Evolution Of Biosynthetic Pathways Research Articles

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA

Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. In this study, we identified the long non-coding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to abscisic acid (ABA). MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel non-coding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

Influence of domestication on specialized metabolic pathways in fruit crops.

During the process of plant domestication, the selection and traditional breeding for desired characters such as flavor, juiciness and nutritional value of fruits, probably have resulted in gain or loss of specialized metabolites contributing to these traits. Their appearance in fruits is likely due to the acquisition of novel and specialized metabolic pathways and their regulation, driven by systematic molecular evolutionary events facilitated by traditional breeding. Plants change their armory of specialized metabolism to adapt and survive in diverse ecosystems. This may occur through molecular evolutionary events, such as single nucleotide polymorphism, gene duplication and transposition, leading to convergent or divergent evolution of biosynthetic pathways producing such specialized metabolites. Breeding and selection for improved specific and desired traits (fruit size, color, taste, flavor, etc.) in fruit crops through conventional breeding approaches may further alter content and profile of specialized metabolites. Biosynthetic routes of these metabolites have been studied in various plants. Here, we explore the influence of plant domestication and breeding processes on the selection of biosynthetic pathways of favorable specialized metabolites in fruit crops. An orderly clustered arrangement of genes associated with their production is observed in many fruit crops. We further analyzed selection-based acquisition of specialized metabolic pathways comparing first the metabolic profiles and genes involved in their biosynthesis, followed by the genomic organization of such genes between wild and domesticated horticultural crops. Domestication of crop plants favored the acquisition and retention of metabolic pathways that enhanced the fruit value while eliminated those which produced toxic or unfavorable metabolites. Interestingly, unintentional reorganization of complex metabolic pathways by selection and traditional breeding processes has endowed us with flavorful, juicy and nutritionally rich fruits.

Microbial production of small peptide: pathway engineering and synthetic biology.

SummarySmall peptides are a group of natural products with low molecular weights and complex structures. The diverse structures of small peptides endow them with broad bioactivities and suggest their potential therapeutic use in the medical field. The remaining challenge is methods to address the main limitations, namely (i) the low amount of available small peptides from natural sources, and (ii) complex processes required for traditional chemical synthesis. Therefore, harnessing microbial cells as workhorse appears to be a promising approach to synthesize these bioactive peptides. As an emerging engineering technology, synthetic biology aims to create standard, well‐characterized and controllable synthetic systems for the biosynthesis of natural products. In this review, we describe the recent developments in the microbial production of small peptides. More importantly, synthetic biology approaches are considered for the production of small peptides, with an emphasis on chassis cells, the evolution of biosynthetic pathways, strain improvements and fermentation.

PlantFAdb: a resource for exploring hundreds of plant fatty acid structures synthesized by thousands of plants and their phylogenetic relationships.

Over 450 structurally distinct fatty acids are synthesized by plants. We have developed PlantFAdb.org, an internet-based database that allows users to search and display fatty acid composition data for over 9000 plants. PlantFAdb includes more than 17000 data tables from >3000 publications and hundreds of unpublished analyses. This unique feature allows users to easily explore chemotaxonomic relationships between fatty acid structures and plant species by displaying these relationships on dynamic phylogenetic trees. Users can navigate between order, family, genus and species by clicking on nodes in the tree. The weight percentage of a selected fatty acid is indicated on phylogenetic trees and clicking in the graph leads to underlying data tables and publications. The display of chemotaxonomy allows users to quickly explore the diversity of plant species that produce each fatty acid and that can provide insights into the evolution of biosynthetic pathways. Fatty acid compositions and other parameters from each plant species have also been compiled from multiple publications on a single page in graphical form. Links provide simple and intuitive navigation between fatty acid structures, plant species, data tables and the publications that underlie the datasets. In addition to providing an introduction to this resource, this report illustrates examples of insights that can be derived from PlantFAdb. Based on the number of plant families and orders that have not yet been surveyed we estimate that a large number of novel fatty acid structures are still to be discovered in plants.

Exploring the geochemical distribution of organic carbon in early land plants: a novel approach.

