Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals.

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.

The production of industrially relevant microbial polysaccharides has recently gained much interest. The capsular polysaccharide of Escherichia coli K4 is almost identical to chondroitin, a commercially valuable biopolymer that is so far obtained from animal tissues entailing complex and expensive extraction procedures. In the present study, the production of capsular polysaccharide by E. coli K4 was investigated taking into consideration a potential industrial application. Strain physiology was first characterized in shake flask experiments to determine the optimal culture conditions for the growth of the microorganism and correlate it to polysaccharide production. Results show that the concentration of carbon source greatly affects polysaccharide production, while the complex nitrogen source is mainly responsible for the build up of biomass. Small-scale batch processes were performed to further evaluate the effect of the initial carbon source concentration and of growth temperatures on polysaccharide production, finally leading to the establishment of the medium to use in following fermentation experiments on a bigger scale. The fed-batch strategy next developed on a 2-L reactor resulted in a maximum cell density of 56 g(cww)/L and a titre of capsular polysaccharide equal to 1.4 g/L, approximately ten- and fivefold higher than results obtained in shake flask and 2-L batch experiments, respectively. The release kinetics of K4 polysaccharide into the medium were also explored to gain insight into the mechanisms underlying a complex aspect of the strain physiology.

BackgroundVaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. A new human isolate named Lactobacillus crispatus L1 was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass.ResultsIn a simulated digestion process 1.8⋅1010 cells∙ml−1 survived the gastric environment with 80% viability, without being affected by small intestine juices. Experiments on six different C sources were performed to analyze growth and organic acids production and, glucose, provided the best performances. A microfiltration strategy was exploited to improve the cellular yield in 2 L-fermentation processes, reaching 27 g · l−1 of dry biomass. Moreover, L. crispatus L1 demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. The specific L. crispatus L1 exopolysaccharide was purified from the fermentation broth and characterized by NMR showing structural features and similarity to exopolysaccharides produced by pathogenic strains. Live L. crispatus L1 cells strongly reduced adhesion of a yeast pathogenic strain, Candida albicans in particular, in adherence assays. Interestingly a higher expression of the human defensin HBD-2 was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with C. albicans growth inhibition.ConclusionsThe paper describes the evaluation of L. crispatus L1 as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells.

Background: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation.Results: E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g • L -1 of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis.

BackgroundThe bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L-1 K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.ResultsThe inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L-1), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.ConclusionThe increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

Bio-based succinic acid production from lignocellulosic biomass is one of the attractive and prominent alternative technologies to overcome issues associated with the utilization of fossil sources. In this context, it is necessary to find new microorganisms that are able to efficiently ferment this recalcitrant feedstock. The ecological approach developed in this study enabled the isolation of Basfia succiniciproducens BPP7 from a complex rumen ecosystem. This new wild-type strain was able to synthesize up to 6.06 ± 0.05 g/L of succinate (corresponding to 0.84 ± 0.017 g of succinate per gram of consumed glucose + xylose and to 0.14 ± 0.001 g of succinate per gram of glucans + xylans present in the biomass before hydrolysis) from Arundo donax hydrolysate in separate hydrolysis and fermentation (SHF) experiments. Higher titers of succinic acid were obtained through the optimization of growth conditions. The optimal medium composition identified on the smaller scale was then used for 2.5-L batch experiments, which used A. donax hydrolysate and yeast extract as the main C and N sources, respectively. A maximal titer of 9.4 ± 0.4 g/L of succinic acid was obtained after 24 h. The overall results clearly demonstrate the potential of B. succiniciproducens BPP7 for succinate production.

In the present work the recently isolated strain Basfia succiniciproducens BPP7 was evaluated for the production of succinic acid up to the pilot fermentation scale in separate hydrolysis and fermentation experiments on Arundo donax, a non-food dedicated energy crop. An average concentration of about 17g/L of succinic acid and a yield on consumed sugars of 0.75mol/mol were obtained demonstrating strain potential for further process improvement. Small scale experiments indicated that the concentration of acetic acid in the medium is crucial to improve productivity; on the other hand, interestingly, short-term (24h) adaptation to higher acetic acid concentrations, and strain recovery, were also observed.