Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha

We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli.     

Effect of 2,4-dichlorophenoxyacetic acid on invertases in chicory root

The invertase present in roots of chicory (Cichoriun intybus) has a pH optimum of 7.5 and a MW of ca 260 000. It requires relatively high ionic strength to remove it from DEAE cellulose. Treatment of chicory root tissue with 2,4-dichlorophenoxyacetic acid gives rise to a highly active invertase with pH optimum of 5.6 and MW of ca 61 000. It is more easily removed from DEAE cellulose.     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

Effect of 2,4-dichlorophenoxyacetic acid on the structure and function of developing chloroplasts

Dark-grown radish seedlings (Raphanus sativus L.) were sprayed with 10^-3 mol·l^-1 2,4-dichlorophenoxyacetic acid and then were exposed to a 14:10 light: dark cycle. Cotyledon samples from these seedlings and unsprayed controls were taken for electron microscopy, chlorophyll determinations, and photosynthetic rate measurements at regular intervals for 72 h. A normal development of etioplasts to chloroplasts with formation of typical grana-fret work system was observed in the control cotyledons. The chloroplasts in the 2,4-D-treated cotyledons showed changes in the organization of the grana thylakoids; these thylakoids being more appressed to each other than in the controls. The chlorophyll content of treated plants was less than that of controls but the rate of chlorophyll biosynthesis was unaffected. The photosynthetic rate/mg chlorophyll was considerably higher for treated plants suggesting that 2,4-D treatment resulted in decreased size of the photosynthetic unit.     

A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil

A bioreporter was made containing a tfdRP(DII)-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 microM to 5.0 mM. This response was linear (R(2) = 0.9825) in the range of 2.0 microM to 1.1 x 10(2) microM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 x 10(2) microM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.     

Determination of the Ks values during the growth of Alcaligenes eutrophus on phenol, 2,4-dichlorophenoxyacetic acid and fructose

The determination of the K_S values presented here is based on the estimation of the stationary substrate concentrations in continuous cultivation experiments. The separation of biomass from the suspension was performed by an ultrafiltration step which succeeded within one second. The decay of substrate concentration during sampling was calculated to amount to less than 6% of the stationary substrate concentration at relevant growth rates. The K_S values derived from these reduced substrate concentrations deviated by only 10% from the theoretical values at a biomass concentration of about 1 g/1. Thus relevant kinetic parameters can be calculated from the data obtained by this procedure. Values of 11, 59 and 14 μM were obtained with 2,4-dichlorophenoxyacetic acid (2,4-D), phenol and fructose, respectively. Similar K_S values were derived with 2,4-D and fructose by using a respirationbased determination for reasons of comparison. With phenol this value was only 7 μM which is as cribed to a physiological background.     

Comparison of a gene probe with classical methods for detecting 2,4-dichlorophenoxyacetic acid (2,4-D)-biodegrading bacteria in natural waters

Colony hybridizations with a gene probe for enumeration of 2,4-dichlorophenoxy-acetic acid (2,4-D)-degrading bacteria were compared with classical enrichment and radiolabel most-probable-number (MPN) assay methods. Two natural water samples (rivers) and raw sewage were tested by each method. UV scans of enrichment cultures revealed 2,4-D degradation with raw sewage occurred in 4–11 days, 4–>22 days with Mary's River water, and 5–>22 days with Willamette River water. [¹⁴C]-2,4-D MPN analysis, measuring release of¹⁴CO₂, yielded estimates of bacteria per milliliter able to degrade 2,4-D. Raw sewage estimates were 1.4 × 10⁵ 2,4-D degraders/ml, Mary's River >1.6 × 10⁵/ml, and Willamette River water 1.6 × 10⁴/ml. Activities noted by UV scan enrichment data supported these results.Autoradiograms of colony blots were also used to estimate numbers of 2,4-D-degrading bacteria. These estimates were also supported by the UV scan data from enrichment cultures. Raw sewage gave counts between 5 × 10⁴ and 2.9 × 10⁵ 2,4-D-degrading bacteria/ml, which correlates well with the estimates obtained by¹⁴C-MPN analyses. River waters, both much lower in total bacterial counts and organic carbon than raw sewage, yielded fewer 2,4-D-degrading bacteria than estimated by¹⁴C-MPN. Media composition and cometabolism may account for discrepancies in estimates for 2,4-D-degrading bacteria observed when colony blot and¹⁴C-MPN analyses were compared.Replica plating made it possible to test for 2,4-D biodegradation from colonies reactive in autoradiograms. Five of 12 (42%) colonies reacting in the colony hybridization exhibited biodegradation activities. Nonreactive colonies failed to degrade 2,4-D.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of¹⁴CO₂ a product of microbial degradation of 2-¹⁴C-2,4-D, was measured during 38-d incubation at 28°C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH₄)₂SO₄ (C:N=5∶1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization, did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent, of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into¹⁴CO₂). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers. The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.     

Influence of temperature on growth, 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in Azotobacter chroococcum

Summary Degradation of 2,4-D by Azotobacter chroococcum is profoundly influenced by incubation temperature. At 30 C the generation time was 1.4 h. Residual 2,4-D was detected when grown at 20 C. At 35 C the plasmid transfer was maximum.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4).

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955, 1987). A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to tfdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.     

Effects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid

Batch experiments were conducted to examine the effects of dissolved oxygen concentration on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by an enrichment culture of 2,4-D-utilizing bacteria. A modified Monod equation was found to describe the relationship between the specific growth rate and the concentrations of both the organic substrate and dissolved oxygen. Values for the maximum specific growth rate, yield, and Monod coefficient for growth on 2,4-D were 0.09 h-1, 0.14 g/g, and 0.6 mg/liter, respectively. The half-saturation constant for dissolved oxygen was estimated to be 1.2 mg/liter. These results suggest that dissolved oxygen concentrations below 1 mg/liter may be rate limiting for the biodegradation of chlorinated aromatic compounds such as 2,4-D, which have a requirement for molecular oxygen as a cosubstrate for metabolism.