RESUMO
(R)-3-Hydroxybutyric acid (R-3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R-3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R-3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R-3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R-3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R-3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R-3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R-3HB.
Assuntos
Ácido 3-Hidroxibutírico , Escherichia coli , Fermentação , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/química , Vias BiossintéticasRESUMO
BACKGROUND: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. RESULTS: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. CONCLUSIONS: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.
Assuntos
Ácido 3-Hidroxibutírico , Acetilcoenzima A , Citrato (si)-Sintase , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Acetilcoenzima A/metabolismo , Citrato (si)-Sintase/metabolismo , Citrato (si)-Sintase/genética , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Engenharia Metabólica/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cetona Oxirredutases/metabolismo , Cetona Oxirredutases/genética , Oxirredutases do ÁlcoolRESUMO
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.
Assuntos
Cupriavidus necator , Poli-Hidroxialcanoatos , Cupriavidus necator/metabolismo , Cupriavidus necator/genética , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/química , Ácido 3-Hidroxibutírico/química , Ácido 3-Hidroxibutírico/biossíntese , Ácidos Pentanoicos/metabolismo , Ácidos Pentanoicos/química , Poliésteres/química , Poliésteres/metabolismoRESUMO
Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.
Assuntos
COVID-19 , Metabolismo Energético , Cetonas , Síndrome do Desconforto Respiratório , SARS-CoV-2 , Linfócitos T , Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/metabolismo , Aminoácidos/biossíntese , Aminoácidos/metabolismo , Animais , COVID-19/complicações , COVID-19/imunologia , COVID-19/patologia , Dieta Cetogênica , Ésteres/metabolismo , Glutationa/biossíntese , Glutationa/metabolismo , Glicólise , Interferon gama/biossíntese , Corpos Cetônicos/metabolismo , Cetonas/metabolismo , Camundongos , Orthomyxoviridae/patogenicidade , Oxirredução , Fosforilação Oxidativa , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Poly-(3-hydroxybutyrate) (PHB) is a polyester with biodegradable and biocompatible characteristics and has many potential applications. To reduce the raw material costs and microbial energy consumption during PHB production, cheaper carbon sources such as sucrose were evaluated for the synthesis of PHB under anaerobic conditions. In this study, metabolic network analysis was conducted to construct an optimized pathway for PHB production using sucrose as the sole carbon source and to guide the gene knockout to reduce the generation of mixed acid byproducts. The plasmid pMCS-sacC was constructed to utilize sucrose as a sole carbon source, and the cascaded promoter P3nirB was used to enhance PHB synthesis under anaerobic conditions. The mixed acid fermentation pathway was knocked out in Escherichia coli S17-1 to reduce the synthesis of byproducts. As a result, PHB yield was improved to 80% in 6.21 g/L cell dry weight by the resulted recombinant Escherichia coli in a 5 L bed fermentation, using sucrose as the sole carbon source under anaerobic conditions. As a result, the production costs of PHB will be significantly reduced.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Anaerobiose , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibutiratos , Poliésteres , Regiões Promotoras Genéticas , Sacarose/metabolismo , Vias Biossintéticas , Fermentação , Engenharia Genética , Engenharia Metabólica , Plasmídeos/genéticaRESUMO
Disordered hepatic glucagon response contributes to hyperglycemia in diabetes. The regulators involved in glucagon response are less understood. This work aims to investigate the roles of mitochondrial ß-oxidation enzyme HADHA and its downstream ketone bodies in hepatic glucagon response. Here we show that glucagon challenge impairs expression of HADHA. Liver-specific HADHA overexpression reversed hepatic gluconeogenesis in mice, while HADHA knockdown augmented glucagon response. Stable isotope tracing shows that HADHA promotes ketone body production via ß-oxidation. The ketone body ß-hydroxybutyrate (BHB) but not acetoacetate suppresses gluconeogenesis by selectively inhibiting HDAC7 activity via interaction with Glu543 site to facilitate FOXO1 nuclear exclusion. In HFD-fed mice, HADHA overexpression improved metabolic disorders, and these effects are abrogated by knockdown of BHB-producing enzyme. In conclusion, BHB is responsible for the inhibitory effect of HADHA on hepatic glucagon response, suggesting that HADHA activation or BHB elevation by pharmacological intervention hold promise in treating diabetes.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Glucagon/metabolismo , Fígado/metabolismo , Mitocôndrias/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Acetilação , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Proteína Forkhead Box O1/metabolismo , Gluconeogênese , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Hidroxibutirato Desidrogenase , Marcação por Isótopo , Corpos Cetônicos/metabolismo , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Oxirredução , Ligação ProteicaRESUMO
