Transforming growth factor ß1 signaling links extracellular matrix remodeling to intracellular lipogenesis upon physiological feeding events.

Adipose tissue dynamically changes its mass in response to external nutritional status, which plays an important role in maintaining the lipid homeostasis. Physiologically, feeding events are associated with the expansion of adipose tissue, but little is known about the detailed molecular mechanisms of this expansion. Here, using comprehensive transcriptome analysis, we found that levels of transforming growth factor ß1 (TGF-ß1), a key regulator of extracellular matrix (ECM) remodeling, were increased in adipose tissue under feeding conditions and associated with the lipogenic pathway. In addition, TGF-ß receptors are highly expressed in adipose tissue, and pharmacological inhibition of TGF-ß1 reduced adipose tissue mass and caused ectopic lipid accumulation in the liver. This reduced fat mass was associated with decreased gene expression in ECM remodeling and lipogenesis. Furthermore, similar results were observed in the adipose tissue of SMAD family member 3 knockout mice or upon systemic TGF-ß neutralization, with significant reductions in both ECM remodeling and lipogenesis-related genes. Mechanistically, we found that insulin-induced TGF-ß1 and cell-autonomous action remodels the ECM of adipocytes, which controls the downstream focal adhesion kinase-AKT signaling cascades and enhances the lipogenic pathway. Of note, destruction of collagens or matrix metalloproteinase/a disintegrin and metalloprotease activities, critical components of ECM remodeling, blocked TGF-ß1-mediated focal adhesion kinase-AKT signaling and the lipogenic pathway. Taken together, this study identifies a previously unknown lipogenic role of TGF-ß1 by which adipocytes can expand to adapt to physiological feeding events.

Hybrid model for daily streamflow and phosphorus load prediction.

Environmental factors, such as climate change and land use changes, affect water quality drastically. To consider these, various predictive models, both process-based and data-driven, have been used. However, each model has distinct limitations. In this study, a hybrid model combining the soil and water assessment tool and the reverse time attention mechanism (SWAT-RETAIN) was proposed for predicting daily streamflow and total phosphorus (TP) load of a watershed. SWAT-RETAIN was applied to Hwangryong River, South Korea. The hybrid model uses the SWAT output as input data for the RETAIN. Spatial, meteorological, and hydrological data were collected to develop the SWAT to generate high temporal resolution data. RETAIN facilitated effective simultaneous prediction. The SWAT-RETAIN exhibited high accuracy in predicting streamflow (Nash-Sutcliffe efficiency (NSE): 0.45, root mean square error (RMSE): 27.74, percent bias (PBIAS): 22.63 for test sets), and TP load (NSE: 0.50, RMSE: 423.93, PBIAS: 22.09 for test sets). This result was evident in the performance evaluation using flow duration and load duration curves. The SWAT-RETAIN provides enhanced temporal resolution and performance, enabling the simultaneous prediction of multiple variables. It can be applied to predict various water quality variables in larger watersheds.

A Nanojunction pH Sensor within a Nanowire.

pH sensors that are nanoscopic in all three dimensions are fabricated within a single gold nanowire. Fabrication involves the formation of a nanogap within the nanowire via electromigration, followed by electropolymerization of pH-responsive poly(aniline) (PANI) that fills the nanogap forming the nanojunction. All fabrication steps are performed using wet chemical methods that do not require a clean room. The measured electrical impedance of the PANI nanojunction is correlated with pH from 2.0 to 9.0 with a response time of 30 s. Larger, micrometer-scale PANI junctions exhibit a slower response. The measured pH is weakly influenced by the salt concentration of the contacting aqueous solution. An impedance measurement at two frequencies (300 kHz and 1.0 Hz) enables estimation of the salt concentration and correction of the measured pH value, preserving the accuracy of the pH measurement across the entire calibration curve for salt concentrations up to 1.0 M. The result is a nanoscopic pH sensor with pH sensing performance approaching that of a conventional, macroscopic pH glass-membrane electrode.

High reliability and accuracy of dynamic magnetic resonance imaging in the diagnosis of cervical Spondylotic myelopathy: a multicenter study.

