Researchers develop high-rate, high-yield bacterial process to convert methane to methanol

Cartoon of the process. Click to enlarge.

Researchers at Columbia University have developed a biological process utilizing autotrophic ammonia-oxidizing bacteria (AOB) for the conversion of methane (CH₄) to methanol (CH₃OH). A paper on their work is published in the ACS journal Environmental Science & Technology.

In fed-batch reactors using mixed nitrifying enrichment cultures from a continuous bioreactor, up to 59.89 ¬± 1.12 mg COD/L (COD = chemical oxygen demand, an indirect measurement of organic compounds in water) of CH₃OH was produced within an incubation time of 7 h-approximately 10x the yield obtained previously using pure cultures of Nitrosomonas europaea. Themaximum specific rate of CH₄ to CH₃OH conversion obtained during this study was 0.82 mg CH₃OH COD/mg AOB biomass COD-d-1.5x times the highest value reported with pure cultures.

There are intense efforts globally to develop biobased fuels, chemicals, and energy. While ethanol has been of primary focus in the past few years, it should be noted that other chemicals and biofuels such as methanol can be also attractive. In addition to being used in gasoline blends, methanol can be used in fuel cells, combined with long-chain fatty acids and lipids to form biodiesel, or chemically dimerized to dimethyl ether (DME, also a fuel). Methanol is also one of the most widely used chemicals for enhancing denitrification in wastewater treatment. Methanol is commonly produced from natural gas, by chemical catalysis. The chemical pathway first involves the oxidation of CH₄ to CO₂ and H₂ and subsequent reduction of CO₂ to CH₃OH, and is quite economically and energy intensive and redundant.

Given that natural gas reserves are finite, it might be more sustainable to look toward alternate sources of CH₄ to produce CH₃OH, such as anaerobic digester gas, biogas, or landfill gas, which in addition contain moisture and CO₂. However, the primary limitation to the more widespread use of such gas mixtures is the cost and energy required to purify the CH₄ present and the challenges of handling a gaseous stream.

On the other hand, ammonia-oxidizing bacteria (AOB) can oxidize CH₄ to CH₃OH via the nonspecific action of the enzyme ammonia monooxygenase (AMO). The other benefit of using bacterial conversion of CH₄ to CH₃OH is that the contaminants such as moisture and CO₂, which need to be removed from anaerobic digestion gas or biogas for chemical conversion to CH₃OH, do not pose a limitation for biological conversion. In fact autotrophic AOB can also utilize the CO₂ contained in gas mixtures for cell synthesis.

-Taher and Chandran

The rationale behind using AOB to oxide methane instead of using methane-oxidizing bacteria (MOB) is rather straightforward, the authors note in their paper: MOB oxidize methane completely to CO₂, which cannot be used readily as a fuel. In other words, if MOB were to be used for methanol production, there would need to be some likely non-trivial engineering to selectively inhibit the metabolic pathways that further process CH₃OH.

AOB only oxidize CH₄ partially to CH₃OH-and possibly to trace amounts of formaldehyde (HCHO), which is highly toxic to AOB. Feedback inhibits any further oxidation of CH₃OH to HCHO.

AOB do not derive any energy or reducing equivalents from this process, and since AMO requires reducing power to function, continued CH₄ oxidation can likely be limited unless reducing power is supplied externally. NH₃ is not an ideal or direct source of reducing power, since it can competitively inhibit methane oxidation. The researchers posited that the use of an alternate reducing power source such as NH₂OH could promote AOB-mediated CH₄ oxidation to CH3OH. They also hypothesized that uncoupling NH₃ and CH₄ feeding strategies could promote CH₄ oxidation to CH₃OH by avoiding competition between these two substrates for AMO.

The results obtained highlight the metabolic versatility of AOB to convert CH₄ to CH₃OH and point to the possibility of developing engineered processes to promote the production and utilization of CH₄ as a chemical needed for enhanced denitrification. Once optimized, the successful implementation of this process could potentially allow wastewater treatment plants to offset some of their CH₃OH costs.

Consequently, the overall greenhouse footprint of wastewater treatment plants (by lowering CH₄ release as well as recovering CH₃OH) could be reduced. At the same time, through this microbially mediated approach, redundancies in currently followed chemical conversion of CH₄ to CH₃OH can be avoided. Further mechanistic and modeling studies are needed to understand the substrate and product fluxes during AOB mediated oxidation of CH₄ to CH₃OH oxidation and to maximize the kinetics and yield of CH₃OH.

