Mixed Sugar Fermentative Strain of Zymomonas mobilis

Technology Marketing SummaryEthanol is the clean, renewable, domestic form of gasoline the U.S. needs to decrease its dependence on foreign oil and mitigate pollution from vehicles. However, the current high cost of ethanol production is preventing this renewable fuel from becoming widespread.

NREL has addressed this cost issue in the fermentation step of ethanol production by developing genetically engineered bacteria known as Zymomonas mobilis 8b. Traditional yeasts have the ability to ferment six-carbon sugars, but are unable to ferment five-carbon sugars. This makes for a low yield of ethanol, or if fermented by two separate organisms in two separate fermentation tanks, a high capital cost for ethanol. In response, NREL scientists have taken the bacterium Zymomonas mobilis, which naturally only ferments six-carbon sugars, and genetically engineered it to express foreign genes encoding enzymes needed to ferment five-carbon sugars. DescriptionZymomonas mobilis is known for its ability to rapidly and efficiently convert cellulosic glucose (six-carbon) substrates into ethanol at a low pH in an anaerobic culture, and in a medium which contains the inhibitory compounds typically associated with lignocellulosic hydrolysis. However, six-carbon sugars only make up 30-40% of a typical biomass feedstock and Z. mobilis cannot ferment the remaining five-carbon sugars, such as the xylose from hemicellulose and arabinose from switchgrass and corn fiber. Thus, achieving a high rate of conversion efficiency in the fermentation of five-carbon sugars is vital to the commercial production of fuels and chemicals from renewable substrates.

In response to this challenge, researchers at NREL have utilized a transposon and a plasmid shuttle vector for stable insertion of four exogenous genes from the yeast Saccharomyces cervisiae into the bacterial genome of Zymomonas mobilis. The transposon is useful for stable insertion of foreign genes into a bacterial genome and comprises of at least one operon having structural genes encoding enzymes from the group xylAsylB, araBAD, and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. The plasmid shuttle vector is useful for the transformation of foreign genes into a bacterial genome.

Previous variants of Z. mobilis are capable of fermenting glucose, xylose, and arabinose at very low conversion efficiency. Additionally, these strains frequently become unstable when grown in the absence of selection pressure or when they have to compete with other organisms such as those in the simultaneous-saccharification-fermentation processes. Z. mobilis 8b exhibits substantially improved stability and can retain native activity for producing pentose and hexose-fermenting enzymes for 80-160 generations, up to 4x more stable than other strains of Zymomonas. BenefitsZymomonas 8b allows for the fermentation of not only glucose and mannose, but also five-carbon (pentose) sugars; xylose and arabinose. Z. mobilis 8b is more stable and retains activity of the pentose-fermenting enzyme for 2-4x as many generations as other strains of Zymomonas mobilis. These factors enhance the fermentation of biomass sugars, leading to greater yields of ethanol at lower costs and more environmentally friendly methods. Applications and IndustriesFermentation technology for the conversion of cellulosic substrates into:
Fuels
Chemicals More InformationU.S. and foreign patent rights available for non-exclusive licensing.
00-14 (7,354,755)
99-01 (6,566,107)
00-14CIP (7,223,575)

NREL is preparing and will be submitting a Microbial Commercial Activity Notice (MCAN) to the Environmental Protection Agency to cover commercial use of this intergenic microorganism. Patents and Patent Applications

ID Number	Title and Abstract	Primary Lab	Date
Patent 6,566,107	Recombinant Zymomonas mobilis with improved xylose utilization A strain derived from Zymomonas mobilis ATCC31821 or its derivative capable of producing ethanol upon fermentation of a carbohydrate medium containing xylose to provide enhanced xylose utilization and enhanced ethanol process yield, the strain or its derivative comprising exogenous genes encoding xylose isornerase, xylulokinase, transaldolase and transketolase, the genes are fused to at least one promotor recognized by Zymomonas which regulates the expression of at least one of the genes.	National Renewable Energy Laboratory	05/20/2003 Issued
Patent 7,223,575	Zymomonas pentose-sugar fermenting strains and uses thereof Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.	National Renewable Energy Laboratory	05/29/2007 Issued
Patent 7,354,755	Stable zymomonas mobilis xylose and arabinose fermenting strains The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.	National Renewable Energy Laboratory	04/08/2008 Issued

Technology Status

Technology ID	Development Stage	Availability	Published	Last Updated
99-01 & 00-14	Production	Available - U.S. and foreign patent rights available for non-exclusive licensing	09/24/2010	10/26/2010