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.
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.
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.
Project Leader of the Hydrocarbon Metagenomic Project
Canadian Synthetic Biology Researchers