-Taher and Chandran

Resources

• Edris Taher and Kartik Chandran (2013) High-Rate, High-Yield Production of Methanol by Ammonia-Oxidizing Bacteria. Environmental Science & Technology doi: 10.1021/es3042912

http://www.greencarcongress.com/2013/03/taher-20130322.htm

MIT team shows targeting metabolic pathways to mitochondria significantly boosts yeast production of isobutanol; potential for other chemicals as well

Researchers from MIT and the Whitehead Institute for Biomedical Research have devised a way to boost significantly isobutanol production in yeast by engineering isobutanol synthesis to take place entirely within mitochondria.

They showed that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway-a three-step catalytic breakdown of valine that produces isobutanol, earlier post-into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. A paper on their work is published in the journal Nature Biochemistry.

Metabolic engineering of cytoplasmic biosynthetic pathways to create industrial strains of S. cerevisiae is commonplace, whereas engineering of biosynthetic pathways that function in mitochondria has largely been ignored. Yet mitochondria have many potential advantages for metabolic engineering, including the sequestration of diverse metabolites, such as heme, tetrahydrofolate, ubiquinone, α-ketoacids, steroids, aminolevulinic acid, biotin and lipoic acid. In addition, mitochondria contain intermediates of many central metabolic pathways, including the tricarboxylic acid (TCA) cycle, amino-acid biosynthesis and fatty-acid metabolism.

The environment in the mitochondrial matrix differs from that in the cytoplasm: it has higher pH, lower oxygen concentration and a more reducing redox potential. This environment may more closely match the optimal for maximal activity of many enzymes such as the iron-sulfur clusters (ISCs), which are essential cofactors of enzymes in diverse pathways including branched-chain amino acid and isoprenoid biosynthetic pathways, and which are synthesized exclusively in mitochondria. Although ISCs can be exported to the cytoplasm, the molecular machinery that loads ISCs onto extramitochondrial enzymes is likely to be incompatible with most exogenous ISC apoenzymes, especially those of bacterial or archaeal origin. The smaller volume of mitochondria could concentrate substrates favoring faster reaction rates and productivity, and confine metabolic intermediates, avoiding repressive regulatory responses, diversion of intermediates into competing pathways or even toxic effects of intermediates to cytoplasmic or nuclear processes.

-Avalos et al.

Though still short of the scale needed for industrial production, the advance suggests that this is a promising approach to engineering not only isobutanol but other useful chemicals as well, says Gregory Stephanopoulos, an MIT professor of chemical engineering and one of the senior authors of the paper.

It's not specific to isobutanol. It's opening up the opportunity to make a lot of biochemicals inside an organelle that may be much better suited for this purpose compared to the cytosol of the yeast cells.

-Gregory Stephanopoulos

Stephanopoulos collaborated with Gerald Fink, an MIT professor of biology and member of the Whitehead Institute, on this research. The lead author of the paper is José Avalos, a postdoc at the Whitehead Institute and MIT.

Yeast typically produce isobutanol in a series of reactions that take place in two different cell locations. The synthesis begins with pyruvate, a plentiful molecule generated by the breakdown of sugars such as glucose. Pyruvate is transported into the mitochondria, where it can enter many different metabolic pathways, including one that results in production of valine, an amino acid. Alpha-ketoisovalerate (alpha-KIV), a precursor in the valine and isobutanol biosynthetic pathways, is made in the mitochondria in the first phase of isobutanol production.

Valine and alpha-KIV can be transported out to the cytoplasm, where they are converted by a set of enzymes into isobutanol. Other researchers have tried to express all the enzymes needed for isobutanol biosynthesis in the cytoplasm. However, it's difficult to get some of those enzymes to function in the cytoplasm as well as they do in the mitochondria.

The MIT researchers took the opposite approach: They moved the second phase, which naturally occurs in the cytoplasm, into the mitochondria. They achieved this by engineering the metabolic pathway's enzymes to express a tag normally found on a mitochondrial protein, directing the cell to send them into the mitochondria.