Several species of novel marine bacteria from the genus Marinobacterium, including M. nitratireducens, M. sediminicola, and M. zhoushanense were found to be capable of producing polyhydroxyalkanoates (PHA) using sugars and volatile fatty acids (VFAs) as the carbon source. M. zhoushanense produced poly-3-hydroxybutytate (PHB) from sucrose, achieving a product titer and PHB content of 2.89 g/L and 64.05 wt%, respectively. By contrast, M. nitratireducens accumulated 3.38 g/L PHB and 66.80 wt% polymer content using butyrate as the substrate. A third species, M. sediminicola showed favorable tolerance to propionate, butyrate, and valerate. The use of 10 g/L valerate yielded 3.37 g/L poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a 3-hydroxyvalerate (3 HV) monomer content of 94.75 mol%. Moreover, M. sediminicola could be manipulated to produce PHBV with changeable polymer compositions by feeding different mixtures of VFAs. Our results indicate that M. sediminicola is a promising halophilic bacterium for the production of PHA.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Oceanospirillaceae/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Ácido 3-Hidroxibutírico/metabolismo , Butiratos , Carbono , Ácidos Graxos Voláteis/metabolismo , Hidroxibutiratos , Poliésteres/química , Poli-Hidroxialcanoatos/metabolismo , Propionatos , Açúcares/metabolismo , ValeratosRESUMO
White adipose tissue (WAT) has the capacity to undergo a white-to-beige phenotypic switch, known as browning, in response to stimuli such as cold. However, the mechanism underlying beige adipocyte formation is largely unknown. Apolipoprotein E (ApoE) is highly induced in WAT and has been implicated in lipid metabolism. Here, we show that ApoE deficiency in mice increased oxygen consumption and thermogenesis and enhanced adipose browning pattern in inguinal WAT (iWAT), with associated characteristics such as increased Ucp1 and Pparγ expression. At the cellular level, ApoE deficient beige adipocytes had increased glucose uptake and higher mitochondrial respiration than wild-type cells. Mechanistically, we showed that ApoE deficient iWAT and primary adipose precursor cells activated the thermogenic genes program by stimulating the production of ketone body ß-hydroxybutyrate (ßHB), a novel adipose browning promoting factor. This was accompanied by increased expression of genes involved in ketogenesis and could be compromised by the treatment for ketogenesis inhibitors. Consistently, ApoE deficient mice show higher serum ßHB level than wild-type mice in the fed state and during cold exposure. Our results further demonstrate that the increased ßHB production in ApoE deficient adipose precursor cells could be attributed, at least in part, to enhanced Cd36 expression and CD36-mediated fatty acid utilization. Our findings uncover a previously uncharacterized role for ApoE in energy homeostasis via its cell-autonomous action in WAT.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Tecido Adiposo Branco , Apolipoproteínas E/deficiência , Metabolismo Energético , Termogênese , Adipócitos , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Células Cultivadas , Fibroblastos , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoERESUMO
Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, ß-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-ß (TGF-ß)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Acetoacetatos/farmacologia , Colesterol/biossíntese , Dieta Cetogênica/efeitos adversos , Cirrose Hepática/metabolismo , Fígado/efeitos dos fármacos , Ácido 3-Hidroxibutírico/biossíntese , Acetoacetatos/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Becaplermina/farmacologia , Tetracloreto de Carbono/administração & dosagem , Catalase/genética , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Colesterol/sangue , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Desmina/genética , Desmina/metabolismo , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Índice de Gravidade de Doença , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Tioacetamida/administração & dosagem , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Aciltransferases/genética , Bacillus cereus/genética , Escherichia coli/genética , Etilenoglicóis/metabolismo , Poliuretanos/química , Poliuretanos/síntese química , Ácido 3-Hidroxibutírico/análise , Ácido 3-Hidroxibutírico/química , Aciltransferases/metabolismo , Cromatografia em Gel , Meios de Cultura , Escherichia coli/metabolismo , Etilenoglicóis/química , Isocianatos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microrganismos Geneticamente Modificados , Via Secretória/genética , Espectroscopia de Infravermelho com Transformada de Fourier , TermografiaRESUMO