BACKGROUND: Cervical spondylotic myelopathy (CSM) is a critical condition that results in significant neurologic deterioration. An accurate diagnosis is essential for determining its outcome and prognosis. The pathology is strongly associated with dynamic factors; therefore, dynamic magnetic resonance (MR) image could be crucial to accurately detect CSM. However, very few studies have evaluated the reliability and accuracy of dynamic MR in CSM. In this study, we aimed to compare intra- and interobserver reliabilities and accuracy of dynamic MR in detecting CSM using sagittal MR scans of the neck in the flexed, neutral, and extended position. METHODS: Out of 131 patients who underwent surgical treatments for CSM, 107 were enrolled in this study. The patient underwent three-types of sagittal MR scans that were obtained separately in different neck positions (neutral, flexion, and extension postures). The MR scans of the cervical spine were evaluated independently by three spine professionals, on the basis of tabled questionnaires. For accuracy, we performed a receiver operator characteristic analysis, and the overall discriminating ability of each method was measured by calculating the area under the ROC curve. The Cohen's kappa coefficient and the Fleiss-generalized kappa coefficient was used to the inter- and intra-observer reliabilities. RESULTS: The intraobserver reliability (using the Cohen's kappa coefficient) and interobserver reliability (using the Fless kappa coefficient) were respectively 0.64 and 0.52 for the neutral sagittal MR. The accuracy of neutral sagittal MR in detecting CSM was 0.735 (95% CI, 0.720 to 0.741) while that of extension sagittal MRI was 0.932 (96% CI, 0.921 to 0.948). CONCLUSIONS: Dynamic MR significantly showed better diagnostic reliability and accuracy in detecting CSM compared to conventional MR. In particular, extension MR scans could provide a more accurate diagnosis than other images.

Enhancing the Sensitivity of the Virus BioResistor by Overoxidation: Detecting IgG Antibodies.

The Virus BioResistor (VBR) is a biosensor capable of rapid and sensitive detection of small protein disease markers using a simple dip-and-read modality. For example, the bladder cancer-associated protein DJ-1 (22 kDa) can be detected in human urine within 1.0 min with a limit of detection (LOD) of 10 pM. The VBR uses engineered virus particles as receptors to recognize and selectively bind the protein of interest. These virus particles are entrained in a conductive poly(3,4-ethylenedioxythiophene) or PEDOT channel. The electrical impedance of the channel increases when the target protein is bound by the virus particles. But VBRs exhibit a sensitivity that is inversely related to the molecular weight of the protein target. Thus, large proteins, such as IgG antibodies (150 kDa), can be undetectable even at high concentrations. We demonstrate that the electrochemical overoxidation of the VBR's PEDOT channel increases its electrical impedance, conferring enhanced sensitivity for both small and large proteins. Overoxidation makes possible the detection of two antibodies, undetectable at a normal VBR, with a limit of detection of 40 ng/mL (250 pM), and a dynamic range for quantitation extending to 600 ng/mL.

An interpretable machine learning method for supporting ecosystem management: Application to species distribution models of freshwater macroinvertebrates.

Species distribution models (SDMs), in which species occurrences are related to a suite of environmental variables, have been used as a decision-making tool in ecosystem management. Complex machine learning (ML) algorithms that lack interpretability may hinder the use of SDMs for ecological explanations, possibly limiting the role of SDMs as a decision-support tool. To meet the growing demand of explainable MLs, several interpretable ML methods have recently been proposed. Among these methods, SHaply Additive exPlanation (SHAP) has drawn attention for its robust theoretical justification and analytical gains. In this study, the utility of SHAP was demonstrated by the application of SDMs of four benthic macroinvertebrate species. In addition to species responses, the dataset contained 22 environmental variables monitored at 436 sites across five major rivers of South Korea. A range of ML algorithms was employed for model development. Each ML model was trained and optimized using 10-fold cross-validation. Model evaluation based on the test dataset indicated strong model performance, with an accuracy of ≥0.7 in all evaluation metrics for all MLs and species. However, only the random forest algorithm showed a behavior consistent with the known ecology of the investigated species. SHAP presents an integrated framework in which local interpretations that incorporate local interaction effects are combined to represent the global model structure. Consequently, this framework offered a novel opportunity to assess the importance of variables in predicting species occurrence, not only across sites, but also for individual sites. Furthermore, removing interaction effects from variable importance values (SHAP values) clearly revealed non-linear species responses to variations in environmental variables, indicating the existence of ecological thresholds. This study provides guidelines for the use of a new interpretable method supporting ecosystem management.

