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Mixed Sugar Fermentative Strain of Zymomonas mobilis

Stage: Production
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.


Zymomonas 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.

For more information, contact Eric Payne at Eric.Payne@nrel.gov

ROI 99-01

Applications and Industries

Fermentation technology for the conversion of cellulosic substrates into:
Fuels
Chemicals

Benefits

Zymomonas 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.

U.S. Patent # 6,566,107

Attachments

US Patent 65...7.pdf

Sep 4, 2019