Terrestrialization depended on the evolution of biosynthetic pathways for biopolymers including lignin, cutin and suberin, which were concentrated in specific tissues, layers or organs such as the xylem, cuticle and roots on the submillimetre scale. However, it is often difficult, or even impossible especially for individual cells, to resolve the biomolecular composition of the different components of fossil plants on such a scale using the well-established coupled techniques of gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Here, we report the application of techniques for surface analysis to investigate the composition of Rhynia gwynne-vaughanii. X-ray photoelectron spectroscopy of two different spots (both 300 µm × 600 µm) confirmed the presence of carbon. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed ‘chemical maps’ (imaging mode with 300 nm resolution) of aliphatic and aromatic carbon in the intact fossil that correlate with the vascular structures observed in high-resolution optical images. This study shows that imaging ToF-SIMS has value for determining the location of the molecular components of fossil embryophytes while retaining structural information that will help elucidate how terrestrialization shaped the early evolution of land plant cell wall biochemistry.This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.

Origin and evolution of transporter substrate specificity within the NPF family.

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

Flower power and the mustard bomb: Comparative analysis of gene and genome duplications in glucosinolate biosynthetic pathway evolution in Cleomaceae and Brassicaceae.

Glucosinolates (GS) are a class of plant secondary metabolites that provide defense against herbivores and may play an important role in pollinator attraction. Through coevolution with plant-interacting organisms, glucosinolates have diversified into a variety of chemotypes through gene sub- and neofunctionalization. Polyploidy has been of major importance in the evolutionary history of these gene families and the development of chemically separate GS types. Here we study the effects of polyploidy in Tarenaya hassleriana (Cleomaceae) on the genes underlying GS biosynthesis. We established putative orthologs of all gene families involved in GS biosynthesis through sequence comparison and their duplication method through calculation of synonymous substitution ratios, phylogenetic gene trees, and synteny comparison. We drew expression data from previously published work of the identified genes and compared expression in several tissues. We show that the majority of gene family expansion in T. hassleriana has taken place through the retention of polyploid duplicates, together with tandem and transpositional duplicates. We also show that the large majority (>75%) is actively expressed either globally or in specific tissues. We show that MAM and CYP83 gene families, which are crucial to GS diversification in Brassicaceae, are also recruited into specific tissue expression pathways in Cleomaceae. Many GS genes have expanded through polyploidy, gene transposition duplication, and tandem duplication in Cleomaceae. Duplicate retention through these mechanisms is similar to A. thaliana, but based on the expression of GS genes, Cleomaceae-specific diversification of GS genes has taken place.

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

BackgroundLarge proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as ‘blooms’, providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects.ResultsA bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ.ConclusionTwo subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 ‘blooms’ in response to environmental pressures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0396-z) contains supplementary material, which is available to authorized users.

Efficient discovery of bioactive scaffolds by activity-directed synthesis.

The structures and biological activities of natural products have often provided inspiration in drug discovery. The functional benefits of natural products to the host organism steers the evolution of their biosynthetic pathways. Here, we describe a discovery approach--which we term activity-directed synthesis--in which reactions with alternative outcomes are steered towards functional products. Arrays of catalysed reactions of α-diazo amides, whose outcome was critically dependent on the specific conditions used, were performed. The products were assayed at increasingly low concentration, with the results informing the design of a subsequent reaction array. Finally, promising reactions were scaled up and, after purification, submicromolar ligands based on two scaffolds with no previous annotated activity against the androgen receptor were discovered. The approach enables the discovery, in tandem, of both bioactive small molecules and associated synthetic routes, analogous to the evolution of biosynthetic pathways to yield natural products.

Evolution of tryptophan biosynthetic pathway in microbial genomes: a comparative genetic study

Biosynthetic pathway evolution needs to consider the evolution of a group of genes that code for enzymes catalysing the multiple chemical reaction steps leading to the final end product. Tryptophan biosynthetic pathway has five chemical reaction steps that are highly conserved in diverse microbial genomes, though the genes of the pathway enzymes show considerable variations in arrangements, operon structure (gene fusion and splitting) and regulation. We use a combined bioinformatic and statistical analyses approach to address the question if the pathway genes from different microbial genomes, belonging to a wide range of groups, show similar evolutionary relationships within and between them. Our analyses involved detailed study of gene organization (fusion/splitting events), base composition, relative synonymous codon usage pattern of the genes, gene expressivity, amino acid usage, etc. to assess inter- and intra-genic variations, between and within the pathway genes, in diverse group of microorganisms. We describe these genetic and genomic variations in the tryptophan pathway genes in different microorganisms to show the similarities across organisms, and compare the same genes across different organisms to find the possible variability arising possibly due to horizontal gene transfers. Such studies form the basis for moving from single gene evolution to pathway evolutionary studies that are important steps towards understanding the systems biology of intracellular pathways.

