2000 Progress Report: Metabolic Engineering of Methylotrophic Bacteria for Conversion of Methanol to Higher Value Added Products

EPA Grant Number: R826729
Title: Metabolic Engineering of Methylotrophic Bacteria for Conversion of Methanol to Higher Value Added Products
Investigators: Lidstrom, Mary E. , Dien, Steven Van
Current Investigators: Lidstrom, Mary E.
Institution: University of Washington
EPA Project Officer: Richards, April
Project Period: October 1, 1998 through September 30, 2001
Project Period Covered by this Report: October 1, 1999 through September 30, 2000
Project Amount: $190,000
RFA: Technology for a Sustainable Environment (1998) RFA Text |  Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development


In the future, environmental concerns will mandate that manufacturing processes shift towards the use of renewable resources and the minimization of wastes, especially hazardous wastes. One approach for achieving this goal is to use nontoxic renewable feedstocks in conjunction with environmentally benign microbial processes, and this combination is increasingly of interest as part of a long-term waste-minimization strategy for industry. One of the major bacterial substrates of interest for developing environmentally benign bioprocesses is methanol. Because methanol is generated from methane commercially, it is a relatively nontoxic renewable chemical feedstock that is soluble, relatively inexpensive (most recent annual average bulk price ~$0.50/gal, or $0.07/lb; http://www.methanex.com/ methanol/current price.htm), and easy to handle. In addition, bacteria exist that grow on C1 compounds (methylotrophs), and at least for some strains, a great deal is known about methylotrophic metabolism. Therefore, the concept that methylotrophic bacteria could serve as nonpolluting multistage catalysts to generate chemicals and materials using methanol as a feedstock is a highly attractive one. Alternatively, it might be of interest to convert a heterotrophic bacterium to use of C1 compounds by genetic engineering. However, dozens of genes are necessary for methylotrophic growth, and many of these appear to be involved in maintaining a balance of the toxic intermediate, formaldehyde. It is unlikely that it would be possible to achieve the desired yields and productivities from C1 compounds by genetically engineering a nonmethylotrophic strain.

Because nature has not designed bacteria to make products under large-scale commercial process conditions, metabolic engineering of methylotrophs will be required to develop strains with more desirable process characteristics. Ideally, for the development of economically viable bioprocesses, the cellular metabolic network should be redirected to synthesizing a specific product from methanol at high efficiency and yield. To achieve such a goal, it is necessary to understand and manipulate central metabolism. In the past, insufficient metabolic information was available to address such goals in methylotrophs. However, the ability to apply genome-wide approaches to the understanding of metabolic pathways significantly has increased our ability to understand whole-cell metabolic networks. As a result, the potential now exists to "re-evolve" bacteria on a genome-wide scale into chemical production strains in a manner conceptually similar to the way chemical manufacturing plants are designed. Because genome sequences are available for methylotrophs, the tools are in hand to create such "re-evolved" production strains for converting methanol to higher value-added products such as vitamins, amino acids, and other bioorganic chemicals.

The long-term goal of this project is to develop optimized strains for converting methanol into higher value-added products, by genome-wide metabolic engineering of a methylotrophic bacterium, Methylobacterium (M.) extorquens AM1. The hypothesis to be tested is that energy metabolism can be optimized and carbon flux can be directed to specific metabolic branches by manipulating central metabolism, specifically, by increasing flux through the assimilatory pathway (the serine cycle) and by increasing available NAD(P)H. The intent is to convert this methylotroph into a platform strain, optimized for process parameters, that could be used for the production of any bioorganic product from methanol. The results of this work will be the foundation for developing other production strains and for the scale-up of methylotrophic bioprocesses.

Progress Summary:

This proposal is funded jointly by EPA and the National Science Foundation (NSF). The goals of the project are to: (1) develop a stoichiometric model for metabolism in M. extorquens AM1; (2) apply the model towards an understanding of methylotrophic energy metabolism; and (3) analyze poly- -hydroxy-butyrate (PHB) metabolism and carry out metabolic engineering to optimize PHB production as a model system. In the 2.5 years of this project, substantial progress has been made in these areas, although a major surprise from the final goal has changed our approach.

