PHB Content Research Articles

Microbial symbiotic electrobioconversion of carbon dioxide to biopolymer (poly (3-hydroxybutyrate)) via single-step microbial electrosynthesis cell

This study proposes an novel single-stage microbial electrosynthesis system (MES) that bioelectrochemically converts CO2 into poly(3-hydroxybutyrate) (PHB). This innovative approach utilizes a symbiotic co-culture of electroactive acetogenic bacteria and PHB-accumulating bacteria within a single reactor, bypassing the need for separate acetate production and extraction. Systematic optimization of key operational parameters, including applied voltage, carbon source, and cathode material, was conducted to maximize PHB production. Results demonstrate that using a voltage of 2.5 V yielded a 7.14-fold increase in PHB content compared to open circuit conditions. Furthermore, functionalizing the cathode with a conductive and hydrophilic PEDOT: PSS polymer coating significantly enhanced the system’s performance, resulting in a 1.5-fold increase in both acetate and PHB production compared to unmodified carbon felt electrodes. The long-term stability and effectiveness of the co-culture system were validated through comprehensive microbial and metagenomic analyses. Results revealed a significant enrichment of CO2-utilizing electrotrophs within the cathode biofilm, including Acetobacterium and Clostridium. Concurrently, a 4 to 14-fold increase in the relative abundance of PHB-biosynthesizing bacteria, such as Pseudomonas, Rhodobacter and Caulobacter was observed in the planktonic phase. This study offers a promising pathway towards a circular bioeconomy by enabling the valorization of CO2 into valuable bio-based products via single-stage MES, utilizing a symbiotic co-culture.

Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes

Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9% lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5%) and purity (98.1%) were obtained with 1,3-dioxolane at 80°C and 6h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor cultures leading to the production of 78.7 gPHB/L, 56.9% (w/w) PHB content and 2.3g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.

Two-step polyhydroxybutyrate production from hydrogenic effluent by freshwater microalgae Coelastrella sp. KKU-P1 and Acutodesmus sp. KKU-P2 under mixotrophic cultivation

This study aimed to produce PHB using hydrogenic effluent discharged from the biohydrogen production process with freshwater microalgae including Coelastrella sp. KKU-P1, and Acutodesmus sp. KKU-P2. Batch experiments explored the influence of initial pH and hydrogenic effluent concentration, revealing optimal conditions at 10 % (v/v) effluent concentration and a pH of 6.5 for both KKU-P1 and KKU-P2. Subsequently, medium formulation and photoperiods were optimized to maximize biomass and PHB accumulation. The results showed that the optimal condition for PHB accumulation with KKU-P1 and KKU-P2 was nitrogen phosphorus (NP)-limited Bold's Basal Medium (BBM) under dark conditions. A two-step PHB accumulation in the upscale bioreactor was investigated under optimal conditions. The results showed that KKU-P1 achieved maximum PHB, protein, carbohydrate, and lipid contents of 4.57 %, 29.37 %, 24.76 %, and 13.21 %, respectively, whereas KKU-P2 achieved 6.35 %, 31.53 %, 16.16 %, and 4.77 %, respectively. Based on these findings, it appears that a mixotrophic approach under nutrient-limiting conditions is effective for PHB production in both KKU-P1 and KKU-P2 strains.

Methylosinus trichosporium OB3b drives composition-independent application of biogas in poly(3-hydroxybutyrate) synthesis

Biological conversion of biogas into poly(3-hydroxybutyrate) (PHB) via Type II methanotrophs holds great promise. However, a prerequisite for widespread biogas utilization is a comprehensive understanding of the effects of biogas composition on methanotrophic PHB synthesis. Herein, we explore the effects of biogas composition on the microbial growth and PHB accumulation of Methylosinus trichosporium OB3b under five different artificial biogas conditions with varying the ratio of methane (CH4) to carbon dioxide (CO2), the two main components of raw biogas. Stoichiometric and kinetic analyses revealed that increased CO2 concentrations correlated with higher maximum specific growth rates (0.021–0.029 h−1) and maximum CH4 utilization rates (0.029–0.042 mg CH4/mg TSS-h). Besides, no significant correlation was found between the CH4-to-CO2 ratio and other growth parameters. Consistent PHB contents (20.2–25.3 wt%) were achieved, irrespective of the CH4-to-CO2 ratio. Furthermore, the ability of M. trichosporium OB3b to grow and synthesize PHB (13.2 wt%) was confirmed using pilot-scale biogas. Our findings highlight the potential of biogas as a feedstock for producing valuable biodegradable polymers, thus contributing to waste-derived fuel (WDF) innovation and environmental sustainability.

