This organism has been known first as Termobacterium mobile and subsequently as Pseudomonas linderi since 1912; although it was first known in Europe as a spoiling agent in cider, its function in the making of potable beverages such as palm wines is well established in Africa, Central[27] and South America, the Middle East, South Asia, and the Pacific islands and can ferment the sugar sap of the Agave cactus to yield pulque.194 The species was described as “undoubtedly one of the most unique bacterium [sic] within the microbial world.”195 Its unusual biochemistry has already

U. S. Patents Awarded for Yeast and Bacterial Ethanol Producer Strains Capable of Utilizing Lignocellulosic Substrates

Date Title Assignee Patent Number January 11, 1983 Direct fermentation of D-xylose to ethanol by a xylose-fermenting yeast mutant Purdue Research Foundation, West Lafayette, IN 4368268 September 18, 1984 Process for manufacturing alcohol by fermentation Kyowa Hakko Kogyo Co. Ltd. 4472501 December 25, 1984 Production of ethanol by yeast using xylulose Purdue Research Foundation, West Lafayette, IN 4490468 April 16, 1985 Direct fermentation of D-xylose to ethanol by a xylose-fermenting yeast mutant Purdue Research Foundation, West Lafayette, IN 4511656 May 5, 1987 Process for enhanced fermentation of xylose to ethanol United States 4663284 June 20, 1989 Process for producing ethanol from plant biomass using the fungus Paecilomyces sp. United States 4840903 March 19, 1991 Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes University of Florida, Gainesville, FL 5000000 December 13, 1994 Combined enzyme mediated fermentation of cellulous [sic] and xylose to ethanol… United States 5372939 May 2, 1995 Xylose isomerase gene of Thermus aquaticus Nihon Shokuhin Kako Co. Ltd., Tokyo, Japan 5411886 June 13, 1995 Ethanol production by recombinant hosts University of Florida, Gainesville, FL 5424202 October 3, 1995 Process for producing alcohol CSIR, New Delhi, India 5455163 January 9, 1996 Ethanol production in Gram-positive microbes University of Florida, Gainesville, FL 5482846 May 7, 1996 Recombinant Zymomonas for pentose fermentation Midwest Research Institute, Kansas City, MO 5514583 January 27, 1998 Pentose fermentation by recombinant Zymomonas Midwest Research Institute, Kansas City, MO 5712133 March 10, 1998 Recombinant Zymomonas for pentose fermentation Midwest Research Institute, Kansas City, MO 5726053 August 4, 1998 Recombinant yeasts for effective fermentation of glucose and xylose Purdue Research Foundation, West Lafayette, IN 5789210 December 1, 1998 Single Zymomonas mobilis strain for xylose and arabinose fermentation Midwest Research Institute, Kansas City, MO 5843760

Xylose utilization by recombinant yeasts

Ethanol production in Gram-positive microbes

Recombinant cells that highly express chromosomally integrated heterologous genes

Recombinant organisms capable of fermenting cellobiose

Stabilization of PET operon plasmids and ethanol production in bacterial strains…

Genetically modified cyanobacteria for the production of ethanol…

SHAM-insensitive terminal-oxidase gene from xylose-fermenting yeast

Pentose fermentation of normally toxic lignocellulose prehydrolysate with strain of Pichia stipitis…

Recombinant Zymomonas mobilis with improved xylose ultilization

Production of ethanol from xylose

Ethanol production in recombinant hosts

Genetically modified cyanobacteria for the production of ethanol…

High-speed, consecutive batch or continuous, low-effluent process…

Transformed microorganisms with improved properties

Xyrofin Oy, Helsinki, Finland University of Florida, Gainesville, FL University of Florida, Gainesville, FL

University of Florida Research Foundation, Inc., Gainesville, FL United States

Enol Energy, Inc., Toronto, Canada

Wisconsin Alumni Research Foundation, Madison, WI

Midwest Research Institute, Kansas City, MO

Midwest Research Institute, Kansas City, MO Xyrofin Oy, Helsinki, Finland University of Florida Research Foundation, Inc., Gainesville, FL

Enol Energy, Inc., Toronto, Canada Bio-Process Innovation, Inc., West Lafayette, IN Valtion Teknillin Tutkimuskeskus, Espoo, Finland

been described (section 3.3.1), but multiple curious features of its metabolism made it a promising target for industrial process development:

• Lacking an oxidative electron transport chain, the species is energetically grossly incompetent, that is, it can capture very little of the potential bioenergy in glucose — in other words, it is nearly ideal from the ethanol fermentation standpoint.

