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Mary E Lidstrom - One of the best experts on this subject based on the ideXlab platform.
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difference in c3 c4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens am1
BMC Microbiology, 2016Co-Authors: David A C Beck, Mary E LidstromAbstract:Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3–C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3–C4 inter-conversion strategies were the major response to the trade-off.
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comprehensive molecular characterization of Methylobacterium extorquens am1 adapted for 1 butanol tolerance
Biotechnology for Biofuels, 2016Co-Authors: Yi Ming Yang, Mary E Lidstrom, David A C Beck, Qian Wen Wang, Wen Jing Chen, Jing Yang, Song YangAbstract:The toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels production. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, has been engineered to generate 1-butanol from cheap carbon feedstocks through a synthetic metabolic pathway. However, M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. Only a few studies have reported the general stress response of M. extorquens AM1 to solvent stress. Therefore, it is highly desirable to obtain a strain with ameliorated 1-butanol tolerance and elucidate the molecular mechanism of 1-butnaol tolerance in M. extorquens AM1 for future strain improvement. In this work, adaptive laboratory evolution was used as a tool to isolate mutants with 1-butanol tolerance up to 0.5 %. The evolved strains, BHBT3 and BHBT5, demonstrated increased growth rates and higher survival rates with the existence of 1-butanol. Whole genome sequencing revealed a SNP mutation at kefB in BHBT5, which was confirmed to be responsible for increasing 1-butanol tolerance through an allelic exchange experiment. Global metabolomic analysis further discovered that the pools of multiple key metabolites, including fatty acids, amino acids, and disaccharides, were increased in BHBT5 in response to 1-butanol stress. Additionally, the carotenoid synthesis pathway was significantly down-regulated in BHBT5. We successfully screened mutants resistant to 1-butanol and provided insights into the molecular mechanism of 1-butanol tolerance in M. extorquens AM1. This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future.
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ethylmalonyl coenzyme a mutase operates as a metabolic control point in Methylobacterium extorquens am1
Journal of Bacteriology, 2015Co-Authors: Nathan M. Good, Cecilia N Martinezgomez, David A C Beck, Mary E LidstromAbstract:The metabolism of one- and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.
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metabolic engineering of Methylobacterium extorquens am1 for 1 butanol production
Biotechnology for Biofuels, 2014Co-Authors: Mary E LidstromAbstract:Background Butanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol.
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metabolic engineering of Methylobacterium extorquens am1 for 1 butanol production
Biotechnology for Biofuels, 2014Co-Authors: Mary E LidstromAbstract:Butanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol. In this work, we engineered a modified CoA-dependent pathway in Methylobacterium extorquens AM1 to produce 1-butanol. Engineered strains displayed different 1-butanol titers using ethylamine as a substrate. A strain overexpressing Treponema denticola trans-enoyl-CoA reductase, Clostridium acetobutylicum alcohol dehydrogenase, and native crotonase was able to generate the highest 1-butanol titer (15.2 mg l-1). In vitro isotopic tracing of metabolic flux and in vivo metabolite analysis showed the accumulation of butyryl-CoA, demonstrating the functionality of the synthetic pathway and identifying targets for future improvement. We demonstrated the feasibility of using metabolic intermediates of the ethylmalonyl-CoA pathway in M. extorquens AM1 to generate value-added chemicals, with 1-butanol as the test case. This will not only establish the biotechnological potential of the ethylmalonyl-CoA pathway, but will also introduce M. extorquens AM1 as a potential platform to produce value-added chemicals.
Christopher J. Marx - One of the best experts on this subject based on the ideXlab platform.
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hopanoid free Methylobacterium extorquens dm4 overproduces carotenoids and has widespread growth impairment
PLOS ONE, 2017Co-Authors: Alexander S Bradley, Emilie E L Muller, Stéphane Vuilleumier, Françoise Bringel, Paige K Swanson, Sean M Caroll, Ann Pearson, Christopher J. MarxAbstract:Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of Methylobacterium extorquens DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids-the other major isoprenoid product of this strain-and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in M. extorquens DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for M. extorquens DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane.
