scholarly journals Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

2022 ◽  
Vol 10 (1) ◽  
pp. 184
Author(s):  
Izumi Orita ◽  
Gento Unno ◽  
Risa Kato ◽  
Toshiaki Fukui
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Negative Impact ◽  
Value Added ◽  
Pha Synthase ◽  
Growth Impairment ◽  
Oxidation Pathway ◽  
Methylotrophic Growth ◽  
Value Added Products

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

2018 ◽  
Vol 28 (5) ◽  
pp. 225-235
Author(s):  
Edmar Ramos Oliveira-Filho ◽  
Linda P. Guamán ◽  
Thatiane Teixeira Mendonça ◽  
Paul F. Long ◽  
Marilda Keico Taciro ◽  
...  
Keyword(s):  
Carbon Source ◽  
Aeromonas Hydrophila ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Medium Chain Length ◽  
Marker Free ◽  
Pha Copolymers ◽  
First Time ◽  
Induced Mutant

Three different polyhydroxyalkanoate (PHA) synthase genes (<i>Ralstonia eutropha</i> H16, <i>Aeromonas</i> sp. TSM81 or <i>Aeromonas hydrophila</i> ATCC7966 <i>phaC</i>) were introduced into the chromosome of two <i>Pseudomonas</i> strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA<sub>MCL</sub>) producer (<i>Pseudomonas</i> sp. LFM046) and a UV-induced mutant strain unable to produce PHA (<i>Pseudomonas</i> sp. LFM461). We reported for the first time the insertion of a chromosomal copy of <i>phaC</i> using the transposon system mini-Tn<i>7</i>. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-<i>co</i>-3HA<sub>MCL</sub>) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring <i>A. hydrophila phaC</i> produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of <i>Pseudomonas</i> sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA<sub>MCL</sub> using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Design of a Synthetic Enzyme Cascade for the in vitro Fixation of a C1 Carbon Source to a Functional C4 Sugar

2021 ◽  
Author(s):  
Samed Güner ◽  
Vanessa Wegat ◽  
André Pick ◽  
Volker Sieber
Keyword(s):  
Carbon Dioxide ◽  
Carbon Source ◽  
Greenhouse Gas ◽  
Value Added ◽  
Waste Stream ◽  
Enzyme Cascade ◽  
Sustainable Future ◽  
Value Added Products

Realizing a sustainable future requires intensifying the waste stream conversion, such as converting the greenhouse gas carbon dioxide into value-added products. In this paper, we focus on utilizing formaldehyde as...

scholarly journals Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

2020 ◽  
Author(s):  
Ashish Prabhu ◽  
Rodrigo Ledesma- Amaro ◽  
Carol Sze Ki Lin ◽  
Frederic Coulon ◽  
Vijay kumar Thakur ◽  
...  
Keyword(s):  
Cell Growth ◽  
Carbon Source ◽  
Succinic Acid ◽  
Yarrowia Lipolytica ◽  
Recombinant Strain ◽  
Sole Carbon Source ◽  
Substrate Inhibition ◽  
Biomass Concentration ◽  
Value Added ◽  
Green Economy

Abstract Background Xylose is a most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast to produce industrially important metabolites, and it is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economically feasible of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising of xylose reductase ( XR ), xylitol dehydrogenase ( XDH ) and xylulose kinase ( XK ) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and accumulated SA (3.8 g/L) with a yield of 0.19 g/g in shake flask studies. Substrate inhibition studies revealed a marked negative impact on cell growth and product formation above 60 g/L xylose concentration. The modelling based on inhibition kinetics revealed that Aiba model showed better fit with experimental data, which resulted the correlation coefficient (R 2 ) of 0.82 and inhibition constant (K I ) 88.9 g/L. The batch cultivation of recombinant strain in bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.18 g/g. Similar results in term of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD 600 : 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main byproduct in all the fermentations. Conclusion The recombinant strain displayed potential bioconversion of xylose to succinic acid. Further this study provided a new insight on conversion of LCB into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

scholarly journals Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

2020 ◽  
Author(s):  
Ashish Prabhu ◽  
Rodrigo Ledesma- Amaro ◽  
Carol Sze Ki Lin ◽  
Frederic Coulon ◽  
Vijay kumar Thakur ◽  
...  
Keyword(s):  
Cell Growth ◽  
Carbon Source ◽  
Succinic Acid ◽  
Lignocellulosic Biomass ◽  
Yarrowia Lipolytica ◽  
Recombinant Strain ◽  
Sole Carbon Source ◽  
Biomass Concentration ◽  
Value Added ◽  
Green Economy