A Comparison of the Effects of Continuous Illumination and Day/Night Regimes on PHB Accumulation in Synechocystis Cells.

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable polymer with properties comparable to polypropylene and therefore has the potential to replace conventional plastics. PHB is intracellularly accumulated by prokaryotic organisms. For the cells PHB functions manly as carbon and energy source, but all possible functions of PHB are still not known. Synechocystis (cyanobacteria) accumulates PHB using light as energy and CO2 as carbon source. The main trigger for PHB accumulation in cyanobacteria is nitrogen and phosphorous depletion with simultaneous surplus of carbon and energy. For the above reasons, obtaining knowledge about external factors influencing PHB accumulation is of highest interest. This study compares the effect of continuous light exposure and day/night (16/8 h) cycles on selected physiology parameters of three Synechocystis strains. We show that continuous illumination at moderate light intensities leads to an increased PHB accumulation in Synechocystis salina CCALA 192 (max. 14.2% CDW - cell dry weight) compared to day/night cycles (3.7% CDW). In addition to PHB content, glycogen and cell size increased, while cell density and cell viability decreased. The results offer new approaches for further studies to gain deeper insights into the role of PHB in cyanobacteria to obtain bioplastics in a more sustainable and environmentally friendly way.

Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

The fundamental role of pH in CH4 bioconversion into polyhydroxybutyrate in mixed methanotrophic cultures

Climate change and plastic pollution are likely the most relevant challenges for the environment in the 21st century. Developing cost-effective technologies for the bioconversion of methane (CH4) into polyhydroxyalkanoates (PHAs) could simultaneously mitigate CH4 emissions and boost the commercialization of biodegradable polymers. Despite the fact that the role of temperature, nitrogen deprivation, CH4:O2 ratio or micronutrients availability on the PHA accumulation capacity of methanotrophs has been carefully explored, there is still a need for optimization of the CH4-to-PHA bioconversion process prior to becoming a feasible platform in future biorefineries. In this study, the influence of different cultivation broth pH values (5.5, 7, 8.5 and 10) on bacterial biomass growth, CH4 bioconversion rate, PHA accumulation capacity and bacterial community structure was investigated in a stirred tank bioreactor under nitrogen deprivation conditions. Higher CH4 elimination rates were obtained at increasing pH, with a maximum value of 50.4 ± 2.7 g CH4·m−3·h−1 observed at pH 8.5. This was likely mediated by an increased ionic strength in the mineral medium, which enhanced the gas-liquid mass transfer. Interestingly, higher PHB accumulations were observed at decreasing pH, with the highest PHB contents recorded at a pH 5.5 (43.7 ± 3.4 %w·w−1). The strong selective pressure of low pH towards the growth of Type II methanotrophic bacteria could explain this finding. The genus Methylocystis increased its abundance from 34 % up to 85 and 90 % at pH 5.5 and 7, respectively. On the contrary, Methylocystis was less abundant in the community enriched at pH 8.5 (14 %). The accumulation of intracellular PHB as energy and carbon storage material allowed the maintenance of high CH4 biodegradation rates during 48 h after complete nitrogen deprivation. The results here obtained demonstrated for the first time a crucial and multifactorial role of pH on the bioconversion performance of CH4 into PHA.

Groundwater denitrification enhanced by a hydrogel immobilized iron/solid carbon source: impact on denitrification and substrate release performance.

