Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products.

Homofermentative production of reduced products requires additional reducing power output (NADH) from glucose catabolism. Anaerobic expression of the pyruvate dehydrogenase complex (PDH, encoded by aceEF-lpd, a normal aerobic operon) is able to provide the additional NADH required for production of reduced products in Escherichia coli fermentation. The multiple promoters (pflBp(1-7)) of pyruvate formate lyase (pflB) were evaluated for anaerobic expression of the aceEF-lpd operon. Four chromosomal constructs, pflBp(1-7)-aceEF-lpd, pflBp(1-6)-aceEF-lpd, pflBp(6,7)-aceEF-lpd, and pflBp6-aceEF-lpd efficiently expressed the PDH complex in anaerobically grown cells. Doubling the reducing power output was achieved when glucose was oxidized to acetyl-CoA through glycolysis and pyruvate oxidation by the anaerobically expressed PDH complex (glucose -->2 acetyl-CoA + 4 NADH). This additional reducing power output can be used for production of reduced products in anaerobic E. coli fermentation.

Metabolic evolution of non-transgenic Escherichia coli SZ420 for enhanced homoethanol fermentation from xylose.

Efficient utilization of pentose sugars (xylose and arabinose) is an essential requirement for economically viable ethanol production from cellulosic biomass. The desirable pentose-fermenting ethanologenic biocatalysts are the native microorganisms or the engineered derivatives without recruited exogenous gene(s). We have used a metabolic evolution (adaptive selection) approach to improve a non-transgenic homoethanol Escherichia coli SZ420 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) for xylose fermentation. An improved mutant, E. coli KC01, was evolved through a 3 month metabolic evolution process. This evolved mutant increased pyruvate dehydrogenase activity by 100%, cell growth rate (h(-1)) by 23%, volumetric ethanol productivity by 65% and ethanol tolerance by 200%. These improvements enabled KC01 to complete 50 g xylose l(-1) fermentations with an ethanol titer of 23 g l(-1) and a yield of 90%. The improved cell growth and ethanol production of KC01 are likely attributed to its three fold increased ethanol tolerance.

Engineering a native homoethanol pathway in Escherichia coli B for ethanol production.

A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (DeltafrdBC DeltaldhA DeltaackA DeltafocA-pflB DeltapdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.

Transformation of sunflower (Helianthus annuus L.) following wounding with glass beads.

A procedure was developed for transformation of Helianthus annuus (sunflower) using Agrobacterium tumefaciens. Cotyledons were removed from young seedlings, and the remaining tissue was uniformly wounded by shaking with glass beads. The wounded tissue was then co-cultivated with a hypervirulent strain of Agrobacterium tumefaciens harboring the binary plasmid pCNL56. Minimal use of defined medium was required, and no callus was observed. The polymerase chain reaction (PCR) followed by DNA hybridization demonstrated the presence of gusA DNA from pCNL56 in total leaf DNA of 6 primary transformants and 2 progeny plants. No Agrobacterium DNA was detected in total DNA from transformed sunflower leaves that was amplified with primers specific to the miaA chromosomal gene of Agrobacterium. Foreign DNA was also detected in the next generation. ß-Glucuronidase (GUS) activity was demonstrated for 5 of the T2 transgenic plants. Grafting was used to increase the number of seeds present on plants that had undergone tissue culture manipulations.

Fatty acid alteration by a delta 9 desaturase in transgenic tobacco tissue.

Nicotiana tabacum tissue was transformed with a rat stearyl-CoA desaturase gene. Gas chromatographic analysis showed an increase in monounsaturated 16 and 18 carbon fatty acids in selected transformed calli and leaves. Fractionation of lipid classes indicated that palmitoleic acid was found in the phosphatidylcholine fraction of desaturase-transformed leaves, but not in leaves transformed with vector sequences. Plant transformation was verified by polymerase chain reaction (PCR) amplification of total leaf DNA.

Expression of soybean-embryo lipoxygenase 2 in transgenic tobacco tissue.

To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. 'KY 14' by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5'-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3' to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1-0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50-529% increase in C6-aldehyde production. The impact on C6-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C6-aldehydes than that of other well-characterized LOX isozymes.

Effects of exogenous auxins on expression of lipoxygenases in cultured soybean embryos.

The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either alpha-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by alpha-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos.

Soybean leaves contain multiple lipoxygenases.

Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pls), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pls of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies.

A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication.

5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.

Variable abundance of a mitochondrial DNA fragment in cultured tobacco cells.

The relative abundance of a cloned 4.5 kilobase (kb) pair mitochondrial DNA sequence in two suspension cultures of tobacco (Nicotiana tabacum cv Turkish samsun and Nicotiana tabacum NT-1) has been examined. This sequence is 70-fold reduced in NT-1 relative to Turkish samsun; the reduction is correlated with an increase in supercoiled mitochondrial DNA. This sequence does not hybridize with mitochondrial DNA from watermelon, maize, or Saccharomyces cerevisiae, nor with several cloned mitochondrial genes and is thus probably not a gene. It may represent most of the plant mitochondrial genome thought to be non-essential for mitochondrial function. The sequence complexity of supercoiled mitochondrial DNA from NT-1 cells is about one-third that found for the entire mitochondrial genome and does not include the cytochrome oxidase subunit II gene.

Bud Induction with Cytokinin : A LOCAL RESPONSE TO LOCAL APPLICATION.

A portion of the surface of detached Graptopetalum paraquayense E. Walther leaves can be used to assay small amounts of reagents in lanolin for their ability to induce shoots only at the site of application. The cytokinins benzyladenine, kinetin, and 6-(gamma,gamma-dimethylallylamino)purine (DMAAP) were tested, and DMAAP was most effective in bud induction at concentrations below 1%. The higher the hormone concentration, the sooner the appearance of leaf primordia and the higher the ultimate yield of buds. Leaves treated with DMAAP for 2 days developed buds as rapidly as those with longer treatments.