Monthly archives of “June 2015

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Biosensor screening of metagenomes for lignin biodegradation genes

 

Coal on the edge of the Bow River, near the old coal mines in Canmore, Alberta.

Coal on the edge of the Bow River, near the old coal mines in Canmore, Alberta.

Second Generation Biofuel

The term ‘second generation biofuel’ refers to the use of lignocellulosic material as the feedstock for fermentation reactions to produce ethanol or other hydrocarbons. The advantage over ‘first generation biofuels’ is that cellulose is an extremely abundant polysaccharide from woody plants and agricultural waste can be used as a source of fermentable sugars, in the place of sugars from food crops, such as wheat, corn, beets or sugar cane.

The challenge is that lignocellulose has evolved towards resisting biodegradation because of its important structural role in plant cell walls.  The polysaccharides in cellulose and hemicellulose are heavily cross-linked to lignin, which explains the recalcitrance.  Therefore, in order to improve the process of making second generation biofuels, we need to improve the ability to remove and isolate cellulose from lignin. Fungi are the best known lignin degraders and most enzymes used for lignin transformation have been characterized from fungi. The focus of this blog is to highlight a new approach to identify bacterial enzymes that are involved in lignin transformation, the early stages of degrading lignin, to aid in producing the most abundant source of fermentable sugars for biofuel production1. The ultimate goal is sustainability, to replace the use of fossil fuels and provide a carbon neutral source of energy.

Most bacteria in nature and on the human body are unculturable. Despite this limitation, microbiologists have developed innovative methods to probe these unknown communities and exploit their genetic potential. While we are also improving our ability to culture these organisms in the lab, a common approach is to recover the total DNA from a sample containing a unique bacterial population and to screen this DNA for novel properties, when it’s expressed in a recombinant bacterial host. This ‘shotgun’ approach is also useful to capture the DNA for metagenomic sequencing projects, where bacterial numbers are low.

Microbial processes are essential for the production of oil in the oilsands and coal beds. The Hydrocarbon Metagenomic Project (http://hydrocarbonmetagenomics.com/) is focussed on understanding the complex microbiology in these important sites. Researchers have isolated DNA from oil sands and coalbed samples, and used 16SrRNA metagenomics to identify the bacteria in these environments2. Given the high abundance of aerobic and anaerobic bacteria in these samples, it was hypothesized that these microorganisms would be excellent sources of bacterial lignin degradation enzymes1.

Biosensor of monoaromatic degradation products of lignin

The genius of microbiology is the creativity of genetic screens to identify new stuff. As touched on in our previous post, biosensors are the use of engineered microbial cells used as a detection kit for almost any kind of small molecule, toxin, antibiotic or compound with any interesting application. This first step in identifying lignin transformation genes was the creation of a biosensor to detect the degradation products of lignin.  A library of strains expressing each intergenic region (E. coli promoter) to GFP was screened to identify genes responsive to the presence of lignin degradation products; the monoaromatic compounds vanillin, vacillate and p-coumarate. The emrRAB promoter was very responsive to these and other structurally related compounds, as well as to lignin treated with solvents and enzymes. This operon encodes a transcriptional regulator, and a multidrug resistance pump EmrAB. This pump extrudes a large variety of antibiotics, and is also responding to exposure to these monoaromatic compounds.

Metagenomic libraries

Samples were obtained from Alberta coal beds  and the total DNA was extracted from these samples. It’s safe to say that the bulk of DNA present in these samples will reflect the microbial diversity in a coal bed.  The total DNA is digested into sizeable fragments and cloned into a fosmid cloning vector. Roughly 60,000 fragments each with a size ~ 40 kb were cloned and about a third of these fragments were sequenced. The exciting part was to employ high throughput approaches screen this library of 60,000 strains for the ability to degrade lignin. Each strain is robotically picked and arrayed into 384-well plates, and grown in very small culture volumes for a few hours in the presence of commercially purified lignin. Using a co-culture system, the biosensor strain was then added to each strain in the arrayed library, and co-cultures were screened for increased fluorescence from the biosensor that detects lignin degradation products. I love putting robots to good use!

This high throughput robotic screen identified 24 fosmids that activated the PemrR-GFP biosensor and 8 were selected for further sequencing and characterization. GC-MS was used to specifically identify the degradation products from many of the recombinant strains expressing lignin degrading genes, as detected with the biosensor. The variety and difference in end products produced by the various lignin transforming strains indicates a variety of enzymatic pathways to transform lignin. A limited characterization of the some of the genes encoded on the fosmid was performed. Mutation of the emrR regulator led to an inability to detect lignin degradation products, as well as slight growth defects in their presence. This observation supports the role of this efflux pump in coping with the toxic affects of these aromatic degradation  products. Random transposon mutagenesis was performed to identify specific genes in the large fragments of DNA that were required for degradation.