Biosynthesis and characterization of poly(d-lactate-co-glycolate-co-4-hydroxybutyrate).

Poly(d-lactate-co-glycolate-co-4-hydroxybutyrate) [poly(d-LA-co-GA-co-4HB)] and poly(d-lactate-co-glycolate-co-4-hydroxybutyrate-co-d-2-hydroxybutyrate) [poly(d-LA-co-GA-co-4HB-co-d-2HB)] are of interest for their potential applications as new biomedical polymers. Here we report their enhanced production by metabolically engineered Escherichia coli. To examine the polymer properties, poly(d-LA-co-GA-co-4HB) polymers having various monomer compositions (3.4-41.0mol% of 4HB) were produced by culturing the engineered E. coli strain expressing xylBC from Caulobacter crescentus, evolved phaC1 from Pseudomonas sp. MBEL 6-19 (phaC1437), and evolved pct from Clostridium propionicum (pct540) in a medium supplemented with sodium 4HB at various concentrations. To produce these polymers without 4HB feeding, the 4HB biosynthetic pathway was additionally constructed by expressing Clostridium kluyveri sucD and 4hbD. The engineered E. coli expressing xylBC, phaC1437, pct540, sucD, and 4hbD successfully produced poly(d-LA-co-GA-co-4HB-co-d-2HB) and poly(d-LA-co-GA-co-4HB) from glucose and xylose. Through modulating the expression levels of the heterologous genes and performing fed-batch cultures, the polymer content and titer could be increased to 65.76wt% and 6.19g/L, respectively, while the monomer fractions in the polymers could be altered as desired. The polymers produced, in particular, the 4HB-rich polymers showed viscous and sticky properties suggesting that they might be used as medical adhesives.

Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation.

Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.

Cyclin-dependent kinase 1 activity coordinates the chromatin associated state of Oct4 during cell cycle in embryonic stem cells.

Cyclin-dependent kinase 1 (Cdk1) is indispensable for embryonic stem cell (ESC) maintenance and embryo development. Even though some reports have described a connection between Cdk1 and Oct4, there is no evidence that Cdk1 activity is directly linked to the ESC pluripotency transcription program. We recently reported that Aurkb/PP1-mediated Oct4 resetting is important to cell cycle maintenance and pluripotency in mouse ESCs (mESCs). In this study, we show that Cdk1 is an upstream regulator of the Oct4 phosphorylation state during cell cycle progression, and it coordinates the chromatin associated state of Oct4 for pluripotency-related gene expression within the cell cycle. Upon entry into mitosis, Aurkb in the chromosome passenger complex becomes fully activated and PP1 activity is inhibited downstream of Cdk1 activation, leading to sustaining Oct4(S229) phosphorylation and dissociation of Oct4 from chromatin during the mitotic phase. Cdk1 inhibition at the mitotic phase abnormally results in Oct4 dephosphorylation, chromosome decondensation and chromatin association of Oct4, even in replicated chromosome. Our study results suggest a molecular mechanism by which Cdk1 directly links the cell cycle to the pluripotency transcription program in mESCs.

Abundance of C-terminal binding protein isoform is a prerequisite for exit from pluripotency in mouse embryonic stem cells.

Unlike lower organisms, mammals have 2 C-terminal binding protein (Ctbp) isoforms, Ctbp1 and Ctbp2. Ctbp2 is revealed as a key factor involved in determining cell fate decisions by regulating the epigenetic state in active embryonic stem cell (ESC) genes. However, the molecular mechanism underlying how Ctbp1 and Ctbp2 have different roles remains elusive. Here we demonstrate that Ctbp isoform abundance is important for mouse embryonic ESCs (mESCs) to exit from pluripotency. Temporal expression patterns of Ctbp isoforms were quite different; Ctbp2 is more highly expressed in mESCs and decreases during differentiation, while Ctbp1 is constantly expressed at a lower level. Ctbp2 knockdown, but not Ctbp1 knockdown, in mESCs resulted in impaired exit from pluripotency. Interestingly, Ctbp1 and Ctbp2 overexpression in Ctbp2-knockdown mESCs leads to exiting from pluripotency in a manner similar to that of wild-type mESCs. Quantification of Ctbp1 and Ctbp2 revealed that differentiation ability correlates with abundance of Ctbp isoform in undifferentiated mESCs, suggesting that a sufficient amount of Ctbp isoform is a prerequisite for exiting from pluripotency. The results support the contention that 2 redundant Ctbp isoforms regulate elaborate differentiation via temporally distinctive regulatory patterns in mESCs.-Suh, M. Y., Kim, T. W., Lee, H.-T., Shin, J., Kim, J.-H., Jang, H., Kim, H. J., Kim, S.-T., Cho, E.-J., Youn, H.-D. Abundance of C-terminal binding protein isoform is a prerequisite for exit from pluripotency in mouse embryonic stem cells.