Stoichiometric Model. A stoichiometric model for serine cycle methylotrophs was developed using a flux balance approach and was used to analyze the growth of methylotrophs with various carbon sources. The model contains 68 mass-balance equations, one on each metabolite, and 65 unknown fluxes, describing the putative central metabolic pathways of M. extorquens AM1. Because some of these central pathways are not yet completely known, several variations of the system were considered. Fifteen key components were identified to be metabolic intermediates and energy-carrying molecules needed by the organism for biomass synthesis, based upon the known biosynthesis pathways of E. coli. E. coli metabolism was used as a starting point, but as more information becomes available regarding metabolic pathways in M. extorquens AM1, the pathways are being modified. The biomass composition during growth on C1 and non-C1 compounds was determined experimentally. Given this biomass composition, these pathways were then used to calculate the requirement of each precursor to synthesize a gram of biomass. Because the energy content and oxidation state of all biomass components are very similar, the model is rather insensitive to small errors in biomass composition or differences between the biosynthesis pathways of M. extorquens AM1 and E. coli.

The model was applied to different possible variations in the metabolic pathways and to growth on C1 and multicarbon substrates. Convex analysis was used to find the elementary flux modes of the stoichiometric matrix. The set of elementary modes represents the vertices of the solution space and contains the optimal solution, which in this case was defined as that maximizing biomass yield. Inspection of the elementary modes indicates that for each case, there are many optimal or near-optimal solutions having only minor differences between them. One general conclusion drawn from these simulations is that during growth on methanol, availability of NAD(P)H for biosynthesis and PHB formation is likely to be a bottleneck, but ATP production is not. This has focused our metabolic reconstruction efforts on understanding NAD(P)H production and consumption.

The model was then applied to the utilization of M. extorquens AM1 as a biocatalyst for the production of useful compounds from methanol. The synthesis of carotenoids was chosen as an example because these pigments are synthesized naturally in this organism and have several industrial uses. The optimal flux distribution was calculated as a function of the desired product/biomass ratio. Changes in the flux distribution were observed with increasing product concentration, thus identifying potential target enzymes for metabolic engineering.

Deciphering the Metabolic Network. In the metabolic network described above, only about half of the reactions have been verified to occur in M. extorquens AM1. Most of these lie in the methylotrophy-specific pathways, leaving the metabolism of C3, C4, and larger molecules relatively unexplored. We have initiated an investigation of this region of the metabolic map by identifying genes involved in growth on succinate or pyruvate. The first approach has been to identify genes predicted to be involved in growth on these compounds in the genome sequence, mutate them in the chromosome by insertional mutagenesis, and test the phenotype of the mutants. So far, four genes of interest were identified: succinate dehydrogenase, citrate synthase (TCA cycle enzymes), malic enzyme, and PEP carboxykinase (possible C3/C4 interconverting anapleurotic enzymes). Enzyme assays in cell extracts demonstrated the presence of each enzyme activity. The insertional mutagenesis system we use allows the generation of both single-crossover mutants that retain a wild-type copy of the gene, and double-crossover null mutants, so we can test for essential genes in this way. The metabolic model predicts that citrate synthase and succinate dehydrogenase should be required for growth on both methylotrophic and heterotrophic substrates. No null mutants were obtained in these genes, suggesting that the model prediction is correct. The model also predicts that some combination of anapleurotic enzymes should be required for growth on heterotrophic substrates, but not for methylotrophic growth. Null mutants were obtained for malic enzyme and PEP carboxykinase. The first mutant shows slow growth on heterotrophic substrates while the second shows no growth defects, suggesting that malic enzyme probably is a key route from C4 to C3 compounds, while PEP carboxykinase may not be as important. We will continue this directed approach as the genome is annotated and as we identify other genes that may be involved in heterotrophic growth.