Encapsulating a solid carbon source and zero-valent iron (ZVI) within a hydrogel can prevent direct contact with groundwater, thereby extending the lifespan of their released active substrates. It is currently unclear whether the solid carbon source and ZVI will mutually influence each other's active substrate release process and the corresponding denitrification patterns, necessitating further investigation. In this study a hydrogel encapsulating different weight ratios of micron-sized zero-valent iron (mZVI, as ZVI) and polyhydroxybutyrate (PHB, as a solid carbon source) was synthesized. The aim was to investigate the influence of PHB on the release of dissolved iron from mZVI and denitrification mechanism. Results indicated that PHB was consumed at a higher rate than mZVI, and more mZVI active sites could be exposed after PHB consumption. Meanwhile, PHB increased the porosity of the hydrogel, allowing more active sites of mZVI to be exposed and thus releasing more dissolved iron. Furthermore, PHB enhanced the rate of microbial corrosion of mZVI, which further increased the release of dissolved iron. Higher PHB content in the hydrogel reduced the oxidation of the released dissolved iron, resulting in a microbial community dominated by heterotrophic microorganisms. Conversely, lower PHB content led to significant Fe(II) oxidation and a considerable relative abundance of mixotrophic microorganisms in the microbial community. Microorganisms with iron reduction potential were also detected. This study provides theoretical support for the precise control of mixed nutrient denitrification based on hydrogel immobilization and lays the foundation for its further practical application in groundwater.

Identification and characterization of a potential strain for the production of polyhydroxyalkanoate from glycerol.

While poly (3-hydroxybutyrate) (PHB) holds promise as a bioplastic, its commercial utilization has been hampered by the high cost of raw materials. However, glycerol emerges as a viable feedstock for PHB production, offering a sustainable production approach and substantial cost reduction potential. Glycerol stands out as a promising feedstock for PHB production, offering a pathway toward sustainable manufacturing and considerable cost savings. The identification and characterization of strains capable of converting glycerol into PHB represent a pivotal strategy in advancing PHB production research. In this study, we isolated a strain, Ralstonia sp. RRA (RRA). The strain exhibits remarkable proficiency in synthesizing PHB from glycerol. With glycerol as the carbon source, RRA achieved a specific growth rate of 0.19 h-1, attaining a PHB content of approximately 50% within 30 h. Through third-generation genome and transcriptome sequencing, we elucidated the genome composition and identified a total of eight genes (glpR, glpD, glpS, glpT, glpP, glpQ, glpV, and glpK) involved in the glycerol metabolism pathway. Leveraging these findings, the strain RRA demonstrates significant promise in producing PHB from low-cost renewable carbon sources.

Construction of cascade circuits for dynamic temporal regulation and its application to PHB production

BackgroundTo maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways.ResultsWe designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening.ConclusionsThe self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.

Enrichment strategies for mixed cultures in valorisation of crude glycerol into polyhydroxyalkanoate bioplastics

This study investigated two different strategies for enrichment using crude glycerol and mixed culture. Coupled feeding allowed simultaneous supplies of glycerol and nutrients at start of feast, whereas uncoupled feeding postponed nutrient supply until start of famine. Adaptation of culture enriched by the uncoupled feeding strategy was faster with higher operational stability. This however resulted in low PHB content of 20% and yield of 0.137 mg PHB/mg glycerol in feast phase, cell growth at yield of 0.298 mgX/mg PHB in famine. With more abundance (29.2%) of PHA storing bacteria in culture enriched by the coupled feeding strategy as revealed from 16 S rRNA analysis, results showed that polymer storage was dominated in feast phase (max. PHB content of 68%, yield of 0.563 mg PHB/mg glycerol), leading to high cell growth yield of 0.745 mg X/mg PHB. PHB accumulation by the enriched culture from coupled feeding has achieved 82% PHB content with 1094 mg PHB/L.h volumetric productivity. Thus, the coupled feeding was more effective to enrich PHA storing culture for PHA production using crude glycerol via two-step process.

