Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.
PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.
Matched MeSH terms: Bryopsida/enzymology*; Bryopsida/genetics; Bryopsida/growth & development
Gunung Senyum Recreational Forest harbours 59 species, two subspecies and five varieties of mosses in 32 genera and 16 families that had been identified from a total of 589 specimens collected from the area. These figures represent 11.8% out of the 558 taxa, 20.2% out of the 158 genera and 34.7% out of the 46 families of mosses reported for Peninsular Malaysia. The total also represents 14.9% of the 442 taxa, 24.0% of the 133 genera and 40.0% of the 40 families of mosses recorded in Pahang. The largest family of mosses found in this limestone forest is Calymperaceae followed by Fissidentaceae. There are two new records for Pahang, Calymperespallidum Mitt. and Taxitheliumbinsteadii Broth. & Dixon. The analysis of species similarities of mosses found in the study area with some other selected areas showed that Gunung Senyum Recreational Forest had a high percentage of species similarity with Perlis State Park at Wang Kelian, another limestone forest, at 38%. Corticol is the main habitat utilised by mosses in Gunung Senyum Recreational Forest with 47 taxa, followed by the lignicol and calcicol each with 35 and 26 taxa, respectively.
This paper reports the mosses from Crocker Range Park (CRP) in Sabah, Malaysian Borneo. In total, 293 species, three subspecies and eight varieties belonging to 118 genera and 36 families are reported. This represents about 40% and 47% of the species and infra-specific taxa reported from Borneo and Sabah, respectively. Out of these, six species are new records for Borneo, namely Barbella horridula, Chaetomitrium lancifolium, Distichophyllum leiopogon, Rhaphidostichum luzonense, Rosulabryum capillare and Taxiphyllum taxirameum and 12 species and one variety are new to Sabah. With these additions, the current number of mosses in Sabah and Borneo are 651 and 766, respectively. The largest family of mosses is Calymperaceae with 35 species and one subspecies, followed by Sematophyllaceae with 32 species and two varieties and Pylaisiadelphaceae with 21 species and one variety. In conclusion, CRP has a very high species richness of mosses which is the second highest in Borneo, after Mount Kinabalu.
Anthropogenic airborne depositions of 210Po,
210Pb and 210Po/210Pb in the mosses and surface soils
collected at the vicinity of a coal-fired power plant were studied. The purpose of the study was to
determine activity concentrations of 210Po,
210Pb and 210Po/210Pb for assessing their variation
accumulation in the mosses and surface soils collected at the vicinity of a coal-fired power plant.
Other purposes were to determine their concentration factor (CF) in relation to track the potential
source of those radionuclides and to identify most suitable moss species as a biological indicator
for atmospheric deposition contaminants. In this study, different species of moss Leucobryum
aduncum, Campylopus serratus, Syrrhopodon ciliates and Vesicularia montagnei were collected in
May 2011 at the area around 15 km radius from Tanjung Bin coal-fired power plant located in
Pontian, Johor. The activity concentrations of 210Po,
210Pb and 210Po/210Pb in mosses were in the
range of 76.81 ± 4.94 – 251.33 ± 16.33 Bq/kg dry wt., 54.37 ± 3.38 – 164.63 ± 11.64 Bq/kg dry wt.
and 1.10 – 2.00, respectively. Meanwhile the ranges for those radionuclides in the surface soil
were 33.53 ± 2.10 – 179.67 ± 12.15 Bq/kg dry wt., 20.55 ± 1.33 – 106.62 ± 6.64 Bq/kg dry wt. and
1.61 – 2.44, respectively. Corresponding high ability of Leucobryum aduncum to accumulate more
210Po and 210Pb, wide geographical distribution, most abundant and high CF, therefore, the
findings can be concluded this species was the most suitable as a biological indicator for
atmospheric deposition contaminants such as 210Po and 210Pb. Furthermore, it is clear the
accumulation of 210Po and 210Pb in mosses might be supplied from various sources of atmospheric
deposition such as coal-fired power plant operation, industrial, agriculture and fertilizer activities,
burned fuel fossil and forest; and other potential sources. Meanwhile, the
: Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.
Biomonitoring of multi-element atmospheric deposition using terrestrial moss is a well-established technique in Europe. Although the technique is widely known, there were very limited records of using this technique to study atmospheric air pollution in Malaysia. In this present study, the deposition of 11 trace metals surrounding the main petroleum refinery plant in Kerteh Terengganu (eastern part of peninsular Malaysia) has been evaluated using two local moss species, namely Hypnum plumaeforme and Taxithelium instratum as bioindicators. The study was also done by means of observing whether these metals are attributed to work related to oil exploration in this area. The moss samples have been collected at 30 sampling stations in the vicinity of the petrochemical industrial area covering up to 15 km to the south, north, and west in radius. The contents of heavy metal in moss samples were analyzed by energy dispersive x-ray fluorescence technique. Distribution of heavy metal content in all mosses is portrayed using Surfer software. Areas of the highest level of contaminations are highlighted. The results obtained using the principal components analysis revealed that the elements can be grouped into three different components that indirectly reflected three different sources namely anthropogenic factor, vegetation factor, and natural sources (soil dust or substrate) factor. Heavy metals deposited mostly in the distance after 9 km onward to the western part (the average direction of wind blow). V, Cr, Cu, and Hg are believed to have originated from local petrochemical-based industries operated around petroleum industrial area.
Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.