Methods for limiting the action of SP3 DNAase and for the determination of the direction of hydrolysis of processive exonucleases.

The action of the exonuclease SP3 DNAase is inhibited by chemical modification of DNA with the cation N-cyclohexyl-N'-beta-(4-methylmorpholinium)-ethylcarbodiimide (CME). The limited activity of the enzyme on CMA-modified DNA makes it possible to demonstrate that the enzyme also initiates its attack on polydeoxyribonucleotides at the 5'-termini. This was determined by the analysis of the products from the digestion of CME-modified DNA containing labeled 5'-terminal phosphate groups. Such procedure can be adopted as a general approach for the determination of the direction of hydrolysis of other processive exonucleases. SP3 DNAase has been shown able to degrade oligo- and polydeoxyribonucleotides with or without 5'-terminal phosphate groups with equal efficiency (Aposhian, H.V., Friedman, N., Nichihara M., Heimer, E.P., and Nussbaum, A.L. (1970) J. Mol. Biol. 49, 367-379). The present work also shows that the enzyme can even hydrolyze oligo- and polynucleotides containing derivatized phosphate groups.

Positive selection vectors based on xylose utilization suppression.

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl- phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site of pUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl- phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl+ phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.

Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Scientific note.

D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeast can ferment xylose and xylulose more than twice as fast as the parent yeast.

Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae.

The intact Pichia stipitis xylose reductase gene (XR) has been cloned and expressed in Saccharomyces cerevisiae. The possible further improvement of the expression of the Pichia gene in the new host was studied. To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5'noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes. It was found that the Pichia genetic signal for transcription of XR is more effective than the yeast TRP5 promoter, but is about half as effective as the yeast strong promoter of the alcohol dehydrogenase gene (ADC1). However, the nucleotide sequence immediately adjacent to the initiation codon of XR, which controls the translation of the gene product, seemed to be five times less effective than the corresponding sequence of the ADC1 gene. By totally replacing its 5'-noncoding sequence with that of the yeast ADC1 gene, the expression of XR in yeast was found to be nearly ten times higher. Furthermore, the cloned Pichia XR described in this article contains very little of its 3'-noncoding sequence. In order to study whether the 3'-noncoding sequence is important to its expression in S. cerevisiae, the intact 3'-noncoding sequences of the yeast xylulokinase gene was spliced to the 3' end of the PADC1-XR structural gene. This latter modification has resulted in a twofold further increase in the expression of the Pichia XR in yeast.

Genetic transformation of xylose-fermenting yeast Pichia stipitis. Scientific note.

A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33).

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400(pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.

Enhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modes.

Agricultural residues, such as grain by-products, are rich in the hydrolyzable carbohydrate polymers hemicellulose and cellulose; hence, they represent a readily available source of the fermentable sugars xylose and glucose. The biomass-to-ethanol technology is now a step closer to commercialization because a stable recombinant yeast strain has been developed that can efficiently ferment glucose and xylose simultaneously (coferment) to ethanol. This strain, LNH-ST, is a derivative of Saccharomyces yeast strain 1400 that carries the xylose-catabolism encoding genes of Pichia stipitis in its chromosome. Continuous pure sugar cofermentation studies with this organism resulted in promising steady-state ethanol yields (70.4% of theoretical based on available sugars) at a residence time of 48 h. Further studies with corn biomass pretreated at the pilot scale confirmed the performance characteristics of the organism in a simultaneous saccharification and cofermentation (SSCF) process: LNH-ST converted 78.4% of the available glucose and 56.1% of the available xylose within 4 d, despite the presence of high levels of metabolic inhibitors. These SSCF data were reproducible at the bench scale and verified in a 9000-L pilot scale bioreactor.

A novel xylB-based positive selection vector.

Expression of a plasmid-borne Escherichia coli xylulokinase gene (xylB) under the control of the lac promoter yields constitutively high levels of xylulokinase activity. When a plasmid containing this lac-xylB fusion (pLEK100) is transformed into a xylB- mutant the Xyl+ phenotype is restored on xylose-containing media. When the same transformants are plated on xylitol medium, growth inhibition is observed. Positive selection is achieved by cloning DNA into the unique restriction sites of pLEK100, to disrupt xylB expression, transforming E. coli, and then plating transformants on xylitol medium. With this protocol only transformants with insert containing plasmids will be obtained. This results in a considerable reduction in the time and effort needed to construct genomic libraries or perform routine DNA cloning experiments. Three unique sites are available which are suitable for positive selection of DNA fragments, via the disruption of translation (BglII) or transcription (HindIII, SalI, and BglII) of the xylB gene.

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis.

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.