Obesity in Yap transgenic mice is associated with TAZ downregulation.

Obesity is characterized by an expansion of white adipose tissue (WAT) mass, which mainly consists of adipocytes. During the commitment and differentiation of adipocytes, PPARγ functions as a key transcriptional factor for adipogenesis, and is associated with its suppressive coregulator, TAZ. Previous studies have shown the importance of TAZ in adipogenesis using an in vitro model; however, the understanding of its role in adipogenesis in vivo remains limited. Here, we report a unique obese mouse model that is associated with TAZ downregulation, which arose from the overexpression of Yap, a Taz paralog. YAP activation facilitated Hippo signaling feedback, which induced a compensatory reduction in YAP, subsequently neutralizing its functional activity. This feedback also induced TAZ suppression and exclusion from the nucleus. In Yap transgenic mice, TAZ downregulation in adipose stem cells activated PPARγ, leading to their differentiation into mature adipocytes and consequently increased adipose tissue. These results highlight the in vivo necessity of TAZ for adipocyte commitment and differentiation, which could provide insight into anti-obesity therapeutics.

OCT4 directly regulates stemness and extracellular matrix-related genes in human germ cell tumours.

Germ cell tumours (GCTs) are one of the most threatening malignancies in young men and women. Although several reports have suggested the importance of OCT4 in human GCTs, its role has not been clearly investigated on a molecular level. In this study, we revealed GCT-specific direct transcriptional target genes of OCT4. Conditional knockdown of OCT4 in GCT cell lines reduced cell proliferation by affecting both cell cycle and death. Knockdown of OCT4 also reduced stemness of GCTs, as assessed by the expression of other stemness factors, alkaline phosphatase staining, and tumour sphere formation ability. Analysis of whole mRNA expression patterns among GCT cells harbouring endogenous, depleted, and rescued OCT4 revealed 1133 OCT4 target genes in GCT. Combined analysis of both the chromatin binding signature of OCT4 and the genes whose expression levels were changed by OCT4 revealed 258 direct target genes of OCT4 in GCTs. In a similar way, 594 direct target genes in normal embryonic stem cells (ESCs) were identified. Among these two sets of OCT4 direct target genes, 38 genes were common between GCTs and ESCs, most of which were related to regulation of pluripotency, and 220 genes were specific to GCTs, most of which were related to focal adhesion and extracellular matrix organisation. These results provide a molecular basis for how OCT4 regulates GCT stemness and will aid our understanding of the role of OCT4 in other cancers.

Regulation of Dipeptidyl Peptidase-4, its Substrate Chemokines, and Their Receptors in Adipose Tissue of ob/ob Mice.

The physiological function of DPP-4 in proteolytic inactivation of incretins has been well established, however, there is limited information on the expression and the significance of DPP-4 in white adipose tissue with regard to obesity. The objective of the work was to reveal the expression and regulation of DPP-4 in adipocytes and compare the expression and activity of DPP-4 in white adipose tissue and several other organs such as the liver, muscle and kidney. We also investigated the gene expression levels of DPP-4 substrate chemokines, and their receptors in white adipose tissue. DPP-4 was mainly expressed in stromal vascular fraction (SVF), and downregulated in adipose tissue of ob/ob compared with C57BL6/J mice. Mimetic conditions of obese fat in vitro showed that differentiation of mouse primary preadipocytes into adipocytes was associated with marked downregulation of DPP-4 expression. Treatment with TNF-α or ROS even decreased DPP-4 expression in mouse primary adipocytes. Various DPP-4 substrate chemokines were expressed in white adipose tissue and regulated by obesity. The expression of receptors for DPP-4 substrate chemokines was markedly high and tightly regulated by obesity in white adipose tissue. Expression of DPP-4 was reduced in adipose tissues of ob/ob mice. Actions of several substrate chemokines might be potentiated by downregulation of DPP-4, synergistically with upregulation of chemokines and their receptors in adipose tissues of obese mice.

