Invented by David E. Fowler, Philip G. Horton, Arie Ben-Bassat, BP Corp North America Inc
The BP Corp North America Inc invention works as followsNovel plasmids containing genes that code for alcohol dehydrogenase or pyruvate-decarboxylase have been described. Recombinant hosts that have been modified with genes for alcohol dehydrogenase or pyruvate are also described. Recombinant hosts can produce significant amounts of alcohol as a fermentation product due to their transformation with these genes. Methods for increasing the growth rate of recombinant host and methods to reduce the accumulation of unwanted metabolic products in these hosts’ growth medium are also disclosed. Recombinant hosts that can produce significant amounts of alcohol as a fermentation product from oligosaccharides are also disclosed. These plasmids contain genes encoding polysaccharases in addition to those genes mentioned above. Methods for making ethanol from oligomeric materials using the recombinant host described above are also described. A method of increasing the production functional proteins in a host recombinant to ethanol is also provided. This involves the overexpression of an adhB gene. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.
Background for Ethanol production using recombinant host
During glycolysis, cells transform simple sugars such as glucose into pyruvic acids, which results in a net production ATP and NADH. Without an electron transport system that can oxidative phosphorylation is functioning, at least 95% pyruvic acids are consumed in short pathways that regenerate NAD+. This is an obligate requirement for glycolysis and ATP production. These NAD+-regeneration systems’ waste products are often referred to as “fermentation products.
Microorganisms can produce a wide range of fermentation products from different genera. These include organic acids such as succinate, lactate, succinate, succinate, and succinate, as well neutral products like ethanol, butanol and acetone. The diversity of fermentation products from bacteria has made them a key determinant in taxonomy. For example, BERGEY’S MANUAL of SYSTEMATIC BACTERIOLOGY Williams & Wilkins Co. Baltimore (1984). (hereafter Bergey’s manual \”).”).
End products from fermentation share several basic features. They are generally non-toxic in the initial production conditions, but can become more toxic when they accumulate. Because their immediate precursors served as electron acceptors in glycolysis, they are less toxic than pyruvate. These fermentation products are the foundation of our most successful and oldest applications of biotechnology. It includes dairy products, meats and beverages as well as fuels. New technologies have allowed researchers to modify the genetic makeup of microorganisms in a way that has led to many advancements in biotechnology.
The Escherichiacoli bacterium is an important host for the cloning, modification and production of genes for biotechnology. The range of hosts for recombinantDNA research has expanded to include yeasts, bacteria, fungi and eukaryotic cell types in recent years. This invention relates to the use recombinantDNA technology to produce specific useful products from a modified host.
The DNA that was used to modify the host can be extracted from Zymomonas mobiliz, a bacterium found in honey and plant saps. Z. mobilis has been used for many years as an inoculum to make palm wines, and for fermentation of Agave Sap to make pulque. Pulque is a Mexican alcohol-containing beverage. This microbe is also used to produce fuel ethanol. It is said to be capable of producing ethanol at rates that are significantly higher than those of yeasts.
Z. mobilis is nutritionally very simple and can synthesize amino acids, nucleotides, and vitamins. However, the organism has a limited range of sugars that it can metabolize. Normally, this includes glucose, fructose, and sucrose. Substrate-level phosphorylation is produced by the fermentation of these sugars and is the only source of energy necessary for biosynthesis. Zymomonas mobiliz is incapable of growing in rich media such as nutrient broth, without fermentable sugar.
Z. mobilis is an obligatively fermentative bacteria that lacks a functional system to oxidativephosphorylation. Z. mobilis, like Saccharomyces cerevisiae yeast, produces carbon dioxide and ethanol as its principal fermentation products. Z. mobilis makes ethanol using a very short pathway that requires only two enzyme activities, pyruvate descarboxylase or alcohol dehydrogenase. This pathway’s key enzyme, Pyruvate Decarboxylase, diverts pyruvate from ethanol. Pyruvate-decarboxylase is responsible for the non-oxidative decarboxylation pyruvate in order to produce carbon dioxide and acetaldehyde. This organism contains two alcohol dehydrogenase enzyme isozymes. They catalyze the reduction in acetaldehyde and ethanol during fermentation. While bacterial alcohol dehydrogenases can be found in many organisms, pyruvate is not a common bacteria. The success of attempts to modify Z. mobilis in order to increase its commercial utility as an Ethanol producer has been very limited.
Most fuel-ethanol is produced using hexose sugars found in corn starch or cane syrup that uses S. cerevisiae and Z. mobilis. These sugars, however, are not as cheap as biomass sugars and can be used in food production. Starch and sugars are only a small fraction of total carbohydrates found in plants. The structural wall polymers, the cellulose, and hemicellulose are the dominant forms of plant carbohydrate found in leaves, stems, hulls and husks as well as cobs and other parts. These polymers are hydrolyzed to release a mixture of neutral sugars, including glucose, xylose and mannose as well as galactose and arabinose. There is no organism that can quickly and efficiently convert all these sugars into ethanol, or any other product of value.