Cloning of the Pachysolen tannophilus Xylulokinase Gene by Complementation in Escherichia coli.

The gene coding for xylulokinase has been isolated from the yeast Pachysolen tannophilus by complementation of Escherichia coli xylulokinase (xylB) mutants. Through subcloning, the gene has been localized at one end of a 3.2-kilobase EcoRI-PstI fragment. Expression of the cloned gene was insensitive to glucose inhibition. Furthermore, the cloned gene did not cross-hybridize with E. coli and Saccharomyces cerevisiae xylulokinase genes.

Esterification of terminal phosphate groups in nucleic acids with sorbitol and its application to the isolation of terminal polynucleotide fragments.

The exposure of mono- and polynucleotides to 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and high concentrations of sorbitol results in the esterification of their monosubstituted phosphate groups. The presence of the sorbitol moiety permits these derivatives to bind strongly at pH 8.7 to columns of chromatographic supports containing the dihydroxyboryl group and to be subsequently released by elution with buffers at pH 5.5. The procedure constitutes a method for the isolation of polynucleotide fragments arising from the terminals of nucleic acids. A new method for the preparation of the chromatographic supports involves the synthesis of the 1,3-propanediol cyclic ester of m-[[3-(N-succinimidoxycarbonyl)propanoyl]amino]benzeneboronic acid and its condensation with aminoethylcellulose or amino-ethylpolyacrylamide. The reagent is readily prepared by reaction of N-[m-(dihydroxyboryl)phenyl)]succinamic acid with 1,3-propanediol to protect the boronate moiety followed by esterification with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose.

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2 microns-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

Plasmid instability kinetics of the yeast S288C pUCKm8 [cir+] in non-selective and selective media.

Plasmid loss kinetics for Saccharomyces cerevisiae transformed with the 2-mum DNA-based-plasmid pUCKm8 were measured in nonselective and selective media. The plasmid pUCKm8 gives the organism two new phenotypes: resistance to the wide spectrum antibiotic G418 sulfate, and the ability to produce the enzyme, beta-lactamase. Plasmid stability was determined using the production of beta-lactamase as a marker. The effect of G418 on the growth rates of all organisms present in the culture and on plasmid stability was also determined. Mathematical models describing plasmid loss kinetics during exponential growth for both nonselective and selective conditions are used to simulate the experimental data. In nonselective medium, over 80% of the cells still exhibited the desired phenotype after 50 doublings. In medium containing G418, improvements in plasmid stability were only marginal due to the appearance of antibiotic-resistant cells.

Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol.

Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline. Furthermore, ethanol can be produced from plentiful, domestically available, renewable, cellulosic biomass. However, cellulosic biomass contains two major sugars, glucose and xylose, and a major obstacle in this process is that Saccharomyces yeasts, traditionally used and still the only microorganisms currently used for large scale industrial production of ethanol from glucose, are unable to ferment xylose to ethanol. This makes the use of these safest, most effective Saccharomyces yeasts for conversion of biomass to ethanol economically unfeasible. Since 1980, scientists worldwide have actively been trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we achieved a historic breakthrough to succeed in the development of the first genetically engineered Saccharomyces yeasts that can effectively ferment both glucose and xylose to ethanol. This was accomplished by carefully redesigning the yeast metabolic pathway for fermenting xylose to ethanol, including cloning three xylose-metabolizing genes, modifying the genetic systems controlling gene expression, changing the dynamics of the carbon flow, etc. As a result, our recombinant yeasts not only can effectively ferment both glucose and xylose to ethanol when these sugars are present separately in the medium, but also can effectively coferment both glucose and xylose present in the same medium simultaneously to ethanol. This has made it possible because we have genetically engineered the Saccharomyces yeasts as such that they are able to overcome some of the natural barrier present in all microorganisms, such as the synthesis of the xylose metabolizing enzymes not to be affected by the presence of glucose and by the absence of xylose in the medium. This first generation of genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts relies on the presence of a high-copy-number 2 mu-based plasmid that contains the three cloned genetically modified xylose-metabolizing genes to provide the xylose-metabolizing capability. In 1995, we achieved another breakthrough by creating the super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts which contain multiple copies of the same three xylose-metabolizing genes stably integrated on the yeast chromosome. This is another critical development which has made it possible for the genetically engineered yeasts to be effective for cofermenting glucose and xylose by continuous fermentation. It is widely believed that the successful development of the stable glucose-xylose-cofermenting Saccharomyces yeasts has made the biomass-to-ethanol technology a step much closer to commercialization. In this paper, we present an overview of our rationales and strategies as well as our methods and approaches that led to the ingenious design and successful development of our genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose to biofuel ethanol.