Interest in Erwinia bacteria for ethanol production dates back at least to the late 1950s; in 1971, the explanation for the unusually high ethanol production by Erwinia species was identified as a PDC/ADH pathway, decarboxylating pyruvate to acetaldehyde fol­lowed by reduction to ethanol, akin to that in Z. mobilis; ethanol is the major fermenta­tive product, accompanied by smaller amounts of lactic acid.224 Soft-rot bacteria secrete hydrolases and lyases to solubilize lignocellulosic polymers, and the PET operon was used to transform E. carotovora and E. chrysanthemi to produce ethanol from cellobi — ose, glucose, and xylose; both strains fermented cellobiose at twice the rate shown by cellobiose-utilizing yeasts.42 The genetically engineered E. chrysanthemi could ferment sugars present in beet pulp but was inferior to E. coli strain KO11 in ethanol production, generating more acetate and succinate in mixed-acid patterns of metabolism.221

Lactococcus lactis is another GRAS organism; its use in the industrial produc­tion of lactic acid is supplemented by its synthesis of the bacteriocin nisin, the only such product approved for food preservation.225 When a PDC-encoding gene from Zymobacter palmae was inserted into L. lactis via a shuttle vector, the enzyme was functionally expressed, but, although a larger amount of acetaldehyde was detected, a slightly higher conversion of glucose to ethanol was measured (although glucose was used more slowly), and less lactic acid was accumulated, no increased ethanol production could be achieved, presumably because of insufficient endogenous ADH activity.226 The same group at USDA’s National Center for Agricultural Utilization Research examined L. plantarum as an ethanologen for genetic improvement; strain TF103, with two genes for lactate dehydrogenase deleted, was transformed with a PDC gene from the Gram-positive bacterium Sarcinia ventriculi to redirect carbon flow toward ethanol production, but only slightly more ethanol was produced (at up to 6 g/l).227 Other attempts to metabolically engineer lactic acid bacteria have been similarly unsuccessful (although more ethanol is produced than by the parental strains and the conversion of glucose to ethanol is increased by nearly 2.5-fold), the bacteria remaining eponymously and predominantly lactic acid producers; although Z. mobilis pdc and adh genes in PET operons are transcribed, the enzyme activities can be very low when compared with E. coli transformants.228 229

Zb. palmae was isolated on the Japanese island of Okinawa from palm sap by sci­entists from the Kirin Brewery Company, Yokohama, Japan. A facultative anaerobe, the bacterium can ferment glucose, fructose, galactose, mannose, sucrose, maltose, melibiose, raffinose, mannitol, and sorbitol, converting maltose efficiently to ethanol with only a trace of fermentative acids.230 Its metabolic characteristics indicate potential as an ethanologen; broadening its substrate range to include xylose followed previous work with Z. mobilis, expressing E. coli genes for xylose isomerase, xylulokinase, transaldolase, and transketolase.231 The recombinant Zb. palmae completely cofer­mented a mixture of 40 g/l each of glucose and xylose simultaneously within eight hours at 95% of the theoretical yield. Introducing a Ruminococcus albus gene for P — glucosidase transformed Zb. palmae to cellobiose utilization; the heterologous enzyme was more than 50% present on the cell surface or inside the periplasm, and the recom­binant could transform 2% cellobiose to ethanol at 95% of the theoretical yield.232 The PDC enzyme of the organism is, as discussed briefly above, an interesting target for het­erologous expression in ethanologenic bacteria; it has the highest specific activity and lowest affinity for its substrate pyruvate of any bacterial PDC, and it has been expressed in E. coli to approximately 33% of the soluble protein. Codon usage for the gene is quite similar to that for E. coli genes, implying a facile recombinant expression.233

Cyanobacteria (blue-green algae) have generally lost their fermentative capabilities, now colonizing marine, brackish, and freshwater habitats where photosynthetic metab­olism predominates; of 37 strains in a German culture collection, only five accumulated fermentation products in darkness and under anaerobic conditions, and acids (glycolic, lactic, formate, and oxalate) were the major products.234 Nevertheless, expression of Z. mobilis pdc and adh genes under the control of the promoter from the operon for the CO2-fixing ribulose 1,5-bis-phosphate carboxylase in a Synechococcus strain synthesized ethanol phototrophically from CO2 with an ethanol:acetaldehyde molar ratio higher than 75:1.235 Because cyanobacteria have simple growth nutrient require­ments and use light, CO2, and inorganic elements efficiently, they represent a sys­tem for longer-term development for the bioconversion of solar energy (and CO2) by genetic transformation, strain and process evolution, and metabolic modeling. The U. S. Department of Energy (DOE) is funding (since 2006) DNA sequencing studies of six photosynthetic bacteria at Washington University in St. Louis, Missouri, and the DOE’s own sequencing facility at Walnut Creek, California, using a biodiversity of organisms from rice paddies and deep ocean sources to maximize biochemical and metabolic potential.