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parallel and divergent evolutionary solutions for the optimization of an engineered central metabolism in Methylobacterium extorquens am1
Microorganisms, 2015Co-Authors: Sean M Carroll, Lon M Chubiz, Deepa Agashe, Christopher J. MarxAbstract:Bioengineering holds great promise to provide fast and efficient biocatalysts for methanol-based biotechnology, but necessitates proven methods to optimize physiology in engineered strains. Here, we highlight experimental evolution as an effective means for optimizing an engineered Methylobacterium extorquens AM1. Replacement of the native formaldehyde oxidation pathway with a functional analog substantially decreased growth in an engineered Methylobacterium, but growth rapidly recovered after six hundred generations of evolution on methanol. We used whole-genome sequencing to identify the basis of adaptation in eight replicate evolved strains, and examined genomic changes in light of other growth and physiological data. We observed great variety in the numbers and types of mutations that occurred, including instances of parallel mutations at targets that may have been “rationalized” by the bioengineer, plus other “illogical” mutations that demonstrate the ability of evolution to expose unforeseen optimization solutions. Notably, we investigated mutations to RNA polymerase, which provided a massive growth benefit but are linked to highly aberrant transcriptional profiles. Overall, we highlight the power of experimental evolution to present genetic and physiological solutions for strain optimization, particularly in systems where the challenges of engineering are too many or too difficult to overcome via traditional engineering methods.
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genetic and phenotypic comparison of facultative methylotrophy between Methylobacterium extorquens strains pa1 and am1
PLOS ONE, 2014Co-Authors: Dipti D Nayak, Christopher J. MarxAbstract:Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains.
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sign epistasis limits evolutionary trade offs at the confluence of single and multi carbon metabolism in Methylobacterium extorquens am1
Evolution, 2014Co-Authors: Sean M Carroll, Mingchun Lee, Christopher J. MarxAbstract:Adaptation of one set of traits is often accompanied by attenuation of traits important in other selective environments, leading to fitness trade-offs. The mechanisms that either promote or prevent the emergence of trade-offs remain largely unknown, and are difficult to discern in most systems. Here, we investigate the basis of trade-offs that emerged during experimental evolution of Methylobacterium extorquens AM1 to distinct growth substrates. After 1500 generations of adaptation to a multi-carbon substrate, succinate (S), many lineages had lost the ability to use one-carbon compounds such as methanol (M), generating a mixture of M(+) and M(-) evolved phenotypes. We show that trade-offs in M(-) strains consistently arise via antagonistic pleiotropy through recurrent selection for loss-of-function mutations to ftfL (formate-tetrahydrofolate ligase), which improved growth on S while simultaneously eliminating growth on M. But if loss of FtfL was beneficial, why were M trade-offs not found in all populations? We discovered that eliminating FtfL was not universally beneficial on S, as it was neutral or even deleterious in certain evolved lineages that remained M(+) . This suggests that sign epistasis with earlier arising mutations prevented the emergence of mutations that drove trade-offs through antagonistic pleiotropy, limiting the evolution of metabolic specialists in some populations.
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development of an optimized medium strain and high throughput culturing methods for Methylobacterium extorquens
PLOS ONE, 2013Co-Authors: Nigel F Delaney, Maryska E Kaczmarek, Lewis M Ward, Paige K Swanson, Mingchun Lee, Christopher J. MarxAbstract:Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.
David A C Beck - One of the best experts on this subject based on the ideXlab platform.
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difference in c3 c4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens am1
BMC Microbiology, 2016Co-Authors: David A C BeckAbstract:Background Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values.
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difference in c3 c4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens am1
BMC Microbiology, 2016Co-Authors: David A C Beck, Mary E LidstromAbstract:Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3–C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3–C4 inter-conversion strategies were the major response to the trade-off.