Functional and bioinformatic analysis revealed the presence of six functional classes of genes that contribute to degrading lignin. These included oxidoreductases, hydrogen peroxide generating enzymes, multidrug efflux systems, protein secretion systems to secrete the degradative enzymes, as well as motility and signal transduction systems that may be required to move toward these nutrient sources in the microscale environments within coal beds. Interestingly, many of the genes identified within the sequenced fosmid clones were associated with genetic elements that facilitate horizontal gene transfer. This evidence suggested that these genes are freely exchanged between microbes in this environment. This is conceptually analogous to pathogens found in hospitals that share advantageous antibiotic resistance genes.

This exciting approach included the construction of a novel biosensor to probe a unique gene pool isolated from lignin-rich coal bed microbial communities and discovered multiple classes of lignin degrading genes. These enzymes may ultimately aid in the refinement of wood waste and lignocellulose to produce fermentable sugars for 2nd generation biofuels. This is the kind of green chemistry discovery that can support new biotechnologies, such as the startup that resulted from this innovative project, MetaMixis Inc. I wish them the best of luck in moving this technology forward.

References

1. Metagenomic scaffolds enable combinatorial lignin transformation.  Strachan CR, Singh R, VanInsberghe D, Ievdokymenko K, Budwill K, Mohn WW, Eltis LD, Hallam SJ. Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10143-8. doi: 10.1073/pnas.1401631111. Epub 2014 Jun 30.

2. Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.  An D, Caffrey SM, Soh J, Agrawal A, Brown D, Budwill K, Dong X, Dunfield PF, Foght J, Gieg LM, Hallam SJ, Hanson NW, He Z, Jack TR, Klassen J, Konwar KM, Kuatsjah E, Li C, Larter S, Leopatra V, Nesbø CL, Oldenburg T, Pagé AP, Ramos-Padron E, Rochman FF, Saidi-Mehrabad A, Sensen CW, Sipahimalani P, Song YC, Wilson S, Wolbring G, Wong ML, Voordouw G. Environ Sci Technol. 2013 Sep 17;47(18):10708-17. doi: 10.1021/es4020184. Epub 2013 Aug 26.

Links

Project Leader of the Hydrocarbon Metagenomic Project

Gerrit Voordouw, Professor, University of Calgary

Canadian Synthetic Biology Researchers

Steven Hallam, Associate Professor, University of British Columbia

Lindsay Eltis, Professor University of British Columbia

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Probiotic biosensor to detect liver cancer

pills_urineOn every visit home, I am greeted and shortly after asked: “how is the cure for cancer coming?” I have yet to find a way to convince my family that I am a microbiologist, I work with bacteria, I don’t try to cure cancer. Well the day has come where microbiologists and virologists have indeed cured cancer. There are numerous examples of viruses that specifically target and kill tumour cells, and several clinical trials to examine this therapy in humans. In fact, my friends in grad school can proudly state they cured cancer with an otherwise, relatively harmless reovirus. One of them went on to start a biotech company using this technology and they are still active (www.oncolyticsbiotech.com).

These cancer killing bacteria and viruses will be the subject of a future post, but here we will focus on the use of bacteria as a cancer detection method. The central observations that motivated this study were that many bacterial species are known to preferentially accumulate and localize in tumours. And not surprisingly, many species of bacteria have been documented to kill diverse types of tumour cells. The general approach to show bacterial growth and localization on tumours is to inject bacteria intravenously into the blood, to facilitate greater access to the organs. This interesting feature of homing in on human tumours is an ability of non-pathogenic bacteria, as well as pathogens. To explain this widespread ability to colonize tumours, it is thought that tumours are sites of suppressed immune surveillance and that are rich in nutrients, both of which support localized bacterial growth.

The authors of this study decided to test the hypothesis that orally administered, harmless probiotic bacteria could pass through the stomach into the normal flow of blood through the hepatic portal vein, to gain access to the liver. The microbiome field has had great success in documenting the abundance and diversity of beneficial bacterial species that consider our body a playground. Not only do they colonize our skin, mouths, respiratory tract, gastrointestinal tracts and the crevices of our toes, but microbiome studies have also reported bacterial DNA signatures in places that were once thought to be sterile, including some organs, the urinary tract, and even the brain. A general conclusion of many microbiome studies is that an altered community of microbes somewhere in the body, is the cause of certain related or unrelated health issues. Although many of these studies identify correlations between healthy and non-healthy microbiomes, and the exact cause of disease is not yet proven, it has definitely opened up the floodgates for the use of probiotics. The well studied probiotic, E coli Nissle 1917 (EcN), was engineered and used as a candidate biosensor for liver tumours, when delivered orally.