The genes coding to alcohol dehydrogenase I and pyruvate oxidase in Z. mobilis were cloned separately, characterized and expressed in E. coli. See Brau & Sahm [1986a] Arch. Microbiol. 144: 296-301, [1986b] Arch. Microbiol. 146: 105-10; Conway et al. [1987a] J. Bacteriol. 169: 949-54; Conway et al. [1987b] J. Bacteriol. 169: 2591-97; Neale et al.  Nucleic Acid. Res. 15: 1753-61, Ingram & Conway  Appl. Environ. Microbiol. 54: 397-404; Ingram et al.  Appl. Environ. Microbiol. 53: 2420-25.
Brau and Sahm (1986a) first showed that ethanol production in recombinant E.coli could be increased by over-expression Z. mobilis pyruvate degradationlase, although low ethanol levels were achieved. This work was further extended by subsequent studies using Klebsiella planticola and Erwinia chlorophyllia, two enteric bacteria. They were able to produce higher levels of ethanol from sugar mixtures, pentoses and hexoses. Tolan & Finn  Appl. Environ. Microbiol. 53: 2033-38, 2039-44.
The aforementioned microbes are generally useful when there is a source of simple sugars. The majority of the world’s renewable, cheap source of biomass is not in monosaccharides, but in lignocellulose. This is a mix of cellulose and hemicellulose. Cellulose is a homopolymer made of glucose. Hemicellulose, however, is a complex heteropolymer that includes not only xylose (which is its primary constituent), but also significant amounts of galactose, arabinose and mannose. The microbial conversion of sugar residues in yard waste and paper from U.S. landfills could produce more than ten billion gallons ethanol, according to estimates. Although microorganisms like those mentioned above can efficiently ferment the monomeric sugars that make up the cellulosic or hemicellulosic compounds in lignocellulose’s cells, improving methods for saccharification remains a major research goal.
The current methods for saccharifying lignocellulose are acidic and enzyme hydrolyses. Acid hydrolysis is usually performed with heat. However, it has several drawbacks. These include the need for energy and the production of acidic wastewater. It can also lead to the formation of toxic substances that could hinder future microbial fermentations. The desirable alternative is enzyme hydrolysis. Enzymes can be added to the medium containing the biolignocellulosic material.
Genetic-engineering methods for adding saccharifying traits to microorganisms in order to produce ethanol or lactate have been directed towards the secretion high levels of enzymes into the medium. This means that the art has focused on modifying microorganisms with the necessary proteins to transport cellularly-produced enzymes into fermentation medium. The enzymes then can act on the substrate polysaccharide to produce mono- and/or oligosaccharides. This is because it has proven difficult to modify organisms that lack the ability to transport these proteins.
It is, therefore, the object of this invention to provide microorganisms capable of effectively diverting Pyruvate to Ethanol during growth under both anaerobic and aerobic conditions.
It is also the object of the present invention that recombinant host growth can be increased and undesirable metabolic products are reduced in the growth medium.
It is another object of this invention to provide recombinant host(s) that can produce sufficient intracellular levels polysaccharase enzyme(s), to effect the breakdown glucose or xylose Oligomers to produce products that the same hosts are capable of fermenting to ethanol.
In order to achieve these and other objectives, there is provided, according to the first major domain, a recombinant hosts other than Escherichiacoli. This host includes heterologous DNA code for the production of alcohol and pyruvate. The host possesses sufficient functional DNA to allow for the production of primary fermentation products of ethanol.
One advantage embodiment uses the Z. mobilis genes that code for alcohol dehydrogenase or pyruvate-decarboxylase in recombinant host.” The first domain also includes plasmids containing genes coding for alcohol and pyruvate, which are useful in providing inventive recombinant host plasmids.
In the second major area of the invention, the recombinant hosts, in addition to DNA coding alcohol dehydrogenase or pyruvate-decarboxylase enzyme, also contains DNA coding proteins that enable the host transport and metabolize oligosaccharides. The host expresses DNA at a level that allows it to produce ethanol from the metabolism of the Oligosaccharide. This domain is preferred by enteric bacteria like Klebsiella and Erwinia. Other preferred hosts will metabolize di- or trisaccharide containing C5 and/orC6 sugar monomers like glucose, xylose, and maltose.
In the third major area of this invention, the DNA required to produce one or more polysaccharises from a recombinant hosts as described above is also included.” The host will produce a polysaccharide and then release that polysaccharide into the medium. This will decrease the amount of commercial enzyme required to degrade the feedstock into fermentable monosaccharides or oligosaccharides.
The DNA of the polysaccharase can be either native or foreign to the host. However, it is more common for the DNA to be heterologous. Cellulolytic, starch-degrading and xylanolytic enzymes are all good examples of polysaccharases. The host can either secrete a small amount of the polysaccharise or accumulate it intracellularly for later release. Advantageously, intracellularly-accumulated enzymes which are thermostable, can be released when desired by heat-induced lysis. The heterologous DNA can encode combinations of enzymes, some of which will be secreted and some that are accumulated.
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