• What little energy production is achieved can be uncoupled from growth by an intracellular wastage (ATPase).

• It shows no Pasteur effect, seemingly oblivious to O2 levels regarding glu­cose metabolism — but acetate, acetaldehyde, and acetoin are accumulated with increasing oxygenation.

During the 1970s, biotechnological interest in Z. mobilis became intense.* A patent for its use in ethanol production from sucrose and fructose was granted in mid-1989 (table 3.6). In the same year, researchers at the University of Queensland, Brisbane, Australia, demonstrated the ability of Z. mobilis to ferment industrial substrates such as potato mash and wheat starch to ethanol, with 95-98% conversion efficiencies at ethanol concentrations up to 13.5% (v/v).196 The Australian process for producing ethanol from starch was scaled up to more than 13,000 l.39

The capability to utilize pentose sugars for ethanol production — with lignocel — lulosic substrates as the goal — was engineered into a strain recognized in 1981 as a superior ethanologen; strain CP4 (originally isolated from fermenting sugarcane juice) exhibited the most rapid rate of ethanol formation from glucose, achieved the highest concentration (>80 g/l from 200 g/l glucose), could ferment both glucose and sucrose at temperatures up to 42°C, and formed less polymeric fructose (levan) from sucrose than the other good ethanol producers. On transfer to high-glucose medium, CP4 had the shortest lag time before growth commenced and one of the shortest doubling times of the strains tested.197 Researchers at the University of Sydney then undertook a series of studies of the microbial physiology and biochemistry of the organism and upscaling fermentations from the laboratory:

• Pilot-scale (500 l)-evaluated mutant strains selected for increased ethanol tolerance while improving ethanol production from sucrose and molasses became targets for strain development.198

• To reduce malodorous H2S evolution by candidate strains, cysteine auxo — trophs were isolated from studies of sulfur-containing amino acids.198

• Technical and engineering developments greatly increased the productiv­ity of selected Z. mobilis strains (as discussed in the context of bioprocess technologies in chapter 4).198

• Direct genetic manipulation was explored to broaden the substrate range.199

• High-resolution 31P nuclear magnetic resonance (NMR) of intracellular phos­phate esters in cells fermenting glucose to ethanol showed that kinetic limita­tions could be deduced early in the ED pathway (figure 3.4), in the conversion of glucose 6-phosphate to 6-phosphonogluconate, and in the glycolytic pathway (phosphoglyceromutase), defining targets for rational genetic intervention.200

By 1993, a review on Z. mobilis could already reference 362 publications.

Strains of Z. mobilis were first engineered to catabolize xylose at the National Renewable Resources Laboratory, Golden, Colorado. Four genes for xylose utilization by E. coli were introduced into Z. mobilis strain CP4 and expressed: xylose isom- erase (xylA), xylulokinase (xylB), transketolase (tktA), and transaldolase (talB) on a plasmid under the control of strong constitutive promoters from Z. mobilis.201202 The transformant CP4 (pZB5) could grow on xylose as the carbon source with an ethanol yield of 86% of the theoretical maximum; crucially, xylose and glucose could be taken up by the cells simultaneously using a permease because no active (energy — expending), selective transport system for glucose exists in Z. mobilis; the transport “facilitator” for glucose is highly specific, and only mannose and (weakly) galactose, xylose, sucrose, and fructose appear to be taken up by this mechanism.203 Using a plasmid containing five genes from E. coli, araA (encoding L-arabinose isomerase), araB (L-ribulose kinase), araD (L-ribulose 5-phosphate-4-epimerase), plus tkta and talB, a strain (ATCC39767[pZB206]) was engineered to ferment l-arabinose and produce ethanol with a very high yield (96%) but at a slow rate, ascribed to the low affinity of the permease uptake mechanism for l-arabinose.204 A third NERL strain was ATCC39767 (identified as a good candidate for lignocellulose conversion based on the evidence of its growth in yellow poplar wood acid hydrolysates) trans­formed with a plasmid introducing genes for xylose metabolism and subsequently adapted for improved growth in the presence of hydrolysate inhibitors by serial sub­culture in progressively higher concentrations of the wood hydrolyate.202205