3,4-Methylenedioxypyrovalerone (MDPV) is consumed worldwide, despite its potential to cause toxicity in several organs and even death. There is a recognized need to clarify the biological pathways through which MDPV elicits general and target-organ toxicity. In this work, a comprehensive untargeted GC-MS-based metabolomics analysis was performed, aiming to detect metabolic changes in putative target organs (brain, heart, kidneys and liver) but also in urine of mice after acute exposure to human-relevant doses of MDPV. Male CD-1 mice received binge intraperitoneal administrations of saline or MDPV (2.5 mg/kg or 5 mg/kg) every 2 h, for a total of three injections. Twenty-four hours after the first administration, target organs, urine and blood samples were collected for metabolomics, biochemical and histological analysis. Hepatic and renal tissues of MDPV-treated mice showed moderate histopathological changes but no significant differences were found in plasma and tissue biochemical markers of organ injury. In contrast, the multivariate analysis significantly discriminated the organs and urine of MDPV-treated mice from the control (except for the lowest dose in the brain), allowing the identification of a panoply of metabolites. Those levels were significantly deviated in relation to physiological conditions and showed an organ specific response towards the drug. Kidneys and liver showed the greatest metabolic changes. Metabolites related with energetic metabolism, antioxidant defenses and inflammatory response were significantly changed in the liver of MDPV-dosed animals, while the kidneys seem to have developed an adaptive response against oxidative stress caused by MDPV. On the other hand, the dysregulation of metabolites that contribute to metabolic acidosis was also observed in this organ. The heart showed an increase of fatty acid biosynthesis, possibly as an adaptation to maintain the cardiac energy homeostasis. In the brain, changes in 3-hydroxybutyric acid levels may reflect the activation of a neurotoxic pathway. However, the increase in metabolites with neuroprotective properties seems to counteract this change. Metabolic profiling of urine from MDPV-treated mice suggested that glutathione-dependent antioxidant pathways may be particularly involved in the compensatory mechanism to counteract oxidative stress induced by MDPV. Overall, this study reports, for the first time, the metabolic profile of liver, kidneys, heart, brain, and urine of MDPV-dosed mice, providing unique insights into the biological pathways of toxicity. Our findings also underline the value of toxicometabolomics as a robust and sensitive tool for detecting adaptive/toxic cellular responses upon exposure to a physiologically relevant dose of a toxic agent, earlier than conventional toxicity tests.
Assuntos
Benzodioxóis/metabolismo , Benzodioxóis/toxicidade , Encéfalo/metabolismo , Rim/metabolismo , Fígado/metabolismo , Miocárdio/metabolismo , Pirrolidinas/metabolismo , Pirrolidinas/toxicidade , Ácido 3-Hidroxibutírico/biossíntese , Animais , Biomarcadores , Análise Química do Sangue , Relação Dose-Resposta a Droga , Ácidos Graxos/biossíntese , Cromatografia Gasosa-Espectrometria de Massas , Homeostase/efeitos dos fármacos , Humanos , Rim/patologia , Fígado/patologia , Masculino , Metaboloma , Camundongos , Urina/química , Catinona SintéticaRESUMO
Naturally occurring thymine (TM) was incorporated into bacterial poly(3-hydroxybutyrate) (PHB) polyester to fabricate a novel and green biocomposite. Both 0.5% and 1% TM exhibit supernucleation effect on PHB, and crystallization kinetics suggests TM significantly increased Tc and Xc, and substantially shortened t1/2 of PHB. Epitaxial nucleation caused by a perfect crystal lattice matching between PHB and TM, was proposed to elucidate nucleation mechanism of PHB. Hydrogen bond interaction exists between CO, C-O-C groups of PHB and -CH3 (or -CH)/-NH- group of TM. TM interacted with CO group of PHB crystalline phase rather than that of amorphous one. In addition, two new IR crystalline bands assigned to C-O-C group of PHB appeared in the presence of TM, which arises from shift of two amorphous ones, respectively. TM enhanced onset thermal degradation temperature of PHB, mainly attributed to increased degree of crystallinity of PHB and flame retardance effect of TM.