Isolation of new Paraburkholderia strains for polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are bioplastics that can serve as substitutes for petroleum-based plastics with the advantages of being biodegradable, biocompatible, and biobased. The microbial production of polyhydroxyalkanoates is generally conducted in the presence of sugar mixes rich in monosaccharides. In this study, molecular and cultural approaches based on forest soils enriched with hydrocarbon complexes led to the identification and isolation of microbial strains affiliated with Paraburkholderia sp. that dominated the microbial communities that are recognized among the top polyhydroxyalkanoates producers. The genome sequencing of those isolated affiliated strains showed that compared to the reference type strain of their species, they harbored more gene copies of the enzymes involved in PHB synthesis. The microbial conversion of sugar mixes for the newly isolated strains showed a higher PHB production (g/L) and content (%) than was exhibited by the reference strain type of that genus Paraburkholderia for PHB production (P. sacchari LMG 19450T).

High production of poly(3-hydroxybutyrate) in Escherichia coli using crude glycerol

Poly(3-hydroxybutyrate) (PHB) is a prominent bio-plastic and recognized as the potential replacement of petroleum-derived plastics. To make PHB cost-effective, the production scheme based on crude glycerol was developed using Escherichia coli. The heterogeneous synthesis pathway of PHB was introduced into the E. coli strain capable of efficiently utilizing glycerol. The central metabolism that links to the synthesis of acetyl-CoA and NADPH was further reprogrammed to improve the PHB production. Key genes were targeted for manipulation, involving those in glycolysis, the pentose phosphate pathway, and the tricarboxylic cycle. As a result, the engineered strain gained a 22-fold increase in the PHB titer. Finally, the fed-batch fermentation was conducted with the producer strain to give the PHB titer, content, and productivity reaching 36.3 ± 3.0 g/L, 66.5 ± 2.8%, and 1.2 ± 0.1 g/L/h, respectively. The PHB yield on crude glycerol accounts for 0.3 g/g. The result indicates that the technology platform as developed is promising for the production of bio-plastics.

Poly(3-hydroxybuyrate) production from industrial hemp waste pretreated with a chemical-free hydrothermal process

In this study, a mild two-stage hydrothermal pretreatment was employed to optimally valorize industrial hemp (Cannabis sativa sp.) fibrous waste into sugars for Poly(3-hydroxybuyrate) (PHB) production using recombinant Escherichia coli LSBJ. Biomass was pretreated using hot water at 160, 180, and 200 °C for 5 and 10 min (15% solids), followed by disk refining. The sugar yields during enzymatic hydrolysis were found to improve with increasing temperature and the yields for hot water-disk refining pretreatment (HWDM) were higher compared to only hot water pretreatment at all conditions. The maximum glucose (56 g/L) and cellulose conversion (92%) were achieved for HWDM at 200 °C for 10 min. The hydrolysate obtained was fermented at a sugar concentration of 20 g/L. The PHB inclusion and concentration of 48% and 1.8 g/L, respectively, were similar to those from pure sugars. A pH-controlled fermentation resulted in a near bi-fold increase in PHB yield (3.46 g/L).

Fermentation development using fruit waste derived mixed sugars for poly(3-hydroxybutyrate) production and property evaluation

Free sugars from fruit wastes were evaluated for the production of poly(3-hydroxybutyrate) (PHB) in Paraburkholderia sacchari fed-batch bioreactor fermentations. Different initial sugar concentration, carbon to inorganic phosphorus (C/IP) ratio, IP addition during feeding and volumetric oxygen transfer coefficient (kLa) were evaluated to promote PHB production. The highest intracellular PHB accumulation (66.6%), PHB concentration (108.3 g/L), productivity (3.28 g/(L·h)) and yield (0.33 g/g) were achieved at 40 g/L initial sugars, C/IP 26.5, 202.6 h−1kLa value and 20% IP supplementation in the feeding solution. The effect of different microbial mass harvesting time on PHB properties showed no influence in weight average molecular weight and thermal properties. The harvest time influenced the tensile strength that was reduced from 28.7 MPa at 22 h to 13.3 MPa at 36 h. The elongation at break and Young’s modulus were in the range 3.6–14.8% and 830–2000 MPa, respectively.

