# Presentation: 948591

## Genomic Sequences of the Ethanol Producing Bacteria Thermoanaerobacter ethanolicus 39E and Thermoanaerobacter ethanolicus X514

-  **C. L. Hemme****1,2****, M. W. Fields****3****, A. Copeland****4****, S. Lucas****4****, K. Barry****4****, J. C. Detter****4,5****, T. Glavina4, N. Hammon****4****, S. Israni****4****, S. Pitluck****4****, M. Land****1****, **
**E. H. Saunders****5****, D. C. Bruce****5****, A. Lapidus****4****, C. S. Han****5****, F. Larimar****1****, P. Richardson****4****, E. Rubin****4**** and J. Zhou****2****Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN****1****; Institute for Environmental Genomics, University of Oklahoma, Norman, OK****2****; **
**Department of Soil & Crop Sciences** **, Department of Microbiology, Miami University, Oxford, OH****3****; DOE Joint Genome Institute, Walnut Creek, CA****4****; Los Alamos National Laboratory, Los Alamos, NM****5**

**Sequencing and assembly of the two *****T. ethanolicus***** strains was performed at the JGI Production Genomics Facility (Walnut Creek, CA)**
**Closure of the genomes is being conducted at JGI-PGF (X514) and JGI-Los Alamos (39E)**
**Automatic annotation of the sequences was performed JGI-ORNL**
**All work was conducted under the auspices of the US Department of Energy Microbial Genome Program**

- Recent instability in the global energy markets due to increased demand coupled with disruption of supply has sparked a renewed interest in the development of alternative energy sources.  One such alternative energy source that has been singled out for particular study is biogenerated ethanol derived from plant cell wall material.  Clostridia bacteria are often employed in such efforts due to their general ability to degrade complex carbohydrates (cellulose, hemicellulose, chitin, etc.) to monomeric sugars (glucose, galactose, xylose, etc.) which can in turn be fermented to commercially valuable end products (ethanol, etc.).  The genomic sequences of two thermophilic ethanol-producing bacteria, *Thermoanaerobacter ethanolicus *39E and *T. ethanolicus *X514, have recently been sequenced and are undergoing finishing.  39E was originally isolated from a thermal spring in Yellowstone National Park that is capable of producing ethanol from a variety of polysaccharides including hemicellulose, xylan, cyclomaltodextrins and starch.  X514 is a deep subsurface, metal-reducing strain of the species that has been geologically separated from 39E for ~200 Mya.  The partially-completed genome sequences of these two strains are compared with each other and with the completed sequence of *T. tengcongensis* MB4T.

**Introduction**

**Genome Statistics**

***T. ethanolicus***** 39E**
**    ****Genome Size (Mb):****	****2.3**
**    ****Contigs:****	****88 (20)**
**    ****Predicted CDS:****	****2254**
**    ****%GC:****	****34.5**
***T. ethanolicus***** X514**
***    *****Genome Size (Mb):****	****2.2**
**    ****Contigs:****	****94 (18)**
**    ****Predicted CDS:****	****2230**
**    ****%GC:****	****34.4%**
***T. tengcongensis********
**    ****Genome Size (Mb):****	****2.7**
**    ****Contigs:****	****1**
**    ****Predicted CDS:****	****2588**
**    ****%GC:****	****37.6%**
***Genome sequenced independently from this project**

**Fig 2.**  Central carbon metabolism and feeder pathways of the three *Thermoanaerobacter* strains.  The presence of the enzymes for a given step are indicated for each organism: *T. tengcongensis* (magenta), 39E (cyan) and X514 (yellow).  The bold red arrows indicate the direction of carbon flow during fermentation of glucose.  Ethanol production is predominantly carried out by the bifunctional secondary alcohol dehydrogenase/acetyl-CoA thioesterase, with the primary alcohol dehydrogenase serving to recycle nicotinamide.

**Fig 1.  **16S rRNA phylogeny of *Thermoanaerobacter*, *Thermoanaerobacterium* and *Clostridium*.  Organisms listed in blue have been or are being sequenced independently of this project.  The two *Thermoanaerobacter ethanolicus* strains sequenced as part of this project are listed in red.

**Fig 3.**  Genome organization for *T. ethanolicus* 39E as reported in the literature (A) and as observed from the genomic sequence (B).

**3 *****Thermoanaerobacter***** species**
** ****Tten    595**
** ****F0F1-ATPase complex**
** ****Hydrogenase maturation factors**
** ****Kdp high-affinity potassium transport system**
** ****X514   207**
** ****39E     128**
**2 *****T. ethanolicus***** strains**
**X514   283**
** ****α****-galactosidase**
** ****glycerate kinase**
** ****cobalamin biosynthesis genes**
** ****multiple phage proteins/integrase**
** ****39E     271**
** ****KDPG permease**
** ****1-phosphofructokinase**

**Fig 5.**  Unique genes present in each *Thermoanaerobacter* when compared by genus and species.  Gene families were determined using the method of Lerat et al., 2005.  Genes from families consisting of only the self-to-self hit were considered to be unique for that species.

**Fig 6.**  Artemis comparison of *T. tengcongensis* (A), *T. ethanolicus* 39E (B) and *T. ethanolicus* X514 (C).  Only hits >500 bp are shown.

**A)**

**B)**

**C)**

***T. ethanolicus *****39E**
** ****Fe-containing alcohol dehydrogenase**
** ****ABC sugar transporters**
** ****KDPG and KHG aldolase**
** ****Altronate dehydratase**
** ****Auxin efflux carrier**
***T. ethanolicus *****X514**
*** *****Heavy metal-transloating P-type ATPase**
** ****Glycosidase**
** ****Ferric iron uptake regulator**
** ****Fe-containing alcohol dehydrogenase**
** ****ABC sugar transporters**
** ****NADH:flavin oxidoreductase**
** ****Xylulokinase**
** ****Transketolase**
** ****Acetate Kinase**

**Fig 7.  Sampling of putative gene duplications/expansions in *****T. ethanolicus***** strains**

**94% ANI Species Cutoff**
**(70% DNA-DNA Reassociation)**

**16S rRNA Phylogeny**

**Putative strain-Specific Gene Expansions**

**Future Work**

**  ****Close both *****T. ethanolicus *****genomes**
**  ****Test metal reduction capabilities of 39E and compare to X514**
**  ****Test ethanol production capabilities of X514 and compare to 39E**
**  ****Design and build microarray for 39E to test gene expression of cultures grown on a variety of carbon sources with the intent to clarify carbon flow from the carbon source to ethanol**