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comprehensive molecular characterization of Methylobacterium extorquens am1 adapted for 1 butanol tolerance
Biotechnology for Biofuels, 2016Co-Authors: Yi Ming Yang, Mary E Lidstrom, David A C Beck, Qian Wen Wang, Wen Jing Chen, Jing Yang, Song YangAbstract:The toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels production. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, has been engineered to generate 1-butanol from cheap carbon feedstocks through a synthetic metabolic pathway. However, M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. Only a few studies have reported the general stress response of M. extorquens AM1 to solvent stress. Therefore, it is highly desirable to obtain a strain with ameliorated 1-butanol tolerance and elucidate the molecular mechanism of 1-butnaol tolerance in M. extorquens AM1 for future strain improvement. In this work, adaptive laboratory evolution was used as a tool to isolate mutants with 1-butanol tolerance up to 0.5 %. The evolved strains, BHBT3 and BHBT5, demonstrated increased growth rates and higher survival rates with the existence of 1-butanol. Whole genome sequencing revealed a SNP mutation at kefB in BHBT5, which was confirmed to be responsible for increasing 1-butanol tolerance through an allelic exchange experiment. Global metabolomic analysis further discovered that the pools of multiple key metabolites, including fatty acids, amino acids, and disaccharides, were increased in BHBT5 in response to 1-butanol stress. Additionally, the carotenoid synthesis pathway was significantly down-regulated in BHBT5. We successfully screened mutants resistant to 1-butanol and provided insights into the molecular mechanism of 1-butanol tolerance in M. extorquens AM1. This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future.
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ethylmalonyl coenzyme a mutase operates as a metabolic control point in Methylobacterium extorquens am1
Journal of Bacteriology, 2015Co-Authors: Nathan M. Good, Cecilia N Martinezgomez, David A C Beck, Mary E LidstromAbstract:The metabolism of one- and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.
Denis Groleau - One of the best experts on this subject based on the ideXlab platform.
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production and characterization of polyhydroxyalkanoates by recombinant Methylobacterium extorquens combining desirable thermal properties with functionality
Biochemical Engineering Journal, 2011Co-Authors: Philipp Höfer, Patrick Vermette, Denis GroleauAbstract:Abstract Recombinant Methylobacterium extorquens ATCC 55366 harboring phaC2 from Pseudomonas fluorescens GK13 ( M. ex-phaC2 ) was capable of producing blends of polyhydroxyalkanoates. By co-feeding methanol and 5-hexenoic acid (C6 ), functionalized PHAs (4 ≤ chain length ≤ 6) containing C–C double bonds were created. Bioreactor studies revealed negative impacts of 5-hexenoic acid on biomass and PHA production by reducing overall yields. In contrast, there was a positive relationship between 5-hexenoic acid supply and abundance of polymeric functional groups, i.e., molar portions of monomeric units bearing double bonds increased with increased 5-hexenoic acid supply. Correlation of C6 consumption to methanol addition resulted in a model that allowed for on-line estimation of toxic co-substrate concentration. The functionalized PHAs were ductile and showed signs of side chain crosslinking, resulting in reduced degrees of crystallinity. Incorporation of 3-hydroxyhex-5-enoate and 3-hydroxyhexanoate into the polymeric chains produced desirable thermal properties with enhanced thermal stability and reduced melting temperatures (139–168 °C). Thermal degradation and melting temperatures obtained suggest a comfortable range for melt-processing of these polymers and allow for autoclaving as convenient sterilization process. Consequently, functionalized PHAs produced in this study are candidates for medical applications as part of biocomposite materials. The use of methanol as main substrate for cultivation of recombinant M. extorquens offers the possibility to reduce production costs and develop new process control strategies.