Assuntos
Ácido 3-Hidroxibutírico/química , Materiais Biocompatíveis/química , Hidroxibutiratos/química , Poliésteres/química , Ácido 3-Hidroxibutírico/biossíntese , Bactérias/química , Bactérias/genética , Ligação de Hidrogênio , Cinética , Polímeros/química , Timina/químicaRESUMO
Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of Cupriavidusnecator transformants harbouring the phaCBP-M-CPF4 gene (including PHB¯4/pBBR1-CBP-M-CPF4) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa, along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of ß-ketothiolase (PhaACn) and NADPH-dependent acetoacetyl-CoA reductase (PhaBCn) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB-co-3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 × 106 to 1.8 × 106 Da. PhaCBP-M-CPF4 displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Aciltransferases/metabolismo , Cupriavidus necator/metabolismo , Fermentação , Óleos de Plantas/metabolismo , Ácido 3-Hidroxibutírico/isolamento & purificação , Caproatos/isolamento & purificação , Ativação Enzimática , Peso Molecular , Oxirredução , Óleo de Palmeira/metabolismo , Plasmídeos/química , Poli-Hidroxialcanoatos/biossíntese , Polímeros/metabolismo , Transformação BacterianaRESUMO
Nitrate accumulation causes long-time threat to aquatic animals in recirculating aquaculture system (RAS); thus, nitrate removal is also required in RASs. However, the lack of carbon sources makes denitrification difficult to function. Nitrate removal performance of an aerobic denitrifying and extracellular polyhydroxyalkanoate depolymerase-producing bacterium, Pseudomonas sp. AOB-7, using polyhydroxyalkanoate (PHA) granules as a solid sustained-release carbon source in RAS was evaluated. With the initial nitrate-N concentration of 140 mg/L, the high denitrification rates of 0.056 g NO3--N L-1 day-1 and 0.035 g NO3--N L-1 day-1 were achieved in denitrification medium containing poly-ß-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), respectively. Significant erosions and pits formed on the surface of the granules made them a good biofilm carrier for AOB-7, and 3-hydroxybutyrate (3-HB) monomer was the major product released to aquatic phase, which was benefit to animals. SEM photos showed that AOB-7 entered and attached on the inside of the PHA particle holes. A 4-week application trial was conducted to reveal the effects of PHB (AOB-7) denitrifying agent and 3-HB produced on growth of zebrafish (Brachydanio rerio) by adding 0.1% (w/v) PHB (AOB-7) denitrifying agent. Result indicated that PHB (AOB-7) denitrifying agent can significantly reduce nitrate-N content in RASs. Compared with the control group, feed coefficient ratio reduced by 18% and weight gain ratio increased by 29% in the PHB (AOB-7) denitrifying agent group. 3-HB monomer produced during the denitrification was speculated to function as a prebiotic and promote zebrafish growth. KEY POINTS: ⢠AOB-7 showed a good aerobic denitrifying ability on PHA granules as sustained-release C source. ⢠PHB (AOB-7) denitrifying agent can significantly reduce nitrate content in RAS. ⢠R-3-HB monomer was the major product released to aquatic phase and function as a prebiotic.
