instead used in anaerobic digestion and mixed microbial culture, creating a significant knowledge gap in their use for higher value biochemical production via pure and single microbial culture. To directly investigate this knowledge gap, various waste streams produced by a single food processor were enzymatically hydrolyzed and characterized, and the degree of variability with regard to substrates, carbon, and nitrogen was quantified. The impact of hydrolysate variability on the viability and performance of polyhydroxyalkanoates biopolymers production using bacteria (Cupriavidus necator) and archaea (Haloferax mediterranei) as well as sophorolipids biosurfactants production with the yeast (Starmerella bombicola) was then elucidated at laboratory-scale. After which, strategies implemented during this experimental proof-of-concept study, and beyond, for improved industrial-scale valorization which addresses the high variability of food waste hydrolysate were discussed in-depth, including media standardization and high non-selective microbial organisms growth-associated product synthesis. The insights provided would be beneficial for future endeavors aiming to utilize food wastes as feedstocks for industrial biotechnology.

Controlled production of a polyhydroxyalkanoate (PHA) tetramer containing different mole fraction of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3 HV), 4 HV and 5 HV units by engineered Cupriavidus necator.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB-4 harboring the gene phaCBP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV.

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

A Review of Current Achievements and Recent Challenges in Bacterial Medium-Chain-Length Polyhydroxyalkanoates: Production and Potential Applications.

Using petroleum-derived plastics has contributed significantly to environmental issues, such as greenhouse gas emissions and the accumulation of plastic waste in ecosystems. Researchers have focused on developing ecofriendly polymers as alternatives to traditional plastics to address these concerns. This review provides a comprehensive overview of medium-chain-length polyhydroxyalkanoates (mcl-PHAs), biodegradable biopolymers produced by microorganisms that show promise in replacing conventional plastics. The review discusses the classification, properties, and potential substrates of less studied mcl-PHAs, highlighting their greater ductility and flexibility compared to poly(3-hydroxybutyrate), a well-known but brittle PHA. The authors summarize existing research to emphasize the potential applications of mcl-PHAs in biomedicine, packaging, biocomposites, water treatment, and energy. Future research should focus on improving production techniques, ensuring economic viability, and addressing challenges associated with industrial implementation. Investigating the biodegradability, stability, mechanical properties, durability, and cost-effectiveness of mcl-PHA-based products compared to petroleum-based counterparts is crucial. The future of mcl-PHAs looks promising, with continued research expected to optimize production techniques, enhance material properties, and expand applications. Interdisciplinary collaborations among microbiologists, engineers, chemists, and materials scientists will drive progress in this field. In conclusion, this review serves as a valuable resource to understand mcl-PHAs as sustainable alternatives to conventional plastics. However, further research is needed to optimize production methods, evaluate long-term ecological impacts, and assess the feasibility and viability in various industries.

Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.