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1.
Metab Eng ; 71: 99-116, 2022 05.
Article in English | MEDLINE | ID: mdl-34547453

ABSTRACT

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.


Subject(s)
Methane , Methanol , Carbon/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , Methane/metabolism , Methanol/metabolism
2.
Front Microbiol ; 12: 638426, 2021.
Article in English | MEDLINE | ID: mdl-33643274

ABSTRACT

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

3.
Appl Microbiol Biotechnol ; 105(2): 869-876, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33404828

ABSTRACT

Recent attempts to create synthetic Escherichia coli methylotrophs identified that de novo biosynthesis of amino acids, in the presence of methanol, presents significant challenges in achieving autonomous methylotrophic growth. Previously engineered methanol-dependent strains required co-utilization of stoichiometric amounts of co-substrates and methanol. As such, these strains could not be evolved to grow on methanol alone. In this work, we have explored an alternative approach to enable biosynthesis of all amino acids from methanol-derived carbon in minimal media without stoichiometric coupling. First, we identified that biosynthesis of threonine was limiting the growth of our methylotrophic E. coli. To address this, we performed adaptive laboratory evolution to generate a strain that grew efficiently in minimal medium with methanol and threonine. Methanol assimilation and growth of the evolved strain were analyzed, and, interestingly, we found that the evolved strain synthesized all amino acids, including threonine, from methanol-derived carbon. The evolved strain was then further engineered through overexpression of an optimized threonine biosynthetic pathway. We show that the resulting methylotrophic E. coli strain has a methanol-dependent growth phenotype with homoserine as co-substrate. In contrast to previous methanol-dependent strains, co-utilization of homoserine is not stoichiometrically linked to methanol assimilation. As such, future engineering of this strain and successive adaptive evolution could enable autonomous growth on methanol as the sole carbon source. KEY POINTS: • Adaptive evolution of E. coli enables biosynthesis of all amino acids from methanol. • Overexpression of threonine biosynthesis pathway improves methanol assimilation. • Methanol-dependent growth is seen in minimal media with homoserine as co-substrate.


Subject(s)
Escherichia coli , Methanol , Amino Acids , Carbon , Escherichia coli/genetics , Laboratories
4.
Metab Eng ; 61: 1-10, 2020 09.
Article in English | MEDLINE | ID: mdl-32360074

ABSTRACT

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.


Subject(s)
Bacterial Proteins , Escherichia coli Proteins , Escherichia coli , Gene Expression Regulation, Bacterial , Methanol/metabolism , Sigma Factor , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Sigma Factor/biosynthesis , Sigma Factor/genetics
5.
Metab Eng ; 60: 45-55, 2020 07.
Article in English | MEDLINE | ID: mdl-32179162

ABSTRACT

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h-1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and 13C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from 13C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.


Subject(s)
Escherichia coli/growth & development , Escherichia coli/metabolism , Glucose/metabolism , Metabolic Engineering/methods , Methanol/metabolism , Biomass , Citric Acid Cycle , Escherichia coli Proteins/genetics , Gene Deletion , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/metabolism , Isocitrate Dehydrogenase/metabolism , Prophages/genetics
6.
Metab Eng ; 52: 168-177, 2019 03.
Article in English | MEDLINE | ID: mdl-30529131

ABSTRACT

Overcoming carbon catabolite repression presents a significant challenge, largely due to the complex regulatory networks governing substrate catabolism, even in microbial cells. In this work, we have engineered an E. coli strain, which we have named X2G, that not only exhibits a reversed substrate preference for xylose over glucose, but also demonstrates an unusual ability to produce significant amounts of glucose. We obtained this non-intuitive phenotype by deleting four genes in upper central metabolism: ptsI, glk, pfkA, and zwf, which respectively encode Enzyme I of the phosphotransferase system, glucokinase, the dominant isozyme of phosphofructokinase, and glucose-6-phosphate dehydrogenase. The deletion of ptsI and glk blocks glucose uptake in E. coli, while the deletion of pfkA and zwf prevents the reassimilation of carbons through glycolysis and the oxidative pentose phosphate pathway, respectively. Our strain X2G is capable of converting 34% of the carbon it takes up as xylose into exported glucose. This corresponds to a glucose production rate of 1.4 ±â€¯0.3 mmol/gDW/h at a specific growth rate of 0.25 ±â€¯0.03 h-1, or about 1.8 ±â€¯0.1 mM of glucose accumulated for every unit increase in OD600. Despite a 22% decrease in xylose uptake rate, a 33% decrease in biomass yield, and a 52% decrease in acetate production rate relative to the wild-type, the intracellular flux profile and cofactor allocation of X2G remain largely unperturbed, as elucidated through 13C-metabolic flux analysis. Further quantification of the pool sizes of key intracellular metabolites revealed that glucose secretion by X2G is likely driven by the substantial accumulation of intracellular glucose 6-phosphate, fructose 6-phosphate, glucose and fructose at levels greater than 20x of that in wild-type E. coli. Combined, our results shed light on the flexibility of central metabolism, and the opportunities this affords for producing value-added pentose- and hexose-derived products from lignocellulosic feedstocks.


Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Gene Deletion , Glucose/metabolism , Xylose/metabolism , Biomass , Catabolite Repression , Fermentation , Fructose/biosynthesis , Glucokinase/metabolism , Glucosephosphate Dehydrogenase/metabolism , Glycolysis , Metabolic Engineering , Metabolic Flux Analysis , Pentose Phosphate Pathway/genetics , Phosphofructokinases/metabolism
7.
Metab Eng ; 50: 173-191, 2018 11.
Article in English | MEDLINE | ID: mdl-30055325

ABSTRACT

Clostridium organisms are of major importance in the development of technologies to produce biofuels and chemicals. They are uniquely capable of utilizing virtually all biomass-derived carbohydrates, as well as waste gases, waste materials, and C1 compounds, and they possess diverse biosynthetic capabilities for producing a broad spectrum of metabolites, including those of C4-C8 chain length. They can also be readily used in synthetic, syntrophic, and other microbial consortia to broaden the biosynthetic repertoire of individual organisms, thus enabling the development of novel biotechnological processes. Engineering Clostridium organisms at the molecular and population level is hampered by genetic engineering, genome engineering, and microbial-population engineering tools. We discuss these challenges, and the promise that derives from their resolution aiming to usher in an era of broader use of Clostridium organisms as biotechnological platforms.


Subject(s)
Clostridium/genetics , Clostridium/metabolism , Metabolic Engineering/methods , Metabolic Engineering/trends , Microbial Consortia , Carbohydrate Metabolism/genetics
8.
Curr Opin Biotechnol ; 50: 81-93, 2018 04.
Article in English | MEDLINE | ID: mdl-29216497

ABSTRACT

Methylotrophy describes the ability of organisms to utilize reduced one-carbon compounds, notably methane and methanol, as growth and energy sources. Abundant natural gas supplies, composed primarily of methane, have prompted interest in using these compounds, which are more reduced than sugars, as substrates to improve product titers and yields of bioprocesses. Engineering native methylotophs or developing synthetic methylotrophs are emerging fields to convert methane and methanol into fuels and chemicals under aerobic and anaerobic conditions. This review discusses recent progress made toward engineering native methanotrophs for aerobic and anaerobic methane utilization and synthetic methylotrophs for methanol utilization. Finally, strategies to overcome the limitations involved with synthetic methanol utilization, notably methanol dehydrogenase kinetics and ribulose 5-phosphate regeneration, are discussed.


Subject(s)
Biofuels , Metabolic Engineering/methods , Methane/metabolism , Methanol/metabolism , Alcohol Oxidoreductases/metabolism , Metabolome
9.
Metab Eng ; 45: 67-74, 2018 01.
Article in English | MEDLINE | ID: mdl-29203222

ABSTRACT

Methane, the main component of natural gas, can be used to produce methanol which can be further converted to other valuable products. There is increasing interest in using biological systems for the production of fuels and chemicals from methanol, termed methylotrophy. In this work, we have examined methanol assimilation metabolism in a synthetic methylotrophic E. coli strain. Specifically, we applied 13C-tracers and evaluated 25 different co-substrates for methanol assimilation, including amino acids, sugars and organic acids. In particular, co-utilization of threonine significantly enhanced methylotrophy. Through our investigations, we proposed specific metabolic pathways that, when activated, correlated with increased methanol assimilation. These pathways are normally repressed by the leucine-responsive regulatory protein (lrp), a global regulator of metabolism associated with the feast-or-famine response in E. coli. By deleting lrp, we were able to further enhance the methylotrophic ability of our synthetic strain, as demonstrated through increased incorporation of 13C carbon from 13C-methanol into biomass.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/metabolism , Gene Deletion , Leucine-Responsive Regulatory Protein/genetics , Methanol/metabolism , Escherichia coli/genetics
10.
Metab Eng ; 45: 75-85, 2018 01.
Article in English | MEDLINE | ID: mdl-29203223

ABSTRACT

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. Introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.


Subject(s)
Bacillus/genetics , Escherichia coli Proteins , Escherichia coli , Gene Expression , Glucose-6-Phosphate Isomerase , Methanol/metabolism , Microorganisms, Genetically-Modified , Pentose Phosphate Pathway , Bacillus/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/metabolism , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/metabolism
11.
Metab Eng ; 39: 49-59, 2017 01.
Article in English | MEDLINE | ID: mdl-27815193

ABSTRACT

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.


Subject(s)
Alcohol Oxidoreductases/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/physiology , Flavanones/biosynthesis , Metabolic Engineering/methods , Metabolic Networks and Pathways/physiology , Methanol/metabolism , Alcohol Oxidoreductases/metabolism , Biosynthetic Pathways/physiology , Escherichia coli Proteins/genetics , Flavanones/genetics , Genetic Enhancement/methods
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