This enzyme relocation boosted the production of isobutanol by 260%, and yields of two related alcohols, isopentanol and 2-methyl-1-butanol, went up even more-370% and 500%, respectively.

There are likely several explanations for the increase, the researchers say. One strong possibility, though difficult to prove experimentally, is that clustering the enzymes together makes it more likely that the reactions will occur, Avalos says.

Another possible explanation is that moving the second half of the pathway into the mitochondria makes it easier for the enzymes to snatch up the limited supply of precursors before they can enter another metabolic pathway.

Enzymes from the second phase, which are naturally out here in the cytoplasm, have to wait to see what comes out of the mitochondria and try to transform that. But when you bring them into the mitochondria, they're better at competing with the pathways in there.

-José Avalos

The findings could have many applications in metabolic engineering. There are many situations where it could be advantageous to confine all of the steps of a reaction in a small space, which may not only boost efficiency but also prevent harmful intermediates from drifting away and damaging the cell.

The researchers are now trying to further boost isobutanol yields and reduce production of ethanol, which is still the major product of sugar breakdown in yeast.

The research was funded by the National Institutes of Health and Shell Global Solutions.

Resources

• Jos√© L Avalos, Gerald R Fink & Gregory Stephanopoulos (2013) Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnology (2013) doi: 10.1038/nbt.2509

http://www.greencarcongress.com/2013/02/chromatin-20130218.htm

http://www.greencarcongress.com/2013/02/mit-20130218.htm

New metabolic engineering tool for microbial cell factories for chemicals, fuels and materials

A South Korean research team led by Sang Yup Lee at the Korea Advanced Institute of Science and Technology (KAIST) has developed a new metabolic engineering tool to construct efficiently microbial cell factories producing desired chemicals, fuels and materials. The new tool allows fine control of gene expression level by employing synthetic small regulatory RNAs; a paper on the work is published in the journal Nature Biotechnology.

Biotechnologists have been working to develop sustainable processes for the production of chemicals, fuels and materials from renewable non-food biomass. One promising technology is the use of microbial cell factories for the efficient production of desired chemicals and materials.

When microorganisms are isolated from nature, their performance in producing desired products is rather poor. Metabolic engineering is performed to improve the metabolic and cellular characteristics to achieve enhanced production of desired product at high yield and productivity. Since the performance of microbial cell factory is very important in lowering the overall production cost of the bioprocess, many different strategies and tools have been developed for the metabolic engineering of microorganisms.

One of the big challenges in metabolic engineering is to find the best platform organism and to find those genes to be engineered so as to maximize the production efficiency of the desired chemical. Even Escherichia coli, the most widely utilized simple microorganism, has thousands of genes, the expression of which is highly regulated and interconnected to finely control cellular and metabolic activities. Thus, the complexity of cellular genetic interactions is beyond our intuition and thus it is very difficult to find effective target genes to engineer.

Together with gene amplification strategy, gene knockout strategy has been an essential tool in metabolic engineering to redirect the pathway fluxes toward our desired product formation. However, experimenting to engineer many genes can be rather difficult due to the time and effort required; for example, a gene deletion experiment can take a few weeks depending on the microorganisms.

Furthermore, as certain genes are essential or play important roles for the survival of a microorganism, gene knockout experiments cannot be performed. Moreover, there are many different microbial strains one can employ. There are more than 50 different E. coli strains that metabolic engineers can consider. Since gene knockout experiments are hard-coded (that is, one should repeat the gene knockout experiments for each strain), the result cannot be easily transferred from one strain to another.

The paper addresses this issue and suggests a new strategy for identifying gene targets to be knocked out or knocked down through the use of synthetic small RNA. Professor Lee's team reported that synthetic small RNA can be employed for finely controlling the expression levels of multiple genes at the translation level. Already well-known for their systems metabolic engineering strategies, Professor Lee's team added one more strategy to efficiently develop microbial cell factories for the production of chemicals and materials.

Here, we report the development of a general strategy for modulating gene expression at the translation stage using synthetic sRNAs that are rationally designed (rather than randomly screened), and we provide proof-of-concept applications to metabolic engineering by increasing the production of tyrosine and cadaverine in E. coli. The synthetic sRNA-based strategy reported here is advantageous over conventional gene-knockout strategies and other large-scale target identification strategies because of its easy implementation and because it does not rely on pre-constructed strain libraries.