Abstract Background : Xylose is a most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast to produce industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economically feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results : In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising of xylose reductase ( XR ), xylitol dehydrogenase ( XDH ) and xylulose kinase ( XK ) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of recombinant strain in bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in term of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD 600 : 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main byproduct in all the fermentations. Conclusion : The recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

scholarly journals Synthetic biology toolkit for engineering Cupriviadus necator H16 as a platform for CO2 valorization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Haojie Pan ◽  
Jia Wang ◽  
Haoliang Wu ◽  
Zhongjian Li ◽  
Jiazhang Lian
Keyword(s):  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Ralstonia Eutropha ◽  
Heterologous Gene Expression ◽  
Value Added ◽  
Cell Factory ◽  
Main Body ◽  
Cell Factories ◽  
Value Added Products

Abstract Background CO2 valorization is one of the effective methods to solve current environmental and energy problems, in which microbial electrosynthesis (MES) system has proved feasible and efficient. Cupriviadus necator (Ralstonia eutropha) H16, a model chemolithoautotroph, is a microbe of choice for CO2 conversion, especially with the ability to be employed in MES due to the presence of genes encoding [NiFe]-hydrogenases and all the Calvin–Benson–Basham cycle enzymes. The CO2 valorization strategy will make sense because the required hydrogen can be produced from renewable electricity independently of fossil fuels. Main body In this review, synthetic biology toolkit for C. necator H16, including genetic engineering vectors, heterologous gene expression elements, platform strain and genome engineering, and transformation strategies, is firstly summarized. Then, the review discusses how to apply these tools to make C. necator H16 an efficient cell factory for converting CO2 to value-added products, with the examples of alcohols, fatty acids, and terpenoids. The review is concluded with the limitation of current genetic tools and perspectives on the development of more efficient and convenient methods as well as the extensive applications of C. necator H16. Conclusions Great progress has been made on genetic engineering toolkit and synthetic biology applications of C. necator H16. Nevertheless, more efforts are expected in the near future to engineer C. necator H16 as efficient cell factories for the conversion of CO2 to value-added products.

scholarly journals Genetically Modified Strains of Ralstonia eutropha H16 with β-Ketothiolase Gene Deletions for Production of Copolyesters with Defined 3-Hydroxyvaleric Acid Contents

2012 ◽  
Vol 78 (15) ◽  
pp. 5375-5383 ◽  
Author(s):  
Nicole Lindenkamp ◽  
Elena Volodina ◽  
Alexander Steinbüchel
Keyword(s):  
Fatty Acids ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Production Capacity ◽  
Wild Type ◽  
Content Type ◽  
Storage Behavior ◽  
Copolymer Poly

ABSTRACTβ-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate,Ralstonia eutrophaH16 produces the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]. InR. eutropha, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373–5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (phaA,bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, andpcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-typeR. eutrophaand the mutants. In contrast, during the growth on oleate of a multiple mutant lackingphaA,bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16ΔphaAand all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.

Polyhydroxyalkanoate production in wild-type and engineered Ralstonia eutropha: carbon flow from central metabolism to storage for value added products

2012 ◽  
Vol 29 ◽  
pp. S9-S10
Author(s):  
Christopher Brigham ◽  
Sebastian Riedel ◽  
Jingnan Lu ◽  
Matthew Neal ◽  
John W. Quimby ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Value Added ◽  
Carbon Flow ◽  
Central Metabolism ◽  
Wild Type ◽  
Value Added Products

scholarly journals From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

2013 ◽  
Vol 79 (12) ◽  
pp. 3582-3589 ◽  
Author(s):  
Daniel Heinrich ◽  
Björn Andreessen ◽  
Mohamed H. Madkour ◽  
Mansour A. Al-Ghamdi ◽  
Ibrahim I. Shabbaj ◽  
...  
Keyword(s):  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Biodiesel Production ◽  
Pha Synthase ◽  
Content Type ◽  
Coa Transferase ◽  
Fed Batch Fermentation ◽  
A Cell ◽  
Cultivation Process

ABSTRACTIn recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) ofPseudomonas putidaKT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) ofClostridium propionicumX2, and polyhydroxyalkanoate (PHA) synthase (phaC1) ofRalstonia eutrophaH16 were cloned and expressed in the 1,3-propanediol producerShimwellia blattae. In a two-step cultivation process, recombinantS. blattaecells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.

scholarly journals Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene fromChromobacterium violaceum

1999 ◽  
Vol 65 (8) ◽  
pp. 3561-3565 ◽  
Author(s):  
Dana Kolibachuk ◽  
Andrea Miller ◽  
Douglas Dennis
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Alcaligenes Eutrophus ◽  
Pha Synthase ◽  
Klebsiella Aerogenes ◽  
E Coli ◽  
Polyhydroxyalkanoic Acid ◽  
Phac Gene ◽  
Fatty Acid Carbon

ABSTRACT The polyhydroxyalkanoic acid synthase gene fromChromobacterium violaceum (phaC Cv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaC Cv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaA Cv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes andRalstonia eutropha (formerly Alcaligenes eutrophus), harboring phaC Cv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. colinor recombinant Pseudomonas putida harboringphaC Cv accumulated significant levels of PHA. Sequence analysis of the phaC Cv product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274).

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