Tiziana Fossati

Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems?

Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why—despite important advances in rDNA applications in higher eukaryotic cells—microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.

Molecular analysis of natural populations of Populus nigra L. intermingled with cultivated hybrids

In this study six simple sequence repeats (SSR or microsatellites) were selected for their ability to fingerprint a total of 60 commercial clones of Populus deltoides Marsh. and Populus × canadensis Moench (typically derived from crosses between Populus nigra L and P. deltoides) and to characterize a natural population of P. nigra growing along the Ticino river in the North of Italy. Out of six SSRs used, four microsatellite loci were found to have alleles which were species‐specific to P. deltoides and could therefore be used as markers for introgression of P. deltoides into P. nigra. In the studied region hybrid poplars and P. deltoides commercial clones are cultivated as monoclonal stands close to the area where black poplar has its natural habitat. SSR analysis was performed to investigate whether there was evidence of introgression between the natural population and the monoclonal plantations of hybrids and P. deltoides clones cultivated in the surrounding area. Three stages of the natural population were analysed: a group of old trees about a hundred years old, a younger population (aged 2–30 years) and the seedlings of three females of this population. Alleles specific to P. deltoides were detected only in the old cohort of the natural population, while no introgression was observed in the younger individuals and their progenies. These results were also confirmed by isozyme analysis of loci PGI‐B, PGM and LAP‐A, which were previously identified as diagnostic for P. nigra, P. deltoides and P.×canadensis.

Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance

Background In industrial large scale bio-reactions micro-organisms are generally exposed to a variety of environmental stresses, which might be detrimental for growth and productivity. Reactive oxygen species (ROS) play a key role among the common stress factors–directly-through incomplete reduction of O2 during respiration, or indirectly-caused by other stressing factors. Vitamin C or L-ascorbic acid acts as a scavenger of ROS, thereby potentially protecting cells from harmful oxidative products. While most eukaryotes synthesize ascorbic acid, yeast cells produce erythro-ascorbic acid instead. The actual importance of this antioxidant substance for the yeast is still a subject of scientific debate. Methodology/Principal Findings We set out to enable Saccharomyces cerevisiae cells to produce ascorbic acid intracellularly to protect the cells from detrimental effects of environmental stresses. We report for the first time the biosynthesis of L-ascorbic acid from D-glucose by metabolically engineered yeast cells. The amount of L-ascorbic acid produced leads to an improved robustness of the recombinant cells when they are subjected to stress conditions as often met during industrial fermentations. Not only resistance against oxidative agents as H2O2 is increased, but also the tolerance to low pH and weak organic acids at low pH is increased. Conclusions/Significance This platform provides a new tool whose commercial applications may have a substantial impact on bio-industrial production of Vitamin C. Furthermore, we propose S. cerevisiae cells endogenously producing vitamin C as a cellular model to study the genesis/protection of ROS as well as genotoxicity.

Ex-situ conservation of Black poplar in Europe: genetic diversity in nine gene bank collections and their value for nature development

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei’s expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (Fst/Gst) was moderate.

The Origin of Clonal Diversity and Structure of Populus alba in Sardinia: Evidence from Nuclear and Plastid Microsatellite Markers

BACKGROUND AND AIMS Populus alba is a thermophilic forest tree present in the Mediterranean basin. Its habitat is highly fragmented and its distribution range has been subject to long-term human interference, resulting in debate surrounding whether certain populations are native or exotic in origin. In particular, populations from the islands of Corsica and Sardinia are of uncertain origin. While populations of P. alba mainly reproduce sexually, clonal reproduction is also common. The aims of this study were to locate and molecularly characterize the poorly studied island populations of P. alba and compare these with samples from various spatial scales, in order to provide information on the genetic structure and phylogeography of this species. This information will provide evidence on whether the species is native to Sardinia, which is important for the development of conservation strategies. METHODS DNA extracts were obtained from the following P. alba trees: 159 from Sardinia, 47 from Ticino regional park (northern Italy), 15 acquired from an Italian Germoplasm Bank (IRC; Italian Reference Collection) and 28 from the Mediterranean basin (MB). Genetic polymorphisms were revealed at nuclear and chloroplast DNA (cpDNA) microsatellite loci, both at the island scale (Sardinia) and at broader scales, for comparative assessment of the genetic and genotypic diversity and phylogeography. KEY RESULTS Based on nuclear microsatellite loci, Sardinian white poplar consists of a small number of genets (26), each of which is represented by several ramets. Despite the uniqueness of the Sardinian haplotypes and the very low value of genetic diversity at the cpDNA level (vK = 0.15), the HT (0.60) and the AR (3.61) values, estimated at the nuclear level for Sardinia, were comparable with those of the other populations and collections. CONCLUSIONS The uniqueness of the cpDNA haplotypes, the prevalence of clonality and the restricted number of genets recorded suggest that Sardinian white poplar could be a floristic relict of the native flora of the island, which has spread through available habitats on the island mainly by means of vegetative propagation and human activities.

