The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the ε-subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”

Thirty microbial phylotypes of microorganisms were found in the gastrointestinal tract of chicken belonging to the Hajseks White breed, and 38 phylotypes were found in the gastrointestinal tract of chicken belonging to the Hajseks Brown breed. The microbiome of the gastrointestinal tract of the chicken embryos of the Hajseks White breed was dominated by the typical representatives of avian intestinal microflora--bacteria of the family Enterobacteriaceae (47.3%), orders Actinomycetales (13.6%) and Bifidobacteriales (20.6%), and the family Lachnospiraceae (1.1%). The microbiome of the gastrointestinal tract of the chicken embryos of the Hajseks Brown breed was dominated by the pathogenic bacteria of the order Rickettsiales (94.8%). The metagenome of gastrointestinal tract of both breeds also contained a small number of genes of unidentified bacteria.

A b s t r a c tMicroorganisms which inhabit gut play great role in providing with nutrients, antibiotics, hormones and vitamins necessary for poultry health and performance. Therefore study of gut microbiome changes during ontogenesis seems to be essential. The structure of gut microflora in poultry embryos is of particular interest and debated because of very few publications on the problem. Despite embryo intestine is commonly considered sterile there are several reports on gut colonization by microorganisms in embryos during ontogenesis. Using T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis to generate a fingerprint of a microbial community we compared gut flora in chick embryos on days 6 and 17 to those in 26-day, 150-day and 300-day old Hisex White layers. Unlike accepted view, a high biodiversity was seen in embryo gut with Enterobacteriaceae (Escherichia coli mainly) predominated. Clostridia, Bacteroides, Negativicutes, Actinomycetales, Bifidocteriales were also found in contrast to earlier reports of their presence only in chicks at hatching and in adult poultry gut. Moreover, in the embryo gut we found the causal agents of dangerous animal disease, Burkholderia sp., Pseudomonas sp., Salmonella sp., Klebsiella sp. and Rickettsiales bacteria. Interestingly, the embryo gut biodiversity on day 6 was higher as compared to day 17 (75±2.75 phylotypes vs 30±1.20 phylotypes). In the layers aged 26, 150 and 300 days the diversity was much higher (over 175±8.12 phylotypes) as compared to embryos due to new members involved into gut bacterial community. Moreover, the poultry aged 300 days was lower both in the total diversity and in the percentage of unidentified microorganisms when compared to 26-day and 150-day old hens. In the adults, the predominating microbial taxa changed, in particular, Clostridia and Negativicutes became more abundant whereas Bacillales and Bifidobacteriales were depressed. Our findings indicate gut colonization by Lactobacilales and pathogenic Listeria sp., Pantoea sp., Enterobacter sp., Mycoplasma sp., Acinetobacter sp., Pasteurellaceae, Campylobacteraceae, Fusobacteria which occurred during ontogenesis. Thus the gut microbiome formation starts in embryo which is important for hatching and growing healthy poultry.

The composition of plant and silage microflora affects the fermentation processes in the silage and its final quality. To date, reports about studying fodder plants and silage microbiota by means of molecular genetic methods are few and limited to descriptions of composition and function of some groups of microorganisms. Moreover, the NGS (next generation sequencing) data on diversity of epiphytic microflora and silage microbiocoenosis are not still reported. We first used this approach in studying phyllosphere and silage microbiom, and reported it to be rather rich in composition and abundance that is in contrast with conventional understanding. At that, the pathogenic and non-culturable microbes were detected in the microbiota, including specific inhabitants of mammalian gastrointestinal tract. So using NGS we examined the structure and diversity of bacterial community of Dactylis glomerata L. harvested plants and the biomass ensilaged with chemical preservative AIV 2000 Plus (KEMIRA OYJ Inc., Finland) composed of mixture of formic, propionic and benzoic acids. Assays were carried out on days 3, 7, 14 and 30 of ensilaging. The results showed that the bacterial community of silage from D. glomerata sharply differed from the composition of foliage microorganisms and varied greatly in the course of successive changes which occurred during maturation of silage preserved by mixture of organic acids. The composition of plant microorganisms and silage were found to be very various in contrast with a traditional view. Among foliar microorganisms of D. glomerata there were mostly the bacteria of phylum Proteobacteria (94.1 %), and in the silage the bacteria of phylums Bacteroidetes and Firmicutes were main representatives (up to 59.5 % and 74.9 %, respectively). In taxonomic diversity of the order Lactobacillales, mainly involved in ensilaging, there were genera Lactobacillus (up to 39.6%), Enterococcus (up to 36.36 %), Lactococcus (up to 14.4 %), Pediococcus (to 1.45 %) and the family Leuconostocaceae (to 3.52 %). Interestingly, in the silage there were the bacteria of phylum Bacteroidetes, families Ruminococcaceae, Lachnospiraceae and Selenomonadales considered the common inhabitants of the mammals' gastrointestinal tract, and also the uncultured and pathogenic microorganisms. Particularly, these were 15 genera of family Enterobacteriaceae, including genera Klebsiella, Salmonella, Yersinia, etc., among which the dangerous mammalian pathogens are frequent. On days 3 and 7, the phylum Bacteroidetes prevailed (59.53 and 48.91 %, respectively). On days 14 and 30, the phylum Firmicutes was dominant (up to 74.85 %) with the facultative aerobic bacteria of order Lactobacillales mostly found (up to 74.76 %). Using NGS, a total of 70 genera were attributed in the plant phyloshere, and in the silage there were 84 genera on day 3, 96 genera on day 7, 51 genera on day 14, and 69 genera on day 30. Classical microbiology methods are not enough to detect these bacteria among silage microbiota.