Metabolic engineering of plant secondary products

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.     

THE POPULATION DYNAMICS OF NORTHERN SEA LIONS, 1975-1985

Abstract: Populations of northern sea lions ( Eumetopias jubatus ) in the vicinity of Marmot Island, Alaska declined during 1975–1985 at about 5% per year (Merrick et al. 1987). The cause of this decline is not known. A life table for the northern sea lion was calculated assuming that life spans follow a Weibull distribution. Samples of northern sea lions taken in the vicinity of Marmot Island, Alaska during 1975–1978 and 1985–1986 indicate that the average age of females older than 3 yr increased about 1.55 yr (SD = 0.35 yr) while the population was declining at about 5% per year. Fecundity rates decreased by 10% over the same period, but the decrease was not statistically significant (Calkins and Goodwin 1988). Possible causes of the population decline and the change in age structure were examined by writing the Leslie matrix population equation in terms of changes in juvenile and adult survival rates and fecundity, and examining the short–term behavior of the trajectories of the average age of adult females, total number of females, and total number of pups with respect to those changes in the vital parameters. From the observed rate of declines of adults and the changes in average age of adult females and fecundity, estimates of the changes in adult and juvenile survival were calculated; estimates of the standard deviations of these changes were estimated via a bootstrap procedure. One purpose of this exercise is to aid in setting priorities for research for determining the cause of the decline. An explanation for the observed declines in numbers of adult sea lions consistent with the observed fecundity rates, a rate of decrease of 5% in the number of adults, and the corresponding increase in average age (of females age 3 yr and older) was a 10%–20% decrease in the survival of juveniles (age 0-3 yr) coupled with an insignificant change in adult survival (0.03%, SD = 1%).     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Immunological determination of digestive rates in the syntopic scorpions Urodacus armatus Pocock and Urodacus novaehollandiae Peters

Scorpions have lengthy periods of inactivity, which may be caused either by a long physiological digestion time or predator avoidance or both. We used a quantitative immunological assay to monitor the amount of prey antigen remaining in the hepatopancreas of Urodacus armatus and U. novaehollandiae for 32 days after a meal to test the hypothesis of a long digestion time. In both species, prey antigen concentration in the hepatopancreas increased between 0.25 and 0.5 days after feeding, probably because of incomplete transfer of prey proteins from the large preoral cavity to the hepatopancreas at this stage of digestion. This was followed by a rapid decline, and by 3 days after feeding less than 25% of ingested antigen remained in each species. Small prey residues could still be detected up to 32 days. Prey decay curves in both species were best fitted by a log-log transformation, and were not significantly different. Since digestion is essentially complete after 3 days, it appears that longer periods of post-feeding inactivity are for predator avoidance, not digestion. However, the 32-day detection period exceeds those reported for other invertebrates, and suggests an unusual digestive physiology in scorptions.     

Spatial pattern,regeneration and growth rates of Brachystegia spiciformis and Julbernardia globiflora

Past work on tree-to-tree interactions in semi-arid savannas, through the use of nearest neighbour analysis, has shown both a regular dispersion pattern and a positive correlation between the size of a tree and the distance to its nearest neighbour. From these results, the importance of competition has been inferred. In the present study, tree-to-tree interactions in a mesic savanna woodland at Marondera, Zimbabwe, dominated by relatively small-sized individuals of Brachystegia spiciformis and Julbernardia globiflora, were examined. Results show that the regular dispersion pattern is uncommon; that there are few significant positive correlations between size of individual and distance to nearest neighbour; and that growth rate is not consistently correlated with size and distance of nearest neighbour. Results of a study of regeneration relative to canopy cover suggest a possible mechanism for these findings: unlike semi-arid savannas, young plants often grow in under-canopy environments so that early spacing of young trees does not occur. Significant positive correlations between the size of a tree and the distance to its nearest neighbour were found in a stand of larger, presumably older, trees, suggesting that such a relationship only develops in mesic miombo woodland through a thinning process as the trees mature. Familial clumping, which is common in the study area, may mask spatial patterns.Abbreviations RBAI Relative basal area increment     

关于一个“基细胞”的疑问

有或没有基细胞是毛鞘藻属(Bulbochaets)与枝鞘藻属(Oedocladium)的区别之一。这里叙述了Mrozinska在其专著(1985)中,将Oedocladium indicum Kama附图(即模式图)上的一个基细胞错误地移置到Oe.PrescottiiIslam上去的情况。     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.     

ABSENCE OF POLLEN DISCOUNTING IN A GENOTYPE OF IPOMOEA PURPUREA EXHIBITING INCREASED SELFING

Throughout southeastern North America, the annual morning glory Ipomoea purpurea exhibits a polymorphism at a locus that influences the intensity of floral pigmentation. Previous studies have shown that when rare, the homozygous white genotype has a greater selfing rate than the homozygous dark genotype. In the absence of pollen discounting (a reduction in transmission of pollen to other plants by genotypes that exhibit increased selfing) and inbreeding depression, this increased selfing rate should favor the white allele. Experiments reported here confirm that the white genotype has elevated selfing rates when rare but indicate pollen discounting is not associated with elevated selfing. Rather, white genotypes contribute more pollen to the outcross pollen pool. The disparity between genotypes in both selfing rates and success at pollen contribution to other plants disappears at intermediate to high frequencies of the white allele. Pollinator movements are consistent with the pattern of selfing. These results suggest that elevated selfing and enhanced success at pollen donation contribute to maintenance of the white allele in natural populations of morning glories.     

A surplus no more? Variation in krill availability impacts reproductive rates of Antarctic baleen whales

The krill surplus hypothesis of unlimited prey resources available for Antarctic predators due to commercial whaling in the 20th century has remained largely untested since the 1970s. Rapid warming of the Western Antarctic Peninsula (WAP) over the past 50 years has resulted in decreased seasonal ice cover and a reduction of krill. The latter is being exacerbated by a commercial krill fishery in the region. Despite this, humpback whale populations have increased but may be at a threshold for growth based on these human-induced changes. Understanding how climate-mediated variation in prey availability influences humpback whale population dynamics is critical for focused management and conservation actions. Using an 8-year dataset (2013–2020), we show that inter-annual humpback whale pregnancy rates, as determined from skin-blubber biopsy samples (n = 616), are positively correlated with krill availability and fluctuations in ice cover in the previous year. Pregnancy rates showed significant inter-annual variability, between 29% and 86%. Our results indicate that krill availability is in fact limiting and affecting reproductive rates, in contrast to the krill surplus hypothesis. This suggests that this population of humpback whales may be at a threshold for population growth due to prey limitations. As a result, continued warming and increased fishing along the WAP, which continue to reduce krill stocks, will likely impact this humpback whale population and other krill predators in the region. Humpback whales are sentinel species of ecosystem health, and changes in pregnancy rates can provide quantifiable signals of the impact of environmental change at the population level. Our findings must be considered paramount in developing new and more restrictive conservation and management plans for the Antarctic marine ecosystem and minimizing the negative impacts of human activities in the region.