A liver cancer model was selected because the liver is the primary site for metastasis of many other tumours. Also, bacteria that are ingested orally, can transit through the stomach or small intestines to access the hepatic portal vein, which supplies the liver with 75% of its blood supply. The resident liver macrophages or Kupffer cells, with help from neutrophils, are the liver’s recycling and filtering system. These resident immune cells clear bacteria present in the blood, and also recycle the iron in red blood cells.

hepatic portal vein_wiki

Designer probiotics

The passage of normal flora microbes from the gut to the liver requires transit across the epithelial barrier of the gastrointestinal tract to the blood, and some researchers have shown that this generally occurs in a disease state. However, in this study using healthy, immunocompetent mice with in tact gut flora, the probiotic E coli Nissle 1917 translocated across the GI tract within 24 hours. EcN is well studied and tolerated as a probiotic.

The authors used a relatively common strategy to engineer EcN to express two different reporter systems: luminescence (luxCDABE) and lacZ. The bacterial-derived lux gene products produce light through enzymatic reactions, which is an ideal reporter system in bacteria. This reporter generates a strong signal in vitro and in vivo during infection or colonization of an animal. LacZ (β-galactosidase) is the work horse of bacterial reporters, and this enzyme cleaves the glycosidic bond between disaccharides into monosaccharides. Numerous substrates have been designed that produce end products upon cleavage that change colour, are fluorescent, luminescent, as well as other output measurements.

True to their hypothesis, oral EcN probiotics indeed targeted the tumours of the liver within 24 hours. The authors employed a unique in vivo imaging application that allowed for the detection of two distinct luminescence signals, one from the firefly luciferase enzyme from the tumour cells, and the other from the localized lux-expressing probiotic strain. The wavelength of light from the firefly luciferase (580-620 nm) varies from that produced by the bacterial luciferase (500-580 nm), allowing for two non-overlapping light signals, very similar to the use of two different fluorophores, such as green fluorescent protein (GFP) and red fluorescent protein (RFP).  The distinct light signals were shown to co-localize, thereby confirming tumour-localized bacterial growth. The oral probiotics did not colonize healthy organs (spleen, kidney, brain, heart, lung) and had no ill health effects for up to 12 months. In contrast, the use in intravenous administration of bacteria could lead to colonization of other organs, the recovery of bacteria in the blood, and likely sepsis.

The engineered EcN expressed the lux and lacZ reporters from either a stable integration on the chromosome, or from potentially unstable expression plasmids. They used additional approaches to ensure plasmid stability without any antibiotics to select for plasmid maintenance, including the  hok-sok (toxin-antitoxin) plasmid maintenance system and the actin-like protein Alp7 from Bacillus subtilis. This protein forms an actin-like filament that physically pushes plasmids to the poles, thereby ensuring segregation of the plasmid during cell division.

And how did this all come together as a diagnostic test for liver cancer? First, tumours were excised and shown to produce the second reporter lacZ at 5X above the background levels. The next step was to determine if by products of the LacZ enzyme could be detected in the urine, which indicates the presence of tumour-specific bacterial growth in the liver. The mice were administered the LacZ substrate intravenously after the tumour was colonized. Very small volumes of urine (1 μl) could be used to detect lacZ activity, in this case the production of light from cleaving the LuGal substrate. They also showed colour changes in the urine when using an alternative LacZ substrate that visibly changes the urine colour in a positive test.

The report concluded that this probiotic biosensor is sensitive, specific and safe to detect liver tumours in mice. The ultimate test was the detection of probiotic LacZ enzyme activity in the urine. The importance of this technology is that early detection of liver cancer is an unmet and pressing clinical need, and it may improve the current diagnostic methods that rely on imaging techniques. Since other tumours can be exposed to high bacterial concentrations, this approach may also work in detecting colorectal cancers. Although this approach has the advantages of being low cost, nonsurgical and nonradioactive, it will require significant regulatory approval and further testing in humans before approval.

Highlights: Oral administration of probiotic and simple urine test next day to detect liver cancer.

Simple synthetic gene circuits were used but could be improved to increase the signal strength and also the specificity.

Programmable probiotics for detection of cancer in urine. Science Translational Medicine. May 27, 2015, Vol 7, Issue 289. Tal Danino et al.