Scalable Plasmid Transfer using Engineered P1-based Phagemids

Dramatic improvements to computational, robotic, and biological tools have enabled genetic engineers to conduct increasingly sophisticated experiments. Further development of biological tools offers a route to bypass complex or expensive mechanical operations, thereby reducing the time and cost of highly parallelized experiments. Here, we engineer a system based on bacteriophage P1 to transfer DNA from one E. coli cell to another, bypassing the need for intermediate DNA isolation (e.g., minipreps). To initiate plasmid transfer, we refactored a native phage element into a DNA module capable of heterologously inducing phage lysis. After incorporating known cis-acting elements, we identified a novel cis-acting element that further improves transduction efficiency, exemplifying the ability of synthetic systems to offer insight into native ones. The system transfers DNAs up to 25 kilobases, the maximum assayed size, and operates well at microliter volumes, enabling manipulation of most routinely used DNAs. The system's large DNA capacity and physical coupling of phage particles to phagemid DNA suggest applicability to biosynthetic pathway evolution, functional proteomics, and ultimately, diverse molecular biology operations including DNA fabrication.

Metamorphic enzyme assembly in polyketide diversification

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a beta-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide beta-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH(1)s), decarboxylases (ECH(2)s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide beta-branching pathway was modified by introduction of a gamma-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), alpha-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH(2) was found to catalyse formation of the alpha,beta enoyl thioester, whereas Jam ECH(2) formed a vinyl chloride moiety by selectively generating the corresponding beta,gamma enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH(2) instead of the canonical alpha,beta C = C saturation reaction. Thus, the combination of chlorination and polyketide beta-branching, coupled with mechanistic diversification of ECH(2) and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

On the origin and evolution of biosynthetic pathways: integrating microarray data with structure and organization of the Common Pathway genes

BackgroundThe lysine, threonine, and methionine biosynthetic pathways share the three initial enzymatic steps, which are referred to as the Common Pathway (CP). In Escherichia coli three different aspartokinases (AKI, AKII, AKIII, the products of thrA, metL and lysC, respectively) can perform the first step of the CP. Moreover, two of them (AKI and AKII) are bifunctional, carrying also homoserine dehydrogenasic activity (hom product). The second step of the CP is catalyzed by a single aspartate semialdehyde dehydrogenase (ASDH, the product of asd). Thus, in the CP of E. coli while a single copy of ASDH performs the same reaction for three different metabolic routes, three different AKs perfom a unique step. Why and how such a situation did emerge and maintain? How is it correlated to the different regulatory mechanisms acting on these genes? The aim of this work was to trace the evolutionary pathway leading to the extant scenario in proteobacteria.ResultsThe analysis of the structure, organization, phylogeny, and distribution of ask and hom genes revealed that the presence of multiple copies of these genes and their fusion events are restricted to the γ-subdivision of proteobacteria. This allowed us to depict a model to explain the evolution of ask and hom according to which the fused genes are the outcome of a cascade of gene duplication and fusion events that can be traced in the ancestor of γ-proteobacteria. Moreover, the appearance of fused genes paralleled the assembly of operons of different sizes, suggesting a strong correlation between the structure and organization of these genes. A statistic analysis of microarray data retrieved from experiments carried out on E. coli and Pseudomonas aeruginosa was also performed.ConclusionThe integration of data concerning gene structure, organization, phylogeny, distribution, and microarray experiments allowed us to depict a model for the evolution of ask and hom genes in proteobacteria and to suggest a biological significance for the extant scenario.

Sterols and other triterpenoids: source specificity and evolution of biosynthetic pathways

The source specificity and biosynthesis of cyclic isoprenoids such as hopanoids, sterols and pentacyclic triterpenoids, which are commonly used as biomarkers in organic geochemistry, is reviewed. Advances in molecular biology have provided much new information about the identity of isoprenoid lipid synthesis genes, which now makes it possible to confirm whether the biosynthetic capability to produce a particular biomarker exists in an organism or not. Such information will allow organic geochemists to use biomarkers to assign sources of organic matter with more confidence and opens up the possibility of using fossil rDNA in younger sediments as the ultimate biomarker. In ancient sediments the presence of a biomarker may only provide information about the existence of a biosynthetic pathway rather than the presence of a particular group of organisms. For such applications, it is vital that we understand how biochemical pathways have evolved over time, in parallel with the well-recognised evolution of species on earth as codified in the three kingdoms of life – Archaea, Bacteria and Eukaryota. It has become clear that the cyclases that produce hopanoids from squalene (so-called SHCs) and sterols and pentacyclic triterpenoids from oxidosqualene (OSCs) are closely related and reflect evolutionary changes in this biosynthetic pathway over time. There has been much debate whether the sterols that have been detected in some cyanobacterial cultures are produced by de novo synthesis, and there is growing evidence that they might be derived from contaminating organisms such as yeasts and fungi or in some cases result from laboratory contamination or sterols in the culture medium. The presence of 4-methylsterols in some bacteria shows that some of the necessary biochemical apparatus is present in a few prokaryotes. The genomes of several cyanobacteria have been fully sequenced and amongst the genes recognized are several that have a close sequence match to genes related to sterol biosynthesis. These include a putative Δ 24 sterol C-methyltransferase gene, another coding a sterol C-methyltransferase and another for a sterol C-5-desaturase (or C-4 methyl sterol oxidase), but this is only a part of the full complement of genes needed for sterol biosynthesis.