A second approach to this problem has been followed, which is to screen a pool of random mutants for growth phenotypes on non-C1 substrates. Such a pool was created by random insertion of a mini-Tn5 cassette into the M. extorquens AM1 chromosome, followed by selection for growth on methanol in the presence of an antibiotic. These insertion mutants were then screened for decreased growth on pyruvate and/or succinate. For those showing a phenotype, the insertion site was mapped by touchdown PCR, a technique using one Tn5-specific and one degenerate primer. After sequencing the PCR product, the site of transposon insertion was located in the M. extorquens AM1 genome sequence. Putative function was assigned based on a BLAST search. To date, over 11,000 mutants have been screened, and 84 of them show some growth defect on pyruvate, succinate, or both. We are beginning to obtain sequence for these; in five cases, the insertion site exhibits high identity to known metabolic genes (Table 1). In each case, the model predicts the correct phenotype. We will continue to screen mutants and obtain sequence until we begin to obtain second mutants in the same gene. In addition, all mutants of interest will be further characterized to confirm that the observed phenotype is a result of the interrupted gene, and to confirm the enzyme activity encoded by the gene.

Table 1. Genes identified to date with specific role in growth on C3 and/or C4 compounds. All mutants grow normally on C1 compounds. pyr-, impaired growth on pyruvate; succ-, impaired growth on succinate
Mutant Phenotype
a ketoglutarate dehydrogenase subunit
pyr- succ-
TCA cycle
a ketoglutarate dehydrogenase subunit
pyr- succ-
TCA cycle
dicarboxylate transporter
succinate transport
glucose-1-P dehydrogenase
pyr- succ-
glucose-6-P isomerase
pyr- succ-

Figure 1. Predicted central metabolic pathways in M. extorquens AM1. Large arrows = major pathways.

Figure 1. Predicted central metabolic pathways in M. extorquens AM1. Large arrows = major pathways.

Metabolism of Poly-b-hydroxybutyrate. M. extorquens AM1 synthesizes about 40 percent of its dry weight as PHB during normal growth on methanol, but only about 15 percent during normal growth on succinate, suggesting a more important role for PHB in methylotrophy than in heterotrophy. The availability of a partial genome sequence made it straightforward to identify by similarity the genes involved in PHB production and degradation, clone them, and generate mutants in them by insertional mutagenesis to test growth phenotypes. The genes were found in multiple clusters, similar to the arrangements common in other bacteria. These included genes predicted to encode two proteins known to be associated with PHB granules (granule-associate proteins; approximately 11 and 21 kDa). In other bacteria, the role of the 21 kDa protein is not known, but the 11 kDa granule-associated protein apparently acts as a repressor of PHB synthesis. By a combination of promoter studies and PHB measurements, we have shown a similar role for this protein in M. extorquens. This protein is expressed at higher levels in succinate-grown cells than in methanol-grown cells, which may be one reason for the difference in PHB production under these conditions. In addition, we have identified a second regulator (meaB), which acts as an activator of PHB synthesis. This work is being written up to be submitted for publication.

We also analyzed the PHB synthesis and degradation genes (Figure 2). Mutants in the degradation genes showed no detectable phenotype for growth or steady-state level of PHB. However, to our surprise, mutants in the PHB synthesis pathway showed growth defects on C1 and C2 compounds. We were able to show that these defects are due to the occurrence of L-hydroxybutyrate (the PHB precursor) as an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate. This conversion is a part of the serine cycle and also is necessary for growth on C2 compounds. The identity of this pathway has been sought for over 30 years, without success, although our earlier work had shown that the conversion of propionyl CoA to succinate was part of this pathway. However, our studies of PHB serendipitously have allowed us to solve this long-standing, central metabolic problem in methylotrophy. We now know that PHB synthesis and methylotrophy are metabolically intertwined, with L-hydroxybutyrate serving as a branch point between assimilatory metabolism (the serine cycle) and PHB synthesis (Figure 2). In the assimilatory route, it is converted to butyryl CoA, and then by an as yet unconfirmed pathway to propionyl CoA. Our hypothesis is that PHB synthesis helps regulate the reducing power balance in the cell (it consumes NADPH) and helps store carbon under conditions of carbon excess, in response to acetyl CoA overproduction. We have generated a transhydrogenase mutant in this bacterium and also have overexpressed E. coli transhydrogenase in the wild-type, and neither strain shows alterations in growth or PHB synthesis. These results suggest that the NADH/NADPH balance is achieved by a mechanism other than transhydrogenase, possibly by passage of carbon through the hydroxybutyrate branch of the serine cycle. This branch has the net effect of converting two NADPH to two NADH. This work has been published (see reprint).

