Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

BACKGROUND: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. RESULTS: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%. CONCLUSIONS: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Microbial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), from lab to the shelf: A review.

Polyhydroxyalkanoates (PHAs) are natural biopolyesters produced by microorganisms that represent one of the most promising candidates for the replacement of conventional plastics due to their complete biodegradability and advantageous material properties which can be modulated by varying their monomer composition. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has received particular research attention because it can be synthesized based on the same microbial platform developed for poly(3-hydroxybutyrate) [P(3HB)] without much modification, with as high productivity as P(3HB). It also offers more useful mechanical and thermal properties than P(3HB), which broaden its application as a biocompatible and biodegradable polyester. However, a significant commercial disadvantage of P(3HB-co-3HV) is its rather high production cost, thus many studies have investigated the economical synthesis of P(3HB-co-3HV) from structurally related and unrelated carbon sources in both wild-type and recombinant microbial strains. A large number of metabolic engineering strategies have also been proposed to tune the monomer composition of P(3HB-co-3HV) and thus its material properties. In this review, recent metabolic engineering strategies designed for enhanced production of P(3HB-co-3HV) are discussed, along with their current status, limitations, and future perspectives.

In vivo study of newly developed albumin-conjugated urate oxidase for gout treatment.

BACKGROUND: Exogenously providing engineered Uox with enhanced half-life is one of the important urate-lowering treatments for gout. The potential of PAT101, a recombinant human albumin (rHA)-conjugated variant, was evaluated and compared as a novel gout treatment through various in vivo studies with PAT101 and competing drugs. METHODS: PAT101 was produced by site-specific conjugation of rHA and Aspergillus flavus Uox (AfUox-rHA) through clickable non-natural amino acid (frTet) and Inverse electron demand Diels-Alder (IEDDA) reaction. In vivo pharmacokinetics, efficacy tests and in vitro immunogenetic assay were performed after single or multiple doses of PAT101 and its competitors in BALB/c mice, transgenic (TG) mice, Sprague-Dawley (SD) rats, and non-human primate (NHP). RESULTS: The half-life of PAT101 in single-dose treated TG mice was more than doubled compared to pegloticase. In SD rats with 4 weeks of repeated administration of rasburicase, only 24% of Uox activity remained, whereas in PAT101, it was maintained by 86%. In the Uox KO model, the survival rate of PAT101 was comparable to that of pegloticase. In addition, human PBMC-based CD4+/CD8+ T-cell activation analysis demonstrated that PAT101 has a lower immune response compared to the original drug, rasburicase. CONCLUSION: All results suggest that this rHA-conjugated AfUox, PAT101, can be provided as a reliable source of Uox for gout treatment.

Chemoautotroph Cupriavidus necator as a potential game-changer for global warming and plastic waste problem: A review.

Cupriavidus necator, a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy.

Recent Advances in Sustainable Plastic Upcycling and Biopolymers.

Advances in scientific technology in the early twentieth century have facilitated the development of synthetic plastics that are lightweight, rigid, and can be easily molded into a desirable shape without changing their material properties. Thus, plastics become ubiquitous and indispensable materials that are used in various manufacturing sectors, including clothing, automotive, medical, and electronic industries. However, strong physical durability and chemical stability of synthetic plastics, most of which are produced from fossil fuels, hinder their complete degradation when they are improperly discarded after use. In addition, accumulated plastic wastes without degradation have caused severe environmental problems, such as microplastics pollution and plastic islands. Thus, the usage and production of plastics is not free from environmental pollution or resource depletion. In order to lessen the impact of climate change and reduce plastic pollution, it is necessary to understand and address the current plastic life cycles. In this review, "sustainable biopolymers" are suggested as a promising solution to the current plastic crisis. The desired properties of sustainable biopolymers and bio-based and bio/chemical hybrid technologies for the development of sustainable biopolymers are mainly discussed.

Biosynthesis of polyhydroxyalkanoates from sucrose by metabolically engineered Escherichia coli strains.

Sucrose utilization has been established in Escherichia coli strains by expression of Mannheimia succiniciproducens ß-fructofuranosidase (SacC), which hydrolyzes sucrose into glucose and fructose. Recombinant E. coli strains that can utilize sucrose were examined for their abilities to produce poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from sucrose. When recombinant E. coli strains expressing Ralstonia eutropha PhaCAB and SacC were cultured in MR medium containing 20 g/L of sucrose, all recombinant E. coli strains could produce P(3HB) from sucrose. Also, recombinant E. coli strains expressing Pseudomonas sp. MBEL 6-19 PhaC1437, Clostridium propionicum Pct540, R. eutropha PhaAB enzymes along with SacC could produce P(3HB-co-LA) from sucrose. Among the examined E. coli strains, recombinant E. coli XL1-Blue produced the highest contents of P(3HB) (53.60 ± 2.55 wt%) and P(3HB-co-LA) (29.44 ± 0.39 wt%). In the batch fermentations, recombinant E. coli XL1-Blue strains completely consumed 20 g/L of sucrose as the sole carbon source and supported the production of 3.76 g/L of P(3HB) and 1.82 g/L of P(3HB-co-LA) with 38.21 wt% P(3HB) and 20.88 wt% P(3HB-co-LA) contents, respectively. Recombinant E. coli strains developed in this study can be used to establish a cost-efficient biorefinery for the production of polyhydroxyalkanoates (PHAs) from sucrose, which is an abundant and inexpensive carbon source.

Enhanced production of poly­3­hydroxybutyrate (PHB) by expression of response regulator DR1558 in recombinant Escherichia coli.

During microbial production of target product, accumulation of by-products and target product itself may be toxic to host strain. Thus, development of abiotic stress tolerant strains are essential to achieve high productivity of target product with sustained metabolism. Expression of DR1558 from Deinococcus radiodurans, a response regulator in two-component signal transduction system, was reported to increase the tolerance against oxidative stress in Escherichia coli. In this study, the effect of overexpression of DR1558 was examined on poly­3­hydroxybutyrate (PHB) production in recombinant E. coli expressing Ralstonia eutropha PHB biosynthesis genes. It was found that dr1558 overexpressing E. coli produced 5.31 g PHB/L and 9.24 g dry cell weight/L, while control strain produced 1.52 g PHB/L and 4.47 g dry cell weight/L in 48 h shake-flask cultivation. Transcriptional analysis of E. coli suggested that DR1558 could improve the expression efficiency of the genes related to central carbon metabolism and threonine bypass pathway in PHB producing E. coli. When thrABC genes were overexpressed, PHB content was increased in recombinant E. coli, which suggests that stress-tolerant genes from extremophiles should be useful in the development of engineered strains for the production of bio-based products.