A strain cofermenting glucose, xylose, and arabinose was constructed by chro­mosomal integration of the genes; this strain (AX101, derived from ATCC39576) was genetically stable, fermented glucose and xylose much more rapidly than it did arabinose, but produced ethanol at a high efficiency (0.46 g/g sugar consumed) and with only minor accumulations of xylitol, lactic acid, and acetic acid.206-208 The major practical drawback for the AX101 strain is its sensitivity to acetic acid (formed in lignocellulosic hydrolysates by the breakdown of acetylated sugars); this sensitivity was demonstrated in trials of the strain with an agricultural waste (oat hulls) sub­strate pretreated by the two-stage acid process developed by the Iogen Corporation in Canada, although the bacterial ethanologen outperformed a yeast in both volumet­ric productivity and glucose to ethanol conversion.209

The University of Sydney researchers have also transformed their best candidate ethanologen with the NERL pZB5 plasmid to introduce xylose utilization; strain ZM4(pZB5) produced 62 g/l of ethanol from a medium of 65 g/l of both glucose and xylose, but its ethanol tolerance was lower than that of the Z. mobilis wild type.210 The recombinant Z. mobilis shares the energy limitation on xylose observed with E. coli.180 NMR examination of strain ZM4(pZB5) growing on glucose-xylose mixtures demonstrated low levels of nucleotide phosphate sugars inside the cells when xylose was mainly supporting metabolism; because these intracellular components are bio­synthetic precursors for cell replication, the energy limitation has a clear biochemical mechanism for growth restriction.211 In addition to the metabolic burden imposed by the plasmids, the production of unwanted by-products (xylitol, acetate, lactate, acetoin, and dihydroxyacetone) and the formation of xylitol phosphate as a possible inhibitor of enzyme-catalyzed processes may all contribute to the poorer fermenta­tion performance on xylose as a carbon source. Further NMR investigations showed that acetic acid at growth-inhibitory concentrations decreased nucleotide phosphate sugars inside the cells and caused acidification of the cytoplasm, both complex bio­chemical factors difficult to remedy by genetic manipulation.212 Taking one step back, a mutant of the ZM4 strain with greater tolerance to acetate was isolated by classical selection procedures; electroporating the pZB5 plasmid into this AcR strain resulted in a xylose-fermenting strain with demonstrably improved resistance to sodium acetate at a concentration of 12 g/l.213 Overexpressing a heterologous xylulokinase gene under the control of a native Z. mobilis promoter did not, however, increase growth or xylose metabolism on a xylose-containing medium, indicating that constraints on xylose uti­lization reside elsewhere in the catabolic pathway or in xylose uptake.214 The xylulo — kinase-catalyzed step was more convincingly rate-limiting for xylose utilization with a Z. mobilis strain constructed at the Forschungszentrum Julich (Germany) with K. pneumoniae XI and XK, as well as E. coli transketolase and transaldolase genes; overexpression of XK was deduced to be necessary and sufficient to generate strains capable of fermenting xylose to ethanol at up to 93% of the theoretical yield.215

The potential impact of the acetate inhibition of Z. mobilis is so severe with com­mercial process that investigations of the effect have continued to explore new molecu­lar targets for its abatement. With starting acetate concentrations in the range 0-8 g/l in fermentations of glucose and xylose mixtures, high acetate slowed the increase in intracellular ATP (as a measure of bioenergetic “health”).216 Expressing a gene from E. coli encoding a 24-amino acid proton-buffering peptide protects Z. mobilis strain CP4 from both low pH (<3.0) and acetic acid; optimization of this strategy may be success­ful with high-productivity strains for lignocellulose hydrolysate fermentation.217