Mechanical and Structural Properties of Polyhydroxybutyrate as Additive in Blend Material in Additive Manufacturing for Medical Applications.

Today, additive manufacturing (AM) is considered one of the vital tenets of the industry 4.0 revolution due to its high productivity, decentralized production and rapid prototyping. This work aims to study the mechanical and structural properties of polyhydroxybutyrate as an additive in blend materials and its potential in medical applications. PHB/PUA blend resins were formulated with 0 wt.%, 6 wt.%, 12 wt.% and 18 wt.% of PHB concentration. Stereolithography or an SLA 3D printing technique were used to evaluate the printability of the PHB/PUA blend resins. Additionally, from FESEM analysis, a change was observed in PUA's microstructure, with an additional number of voids spotted. Furthermore, from XRD analysis, as PHB concentration increased, the crystallinity index (CI) also increased. This indicates the brittleness properties of the materials, which correlated to the weak performance of the tensile and impact properties. Next, the effect of PHB loading concentration within PHB/PUA blends and aging duration towards the mechanical performance of tensile and impact properties was also studied by using analysis of variance (ANOVA) with a two-way method. Finally, 12 wt.% of PHB/PUA was selected to 3D print the finger splint due to its characteristics, which are compatible to be used in finger bone fracture recovery.

Engineering Escherichia coli for Poly-β-hydroxybutyrate Production from Methanol.

The naturally occurring one-carbon assimilation pathways for the production of acetyl-CoA and its derivatives often have low product yields because of carbon loss as CO2. We constructed a methanol assimilation pathway to produce poly-3-hydroxybutyrate (P3HB) using the MCC pathway, which included the ribulose monophosphate (RuMP) pathway for methanol assimilation and non-oxidative glycolysis (NOG) for acetyl-CoA (precursor for PHB synthesis) production. The theoretical product carbon yield of the new pathway is 100%, hence no carbon loss. We constructed this pathway in E. coli JM109 by introducing methanol dehydrogenase (Mdh), a fused Hps-phi (hexulose-6-phosphate synthase and 3-phospho-6-hexuloisomerase), phosphoketolase, and the genes for PHB synthesis. We also knocked out the frmA gene (encoding formaldehyde dehydrogenase) to prevent the dehydrogenation of formaldehyde to formate. Mdh is the primary rate-limiting enzyme in methanol uptake; thus, we compared the activities of three Mdhs in vitro and in vivo and then selected the one from Bacillus methanolicus MGA3 for further study. Experimental results indicate that, in agreement with the computational analysis results, the introduction of the NOG pathway is essential for improving PHB production (65% increase in PHB concentration, up to 6.19% of dry cell weight). We demonstrated that PHB can be produced from methanol via metabolic engineering, which provides the foundation for the future large-scale use of one-carbon compounds for biopolymer production.

Microwave-assisted cassava pulp hydrolysis as food waste biorefinery for biodegradable polyhydroxybutyrate production.

Cassava pulp is one of the most abundant agricultural residues that can cause serious disposal problems. This study aimed to apply a biorefinery approach by examining the feasibility of microwave-assisted cassava pulp hydrolysis to attain sustainable management and efficient use of natural resources. Four factors, namely, the liquid-to-solid ratio (20mL/g, 10mL/g, 7.5mL/g, and 5mL/g), types of acids (H2SO4 and H3PO4), watt power (600W, 700W, and 800W) and time (3, 5 and 8min), were carefully investigated. The highest fermentable sugar content of 88.1g/L ± 0.7g/L (0.88g fermentable sugars/g dry cassava pulp) was achieved when 20mL/g cassava pulp was hydrolyzed with 2.5% (v/v) H2SO4 under microwave irradiation at 800W for 8min. Glucose was a major product (82.0g/L ± 5.2g/L). The inhibitor concentration was 5.17g/L ± 0.01g/L, and the levulinic acid concentration was 5.15g/L ± 0.01g/L. The results indicated that the liquid-to-solid ratio, diluted acid concentration, irradiation watt power and time were important factors in producing fermentable sugars from acid hydrolysis under microwave irradiation. The crude hydrolysate was used for PHB production by Cupriavidus necator strain A-04. The hydrolysate to nutrients ratio of 30:70 (v/v) yielded a cell dry weight of 7.5g/L ± 0.1g/L containing PHB content of 66.8% ± 0.3% (w/w), resulting in a yield (g-PHB/g- ) of 0.35g/g. This study demonstrated that the microwave-assisted cassava pulp hydrolysate developed in this study provided a high amount of glucose (88.1% conversion) and resulted in a low concentration of inhibitors without xylose; this was successfully achieved without pregelatinization, alkaline pretreatment or detoxification.