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Introducing a new bioengineered bug: Methylobacterium extorquens tuned as a microbial bioplastic factory
Bioengineered Bugs, 2011Co-Authors: Philipp Höfer, Patrick Vermette, Denis GroleauAbstract:Discussion on and use of methanol as chemical feedstock and as alternative fuel has gained momentum during the past years. Consequently, microorganism and product design based on ''methylotrophism'' is in vogue as reflected by increasing research and development activities in methanol-related areas. A recent example of microorganism and product development is the use of recombinant Methylobacterium extorquens ATCC 55366 strains in the production of second generation biopolyesters. Feeding n-alkenoic acids in addition to methanol yielded functionalized polyhydroxyalkanoates (PHAs) and uncovered how M. extorquens copes with fatty acids. While some parts of the degradation pathway remain unclear, possible metabolic routes are suggested that may explain the significant loss of double bonds prior to polymerization of PHA precursors and occurrence of odd-numbered monomers derived from even-numbered n-alkenoic acids. In addition, microbial discoloration upon fatty acid feeding is discussed and future directions for further genetic modification of M. extorquens are provided.
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Introducing a new bioengineered bug: Methylobacterium extorquens tuned as a microbial bioplastic factory.
Bioengineered bugs, 2011Co-Authors: Philipp Höfer, Patrick Vermette, Denis GroleauAbstract:Discussion on and use of methanol as chemical feedstock and as alternative fuel has gained momentum during the past years. Consequently, microorganism and product design based on ‘’methylotrophism’’ is in vogue as reflected by increasing research and development activities in methanol-related areas. A recent example of microorganism and product development is the use of recombinant Methylobacterium extorquens ATCC 55366 strains in the production of second generation biopolyesters. Feeding n-alkenoic acids in addition to methanol yielded functionalized polyhydroxyalkanoates (PHAs) and uncovered how M. extorquens copes with fatty acids. While some parts of the degradation pathway remain unclear, possible metabolic routes are suggested that may explain the significant loss of double bonds prior to polymerization of PHA precursors and occurrence of odd-numbered monomers derived from even-numbered n-alkenoic acids. In addition, microbial discoloration upon fatty acid feeding is discussed and future directions ...
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production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains
Microbial Cell Factories, 2010Co-Authors: Philipp Höfer, Patrick Vermette, Carlos B. Miguez, Young J. Choi, Michael J Osborne, Denis GroleauAbstract:Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch), yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase appeared superior to the PhaC1 synthase at utilizing C8= as source of C-C double bonds and at incorporating C-C double bonds into PHA from either C6= or C8=. The M. ex-phaC2 strain is, therefore, a promising biocatalyst for generating advanced (functionalized) PHAs for future high value applications in various fields.
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multicopy integration and expression of heterologous genes in Methylobacterium extorquens atcc 55366
Applied and Environmental Microbiology, 2006Co-Authors: Young J. Choi, Denis Groleau, Denis Bourque, Lyne Morel, Carlos B. MiguezAbstract:High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (PmxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [beta-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.
Markus Laukel - One of the best experts on this subject based on the ideXlab platform.
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comparison of the proteome of Methylobacterium extorquens am1 grown under methylotrophic and nonmethylotrophic conditions
Proteomics, 2004Co-Authors: Markus Laukel, Michel Rossignol, Gisele Borderies, Uwe VolkerAbstract:Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C 2 , C 3 , and C 4 compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM 1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO 2 and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.
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purification of the formate tetrahydrofolate ligase from Methylobacterium extorquens am1 and demonstration of its requirement for methylotrophic growth
Journal of Bacteriology, 2003Co-Authors: Christopher J. Marx, Markus LaukelAbstract:The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C1 transfers, the tetrahydrofolate (H4F) pathway and the tetrahydromethanopterin (H4MPT) pathway, and both are required for growth on C1 compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H4F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C1 compounds. Unlike mutants defective for the H4MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [14C]methanol to [14C]CO2. Collectively, these data confirm our previous presumptions that the H4F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H4F pathway in this organism in which it functions to convert formate to methylene H4F for assimilatory metabolism.
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the tungsten containing formate dehydrogenase from Methylobacterium extorquens am1 purification and properties
FEBS Journal, 2003Co-Authors: Markus Laukel, Ludmila ChistoserdovaAbstract:NAD-dependent formate dehydrogenase (FDH1) was isolated from the α-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (α1β1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme ≈ 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and ≈ 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the α-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron–sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The β-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron–sulfur cluster binding motif. The β-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.