-Na et al.

Gene expression works like this: the hard-coded blueprint (DNA) is transcribed into messenger RNA (mRNA), and the coding information in mRNA is read to produce protein by ribosomes. Conventional genetic engineering approaches have often targeted modification of the blueprint itself (DNA) to alter organism's physiological characteristics. Again, engineering the blueprint itself takes much time and effort, and in addition, the results obtained cannot be transferred to another organism without repeating the whole set of experiments.

Professor Lee and his colleagues aimed at controlling the gene expression level at the translation stage through the use of synthetic small RNA. They created novel RNAs that can regulate the translation of multiple messenger RNAs (mRNA), and consequently varying the expression levels of multiple genes at the same time. Briefly, synthetic regulatory RNAs interrupt gene expression process from DNA to protein by destroying the messenger RNAs to different yet controllable extents. The advantages of taking this strategy of employing synthetic small regulatory RNAs include simple, easy and high-throughput identification of gene knockout or knockdown targets, fine control of gene expression levels, transferability to many different host strains, and possibility of identifying those gene targets that are essential.

As proof-of-concept demonstration of the usefulness of this strategy, Professor Lee and his colleagues applied it to develop engineered E. coli strains capable of producing an aromatic amino acid tyrosine, which is used for stress symptom relief, food supplements, and precursor for many drugs. They examined a large number of genes in multiple E. coli strains, and developed a highly efficient tyrosine producer. Also, they were able to show that this strategy can be employed to an already metabolically engineered E. coli strain for further improvement by demonstrating the development of highly efficient producer of cadaverine, an important platform chemical for nylon in the chemical industry.

The design principles and the engineering strategy using synthetic sRNAs reported here are generalizable to other bacteria and applicable in developing superior producer strains. The ability to fine-tune target genes with designed sRNAs provides substantial advantages over gene-knockout strategies and other large-scale target identification strategies owing to its easy implementation, ability to modulate chromosomal gene expression without modifying those genes and because it does not require construction of strain libraries.

-Na et al.

This new strategy, being simple yet very powerful for systems metabolic engineering, could facilitate the efficient development of microbial cell factories capable of producing chemicals, fuels and materials from renewable biomass.

This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.

Resources

• Dokyun Na, Seung Min Yoo, Hannah Chung, Hyegwon Park, Jin Hwan Park, and Sang Yup Lee (2013) Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology doi: 10.1038/nbt.2461

http://www.greencarcongress.com/2013/01/na-20130122.htm

Microbes produce fuels directly from biomass

This is an exciting time when so many breakthroughs are being made which beckon with a bright future of limitless clean energy. There is also this bleak future we're facing where dependency on fossil fuels will doom our society to death and destruction of the wonderful lifestyle we currently enjoy. Somewhere between those two extremes is where our actual future is. If a new biotech company, LS9, has anything to say about it our future is closer to the brightness of limitless clean energy than the darkness of peak-oil-post-apocalyptic doom. LS9 has developed a microbe that can produce an advanced biofuel directly from biomass. Deploying the tools of synthetic biology, the JBEI researchers engineered a strain of Escherichia coli (E. coli) bacteria to produce biodiesel fuel and other important chemicals derived from fatty acids.

What that means is taking pretty much any kind of plant material, feeding it to specially engineered bacteria, who then produce fuels.

Fuels and chemicals have been produced from the fatty acids in plant and animal oils for more than a century. These oils now serve as the raw materials not only for biodiesel fuel, but also for a wide range of important chemical products including surfactants, solvents and lubricants.

The researchers engineered a new strain of E. coli to produce hemicellulases - enzymes that are able to ferment hemicellulose, the complex sugars that are a major constituent of cellulosic biomass and a prime repository for the energy locked within plant cell walls. The work identifies a potentially cost-effective way of converting grass or crop waste directly into fuel, filling gas tanks without raising global food prices or increasing hunger and deforestation in far-flung locales. Moreover, the process is much more climate friendly than manufacturing ethanol from maize, and produces higher-energy fuels that are interchangeable with current petroleum products. The next step is to scale the process up and adapt it to cellulose, which makes up the bulk of plant material.

Article Reference: 

Microbes produce fuels directly from biomass

http://www.ls9.com/

Altered microbe makes biofuel