Rapid combinatorial rewiring of metabolic networks for enhanced poly(3-hydroxybutyrate) production in Corynebacterium glutamicum

BackgroundThe disposal of plastic waste is a major environmental challenge. With recent advances in microbial genetic and metabolic engineering technologies, microbial polyhydroxyalkanoates (PHAs) are being used as next-generation biomaterials to replace petroleum-based synthetic plastics in a sustainable future. However, the relatively high production cost of bioprocesses hinders the production and application of microbial PHAs on an industrial scale.ResultsHere, we describe a rapid strategy to rewire metabolic networks in an industrial microorganism, Corynebacterium glutamicum, for the enhanced production of poly(3-hydroxybutyrate) (PHB). A three-gene PHB biosynthetic pathway in Rasltonia eutropha was refactored for high-level gene expression. A fluorescence-based quantification assay for cellular PHB content using BODIPY was devised for the rapid fluorescence-activated cell sorting (FACS)-based screening of a large combinatorial metabolic network library constructed in C. glutamicum. Rewiring metabolic networks across the central carbon metabolism enabled highly efficient production of PHB up to 29% of dry cell weight with the highest cellular PHB productivity ever reported in C. glutamicum using a sole carbon source.ConclusionsWe successfully constructed a heterologous PHB biosynthetic pathway and rapidly optimized metabolic networks across central metabolism in C. glutamicum for enhanced production of PHB using glucose or fructose as a sole carbon source in minimal media. We expect that this FACS-based metabolic rewiring framework will accelerate strain engineering processes for the production of diverse biochemicals and biopolymers.

Polylactide/poly[(R)‐3‐hydroxybutyrate] (PHB) blend fibers with superior heat‐resistance: Effect ofPHBon crystallization

AbstractBlending low content of poly[(R)‐3‐hydroxybutyrate‐co‐4‐hydroxybutyrate] (P34HB ≤8 wt %) with poly(L‐lactide) (PLLA) has proven effective in enhancing heat resistance of PLLA fibers. Unfortunately, the enhancement is relatively limited with boiling water shrinkage of PLLA/P34HB blend fibers reduced to 9.9% at most. In this study, a series of PLLA/poly[(R)‐3‐hydroxybutyrate] (PHB) blend fibers with low PHB content (≤8 wt %) was prepared with sound spinnability. Surprisingly, the PLLA/PHB blend fibers showed much lower boiling water shrinkage than the PLLA/P34HB blend fibers with the same blend ratio. In particular, the boiling water shrinkage was dropped from ca. 80% for neat PLLA fibers to 9.9% for the PLA/P34HB blend fibers and further to 3.8% for the PLA/PHB blend fibers with 8 wt % P34HB or PHB added. Such an exceptionally improved heat resistance of the PLLA/PHB blend fibers could be closely related to an increase in crystallinity (Xc,PLLA) of the PLLA phase. Specifically, the mobility of the PLLA segments was promoted in the presence of PHB, along with notably improved orientation of the PLLA phase in the PLLA/PHB blend fibers. More importantly, investigation on crystallization behaviors revealed that the PHB phase served as an effective nucleating agent to accelerate the overall crystallization of PLLA. By contrast, the P34HB phase only acted as spherulite growth‐accelerating and/or nucleation‐assisting agents. These findings help provide a facile and effective strategy to improve heat resistance of PLLA fibers more significantly.