Chemotaxonomy and evolution of Plantago L.

In continuation of our investigations of the genus Plantago L. (Plantaginaceae), sixteen species were investigated with respect to water-soluble glycosides. The iridoids auroside, strictoloside and globularicisin, as well as poliumoside, 3-[(4-β-D-glucopyranosyloxy)phenyl]propionic acid and 2-[4-(β-D-glucopyranosyloxy)phenyl]acetic acid were isolated from Plantago for the first time. The latter compound has not previously been isolated as a natural product. Sorbitol was the main carbohydrate in all the species investigated. The distribution of iridoids correlates well with the morphological classification of Rahn and also with a recently published molecular phylogenetic study of nuclear ribosomal and plastid DNA sequences. A new chemotaxonomic finding is an abundance of iridoid glucosides present in one of the two groups within subgenus Coronopus section Coronopus, while the other group lacks iridoids, supporting a subdivision of this section. Moreover, in subgenus Albicans, 10-benzoylcatalpol is a characteristic constituent of section Gnaphaloides, while the corresponding cinnamoyl ester globularin occurs in section Lanceifolia. In biosynthetic experiments, labelled epideoxyloganic acid and deoxygeniposidic acid were incorporated into aucubin and geniposidic acid in Plantago ovata, consistent with earlier findings, but no incorporation into asperuloside was observed. The evolution of biosynthetic pathways in Plantago is discussed.

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

Future Directions in Lichen Chemistry

ficult to explore. Future chemical studies will use newly developing methods of separation and structural analysis to study the more complex compounds, including pigments and polysaccharides, terpenoids, and other aliphatic products. Future biological studies will clarify the genetics and evolution of biosynthetic pathways and explain how these compounds contribute to the success of the lichen symbiosis and of the lichen as a functional unit in nature.

Multifunctional enzymes and evolution of biosynthetic pathways: retro-evolution by jumps.

A likely scenario of evolution of biosynthetic pathways is believed to have occurred by retro-evolution through recruitment of existing enzymes rather than generation of de novo classes. It had been proposed that such retro-evolution occurred in steps as a response to depletion of an essential metabolite and availability of another related substance in the environment. In this article, I argue that because of instability of many such extant intermediates, it is unlikely that retro-evolution had occurred in steps. I further propose that such evolution in many cases has taken place by jumps, i.e., by recruitment of a multifunctional enzyme capable of catalyzing several steps at a time, albeit inefficiently. I further speculate that in some cases one primordial multienzyme may have catalyzed the whole sequence of reaction of a biosynthetic pathway, i.e., the pathway may have evolved by a single leap. Gene duplications and further evolution to more efficient enzymes led to extant pathways. Such a mechanism predicts that some or all enzymes of a pathway must have descended from a common ancestor. Sequence and structural homologies among extant enzymes of a biosynthetic pathway have been examined.

Directed evolution of biosynthetic pathways. Recruitment of cysteine thioethers for constructing the cell wall of Escherichia coli.

We report that expansion of thioether biosynthesis in Escherichia coli generates sulfur-containing amino acids that can replace meso-diaminopimelate, the essential amino acid used for cross-linking the cell wall. This was accomplished by jointly overexpressing the metB gene coding for L-cystathionine gamma-synthase and disrupting the metC gene, whose product, L-cystathionine beta-lyase, is responsible for the destruction of L-cystathionine and other L-cysteine thioethers. As a result, meso-lanthionine and L-allo-cystathionine were produced endogenously and incorporated in the peptidoglycan, thereby enabling E. coli strains auxotrophic for diaminopimelate to grow in its absence. Thus, current techniques of metabolic engineering can be applied to evolving the chemical constitution of living cells beyond its present state.