We are completing our understanding of this pathway by determining the route whereby butyryl CoA is converted to propionyl CoA. We have used thin-layer chromatography of specific mutants exposed to 14C-butyrate and GC-MS of accumulated compounds to identify some of the intermediates. Our current data suggest that butyryl CoA is converted to propionyl CoA via methacrylyl CoA and ketobutyrate, possibly with a-hydroxybutyrate as an intermediate (Figure 2). All of the predicted genes for this pathway have been identified in the chromosome, and we are in the process of generating insertion mutants for each. If these mutants confirm both growth and enzyme activity phenotypes, then this work will have solved this long-standing metabolic problem. Although a complete understanding of this pathway and the reduced cofactors involved is essential for understanding energy balance and carbon flow in serine cycle methylotrophs (one-third of the carbon is predicted to flow through this branch of the pathway during methylotrophic growth), it also is of interest in Streptomyces and several photosynthetic bacteria that apparently use this pathway for growth on C2 compounds. Therefore, this work has been important for our long-term goals in this project, but it also has shown that the manipulation of PHB levels during growth on methanol is complicated by the interconnection of these pathways. So far, all mutants with decreased PHB synthesis exhibit poorer growth on methanol than wild-type strains. Therefore, we are focusing on alternate targets.

Our project work has laid the groundwork for a continuing understanding of methylotrophic metabolism and how to manipulate it by metabolic engineering. We have: developed a stoichiometric model and used it to assess our initial understanding of metabolic pathways in M. extorquens AM1; used a combined genomic and mutant approach to identify pathways involved in heterotrophic metabolism and in PHB metabolism; and discovered a previously unknown portion of methylotrophy and its unexpected overlap with PHB metabolism. With this new level of insight into metabolism in this methylotroph, we have been able to identify preliminary bottlenecks for overproduction of organic products from methanol. For example, our results suggest that ATP production capacity is high in this bacterium, but the generation of NAD(P)H for biosynthesis is predicted to be more restrictive.

Future Activities:

NAD(P)H production and consumption pathways are important targets for further studies. In addition, the serine cycle is likely to be a bottleneck for carbon flux, rather than methanol oxidation or formaldehyde/folate condensation. These results form the foundation for the next year of studies.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 3 publications 2 publications in selected types All 2 journal articles
Type Citation Project Document Sources
Journal Article Korotkova N, Lidstrom ME. Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C-1 and C-2 compounds in the methylotroph Methylobacterium extorquens AM1. Journal of Bacteriology 2001;183(3):1038-1046 R826729 (2000)
R826729 (Final)
not available

Supplemental Keywords:

clean technologies, innovative technology, waste reduction, environmentally conscious manufacturing, genetics., RFA, Scientific Discipline, Sustainable Industry/Business, cleaner production/pollution prevention, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, Environmental Engineering, bioprocessing, renewable feedstocks, cleaner production, sustainable development, waste minimization, waste reduction, environmentally conscious manufacturing, consumption pathways, methanol, biotechnology, formaldehyde pollution, methylotrophic bacteria, chemical manufacturing, energy technology, metabolic engineering, carbon flux, pollution prevention, alternative chemical synthesis, environmentally-friendly chemical synthesis, green chemistry, renewable resource

Progress and Final Reports:

Original Abstract
  • 1999
  • Final Report