Assuntos
Biofilmes/crescimento & desenvolvimento , Desnitrificação , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Aerobiose , Aquicultura , Biodegradação Ambiental , Carbono/metabolismo , Nitratos/isolamento & purificação , Pseudomonas/crescimento & desenvolvimento , Poluentes Químicos da Água/isolamento & purificação , Purificação da ÁguaRESUMO
The biodegradable polyester 3-hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo-oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)-deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end-capped forms may be used as physiologically active substances and building blocks for polymeric materials.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/química , Aciltransferases/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/química , Ácido 3-Hidroxibutírico/isolamento & purificação , Aciltransferases/genética , Aeromonas caviae/enzimologia , Aeromonas caviae/genética , Álcool Desidrogenase/genética , Bacillus cereus/enzimologia , Bacillus cereus/genética , Biodegradação Ambiental , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Hidroxibutiratos/química , Peso Molecular , Poliésteres/química , Polimerização , Proteínas Recombinantes , Recombinação Genética , Fatores de TempoRESUMO
BACKGROUND: Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial polyester with high biodegradability, even in marine environments. Ralstonia eutropha has been engineered for the biosynthesis of P(3HB-co-3HHx) from vegetable oils, but its production from structurally unrelated carbon sources remains unsatisfactory. RESULTS: Ralstonia eutropha strains capable of synthesizing P(3HB-co-3HHx) from not only fructose but also glucose and glycerol were constructed by integrating previously established engineering strategies. Further modifications were made at the acetoacetyl-CoA reduction step determining flux distribution responsible for the copolymer composition. When the major acetoacetyl-CoA reductase (PhaB1) was replaced by a low-activity paralog (PhaB2) or enzymes for reverse ß-oxidation, copolyesters with high 3HHx composition were efficiently synthesized from glucose, possibly due to enhanced formation of butyryl-CoA from acetoacetyl-CoA via (S)-3HB-CoA. P(3HB-co-3HHx) composed of 7.0 mol% and 12.1 mol% 3HHx fractions, adequate for practical applications, were produced at cellular contents of 71.4 wt% and 75.3 wt%, respectively. The replacement by low-affinity mutants of PhaB1 had little impact on the PHA biosynthesis on glucose, but slightly affected those on fructose, suggesting altered metabolic regulation depending on the sugar-transport machinery. PhaB1 mostly acted in the conversion of acetoacetyl-CoA when the cells were grown on glycerol, as copolyester biosynthesis was severely impaired by the lack of phaB1. CONCLUSIONS: The present results indicate the importance of flux distribution at the acetoacetyl-CoA node in R. eutropha for the biosynthesis of the PHA copolyesters with regulated composition from structurally unrelated compounds.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Acil Coenzima A/metabolismo , Plásticos Biodegradáveis/metabolismo , Cupriavidus necator/metabolismo , Caproatos , Oxirredução , Poliésteres/metabolismoRESUMO
Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L-1 butyrate, 8.95 g L-1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Butiratos/metabolismo , Escherichia coli/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Biomassa , Biotransformação , Escherichia coli/genética , Etanol , Fermentação , Glucose , Redes e Vias Metabólicas , Populus , XiloseRESUMO
Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB), and/or the isocitrate lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110, and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate-forming background. Despite low 3HB titers in low-cell-density screening, 3HB-producing BL21 produced five times less acetic acid per mole of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Técnicas de Cultura Celular por Lotes , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Reatores Biológicos , Proteínas de Escherichia coli/genética , Deleção de Genes , Ácido PirúvicoRESUMO
Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.
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Ácido 3-Hidroxibutírico/biossíntese , Bactérias Anaeróbias/metabolismo , Clostridium/metabolismo , Hidroxibutiratos/química , Poliésteres/química , Ácido 3-Hidroxibutírico/química , Processos Autotróficos , Bactérias Anaeróbias/química , Clostridium/química , Gases/química , Gases/metabolismo , Hidroxibutiratos/síntese química , Poliésteres/síntese químicaRESUMO
About 24 h incubation of Azomonas (A.) macrocytogenes isolate KC685000 in 14L fermenter produced 22% poly (3-hydroxybutyrate) (PHB) per cell dry weight (CDW) biopolymer using 1 vvm aeration, 10% inoculum size, and initial pH of 7.2. To control the fermentation process, Logistic and Leudeking-Piret models were used to describe the cell growth and PHB production, respectively. These two models were in good agreement with the experimental data confirming the growth associated nature of PHB production. The best method for recovery of PHB was chemical digestion using sodium hypochlorite alone. The characterization of the produced polymer was carried out using FT-IR, 1HNMR spectroscopy, gel permeation chromatography and transmission electron microscope. The analysis of the nucleotide sequences of PHA synthase enzyme revealed class III identity. The putative tertiary structure of PHA synthase enzyme was analyzed using Modular Approach to Structural class prediction software, Tied Mixture Hidden Markov Model server, and Swiss model software. It was deduced that PHA synthases' structural class was multidomain protein (α/ß) containing a conserved cysteine residue and lipase box as characteristic features of α/ß hydrolase super family. Taken together, all the results of molecular characterization and transmission electron microscope images supported that the PHB formation was attained by the micelle model. To the best of our knowledge, this is the first report on production of growth associated PHB polymer using A. macrocytogenes isolate KC685000, and its class III PHA synthase.