Fanciers of Mexican tequila owe thanks to an anaerobic bacterium,Zymomonas mobilis, which makes its living by fermenting thesugary sap of the century plant (Agave tequilana) into alcohol. Nowgenetic engineering has put Z. mobilis in line for a much bigger job,breaking down the sugar xylose from woody biomass to makeethanol for automotive fuel.
Xylose and glucose are two of the main sugars laid down in growingplants. Glucose, a hexose sugar, has six carbons in its molecule,which makes it a likely alcohol fermentation target for yeast, Z.mobilis, and other microorganisms. Xylose, a pentose with only fivecarbons, resists being fermented.
But because xylose can comprise 25 to 40 percent of all biomass _from trees to corn cobs to municipal garbage _ microbiologists havebeen trying for years to make this oddball sugar edible for ethanol-fermenting microbes. One who claims success is molecular biologistStephen Picataggio of the Department of Energy's NationalRenewable Energy Laboratory (NREL) in Golden, Colo.
A senior scientist in the NREL's Applied Biological SciencesBranch, Picataggio is principal author of a paper in this week'sScience, dated Jan. 13, titled, "Metabolic engineering of a pentosemetabolism pathway in ethanologenic Zymomonas mobilis."
"Native Zymomonas mobilis," Picataggio told BioWorld Today, "isvery effective at fermenting glucose, fructose and sucrose to ethanol,but it lacks the pentose metabolism pathways necessary forfermenting xylose." Starting in mid-1993, he and his team surveyedseveral likely industrial microorganisms with potential for aneconomical biomass-to-ethanol process. They hit on Z. mobilis, butfound that wild strains lacked four enzymes necessary formetabolizing xylose.
Moving Four Genes From One Microbe To Another
"So first," Picataggio said, "we isolated the xylose isomerase andxylulokinase genes from the Escherichia coli genome, and preciselyfused them to a strong constitutive gap promoter from Zymomonasby a PCR [polymerase chain reaction]-mediated overlap extensiontechnique."
He continued, "Next, we took two more E. coli genes, transaldolaseand transketolase, which encode pentose phosphate pathwayenzymes, and fused them to a second Zymomonas promoter.
"Finally, we subcloned both of these operons into a shuttle vectorthat allowed us simultaneously to transfer all four E. coli genes intoZ. mobilis."
The resulting transgenic microbe, he said, "produces ethanol fromxylose very efficiently, at 86 percent of theoretical yield. But moreimportantly, in the combined presence of both glucose and xylose,the engineered strain fermented the two sugars simultaneously at 95percent theoretical yield."
These numbers, Picataggio added, "report performance in laboratorymedia. Now we're optimizing yields of our xylose-fermentingrecombinant strain in commercial feed stocks." Sometime this year,he hopes to scale uptrials to NREL's new $14-million pilot plant, which can ferment aton of biomass a day.
Ethanol from biomass is nothing new. Brazil runs half of its trucksand cars on alcohol fermented from sugarcane. During the oil crisisof the 1970s, many processes were developed in the U.S. forconverting agricultural and timber wastes into ethyl alcohol. Gasoholis still a minor option at the pump.
A major drawback has been the cost of transporting biomass fromfield, forest or saw mill to a central ethanol-fermenting facility. "Tosome extent," Picataggio observed, "our Z. mobilis process helps.The more you can lower the production cost of ethanol at the plant,the wider the area from which you can draw feed stock into thatplant."
In the near term, he foresees that enlisting recombinant Zymononaswill be economically viable at a plant that is already producingethanol, say from agricultural wastes such as corn fiber. In the longrun, "we can use energy plants, grown specifically and exclusivelyfor ethanol production, as an alternative transportation fuel."
A candidate energy crop, officially nominated by NREL and otherfederal agencies, is switchgrass (Panicum virgatum). This toughperennial weed grows three to six feet tall, is a major inhabitant ofAmerican tall-grass prairie, and has a very high yield per acre.
Performing Converse Gene Switch
"E. coli doesn't normally produce ethanol," Picataggio observed. "Itnormally converts sugars to organic acids." But he added that"strains of E. coli have been effectively engineered for ethanolproduction by Lonnie Ingram at the University of Florida [inGainesville]," the converse of his own gene transfer from E. coli toZ. mobilis.
Microbiologist Ingram has read the NREL team's paper in thecurrent Science. Ingram told BioWorld Today, "It is particularlysatisfying to see that one of my former post-doctoral associates, Dr.Christina Eddy, was a primary contributor to this [NREL] effort, andthat our DOE-sponsored work on the glycolytic enzymes in Z.mobilis provided essential tools for the high-level expression of E.coli genes."
In 1991, with suitable ceremony, the U.S. Office of Patents andTrademarks awarded first inventor Ingram patent number 5,000,000,"Ethanol Production by Escherichia coli Strains Co-expressingZymomonas pdc and adh Genes."
The University of Florida, to whom the patent, and two others since,is assigned, has licensed them to a newly created company inGainesville, BioENergy International Inc., which, Ingram said, isnow scaling up pilot ethanol production in view ofcommercialization. n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.