Core Pluripotency Factors Directly Regulate Metabolism in Embryonic Stem Cell to Maintain Pluripotency.

Pluripotent stem cells (PSCs) have distinct metabolic properties that support their metabolic and energetic needs and affect their stemness. In particular, high glycolysis is critical for the generation and maintenance of PSCs. However, it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulate glycolysis directly by controlling the expression of glycolytic enzymes. Specifically, Oct4 directly governs Hk2 and Pkm2, which are important glycolytic enzymes that determine the rate of glycolytic flux. The overexpression of Hk2 and Pkm2 sustains high levels of glycolysis during embryonic stem cell (ESC) differentiation. Moreover, the maintenance of high glycolysis levels by Hk2 and Pkm2 overexpression hampers differentiation and preserves the pluripotency of ESCs in the absence of leukemia inhibitory factor. Overall, our study identifies a direct molecular connection between core pluripotency factors and ESC metabolic signatures and demonstrates the significance of metabolism in cell fate determination.

Ctbp2 Modulates NuRD-Mediated Deacetylation of H3K27 and Facilitates PRC2-Mediated H3K27me3 in Active Embryonic Stem Cell Genes During Exit from Pluripotency.

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment.

Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes ß-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that ß-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

Thermoset elastomers derived from carvomenthide.

Renewable thermoset elastomers were prepared using the plant-based monomer carvomenthide. Controlled ring-opening transesterification polymerization of carvomenthide using diethylene glycol as an initiator gave α,ω-dihydroxyl poly(carvomenthide) (HO-PCM-OH), which was subsequently converted to carboxy-telechelic poly(carvomenthide) (HOOC-PCM-COOH) by esterification with excess succinic anhydride through a one-pot, two-step process, leading to no crystallinity, high viscosity, strong thermal resistance, and low glass transition temperature of the resulting functionalized polyester. Thermal curing processes of the resulting 3, 6, and 12 kg mol(-1) prepolymers were achieved with trifunctional aziridine to give cross-linked PCM elastomers. The thermal properties, mechanical behavior, and biocompatibility of the rubbery thermoset products were investigated by differential scanning calorimetry, thermal gravimetric analysis, dynamic mechanical analysis, tensile tests under static and cyclic loads, and cell adherence. These new materials are useful candidates to satisfy the design objective for the engineering of a variety of soft tissues.

Deep learning-based efficient drone-borne sensing of cyanobacterial blooms using a clique-based feature extraction approach.

Recent advances in remote sensing techniques provide a new horizon for monitoring the spatiotemporal variations of harmful algal blooms (HABs) using hyperspectral data in inland water. In this study, a hierarchical concatenated variational autoencoder (HCVAE) is proposed as an efficient and accurate deep learning (DL) based bio-optical model. To demonstrate its usefulness in retrieving algal pigments, the HCVAE is applied to bloom-prone regions in Daecheong Lake, South Korea. By abstracting the similarity between highly related features using layer-wise clique-based latent-feature extraction, HCVAE reduces the computational loads in deriving outputs while preventing performance degradation. Graph-based clique-detection uses information theory-based criteria to group the related reflectance spectra. Consequently, six latent features were extracted from 79 spectral bands to consist of a multilevel hierarchy of HCVAE that can simultaneously estimate concentrations of chlorophyll-a (Chl-a) and phycocyanin (PC). Despite the parsimonious model architecture, the Chl-a and PC concentrations estimated by HCVAE closely agree with the measured concentrations, with test R2 values of 0.76 and 0.82, respectively. In addition, spatial distribution maps of algal pigments obtained from HCVAE using drone-borne reflectance successfully capture the blooming spots. Based on its multilevel hierarchical architecture, HCVAE can provide the importance of latent features along with their individual wavelengths using Shapley additive explanations. The most important latent features covered the spectral regions associated with both Chl-a and PC. The lightweight neural network DNNsel, which uses only the spectral bands of highest importance in latent-feature extraction, performed comparably to HCVAE. The study results demonstrate the utility of the multilevel hierarchical architecture as a comprehensive assessment model for near-real-time drone-borne sensing of HABs. Moreover, HCVAE is applicable to a wide range of environmental big data, as it can handle numerous sets of features.

Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.