Genetic relationships and clonal identity in a collection of commercially relevant poplar cultivars assessed by AFLP and SSR

A collection of 66 poplar commercial clones widely cultivated in Italy, China and in other countries of southern Europe and belonging to various poplar species and hybrids, have been fingerprinted using both amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) techniques. Three AFLP primer combinations and six SSRs unambiguously genotyped the analysed poplar collection, with the exception of three groups of six, four and two individuals, which turned out to be indistinguishable even if they met the standards currently applied for distinctness, uniformity and stability (DUS) testing when registered. High levels of variation were detected with both molecular techniques; a total of 201 AFLP bands were amplified of which 96% turned out to be polymorphic and up to 15 SSR alleles were identified at a single locus, with a mean of 9.3 alleles per locus in the case of Populus × canadensis. The probability of matching fortuitously any two genotypes at all the SSR loci in the case of P. × canadensis was less then 7.5×10−9. The AFLP-derived dendrogram and principal coordinate analysis (PCOORDA) clustered the clones with respect to their taxonomic classification, and allowed their genetic interrelationships to be established. Correct identification of poplar varieties is essential for ensuring the effective correspondence between the real and the declared identity of a clone, to avoid commercial frauds, and to establish breeding programmes. Molecular markers may play a major role to satisfy all these needs.

The use of AFLP and SSR molecular markers to decipher homonyms and synonyms in grapevine cultivars: the case of the varietal group known as Schiave

Abstract ’’Schiave’’ collectively refers to grapevine cultivars presently grown on the Southern and Northern slopes of the Eastern Alps and bearing different names (Schiava, Trollinger, Rossara, Rossola, Geschlafene, Gansfüsser, Urban and others). Their common origin has been suggested by historic, linguistic and ampelographic considerations. This hypothesis has now been assayed by using more direct approaches based on AFLP and SSR analysis. The present paper shows: (1), that AFLP and SSR give comparable results when used to study genomic similarity among the Schiave grapevine cultivars, and (2) that ’’Schiave’’ is used to group grapevine cultivars that are genetically heterogeneous. In fact, a dendrogram constructed from an AFLP analysis of the 33 best-known Schiave (or correlated) cultivars, shows different, and in some cases relevant, degrees of genomic dissimilarity. The analysed cultivars cluster into at least five taxonomic groups with specific geographic distribution along the valleys of Valtellina, Bergamo and Brescia and those of South Tyrol and Swabia. It is concluded that the common definition ’’Schiave’’ refers to a similar cultivation practice in contiguous regions rather than to a common genetic background.

Genetic structure and introgression in riparian populations of Populus alba L.

Abstract White poplar (Populus alba) is a widespread species of the northern hemisphere. Introgressed populations or hybrid zones with the related species of the European aspen (Populus tremula) have been suggested as potential venues for the identification of functionally important variation for germplasm conservation, restoration efforts and tree breeding. Data on the genetic diversity and structure of introgressed P. alba are available only for sympatric populations from central Europe. Here, clonality, introgression and spatial genetic patterns were evaluated in three riparian populations of P. alba along the Ticino, Paglia‐Tevere and Cesano river drainages in Italy. Samples of all three populations were typed for five nuclear microsatellite markers and 137 polymorphic amplified fragment length polymorphisms. Microsatellite‐based inbreeding co‐efficients (F IS) were significantly positive in all three populations. Genetic diversity was consistently highest in Ticino, the population with the highest level of introgression from P. tremula. Population differentiation (F ST) was low between the Ticino valley in northern Italy and the Cesano valley in central Italy and between the central Italian populations of Cesano and Paglia‐Tevere, consistent with a role of the Appenine mountain range as a barrier to gene flow between adjacent drainage areas. Introgression was not the primary determinant of within‐population spatial genetic structure (SGS) in the studied populations.

Microbial stress: from molecules to systems (Belgirate, May 2012).

M e ti n g R e o rt The meeting ‘‘Microbial Stress: from Molecules to Systems’’ held in Belgirate (Italy) in May 2012 focussed on how organisms cope with intracellular and environmental stresses, how signal detection is turned into a response; the effects of stress on energy metabolism; and the effect of diverse stress responses on industrial biotechnology. Key conclusions included that stress responses are complex and far from being completely understood even for model organisms. Examples were given of how a combination of tools developed for systems and synthetic biology can be exploited to close knowledge gaps. Microbes must be able to balance energy demand and supply in order to survive. Mechanism exploited to achieve this vary depending on the lifestyle of the microbe and the physiological conditions encountered, especially stresses imposed by the artificial conditions in industrial fermenters. Obvious examples include solvent tolerance that must be developed by organisms used for biofuel production; the potential of using high hydrostatic pressure as a bioprocessing parameter; and opportunities to exploit natural diversity gleaned from metagenomic sequencing to find organisms with natural traits suitable for the development of new biotechnologies. The meeting is now part of a biennial series that will be advertised on the EFB website: www.efb-central.org.