http://www.microbialcellfactories.com The latest research articles published by Microbial Cell Factories 2013-05-24T00:00:00Z Background: FK506 is an important immunosuppressant, which can be produced by Streptomyces tsukubaensis. However, the production capacity of the strain is very low. Hereby, a computational guided engineering approach was proposed in order to improve the intracellular precursor and cofactor availability of FK506 in S. tsukubaensis. Results: First, a genome-scale metabolic model of S. tsukubaensis was constructed based on its annotated genome and biochemical information. Subsequently, several potential genetic targets (knockout or overexpression) that guaranteed an improved yield of FK506 were identified by the recently developed methodology. To validate the model predictions, each target gene was manipulated in the parent strain D852, respectively. All the engineered strains showed a higher FK506 production, compared with D852. Furthermore, the combined effect of the genetic modifications was evaluated. Results showed that the strain HT-DeltaGDH-DAZ with gdhA-deletion and dahp-, accA2-, zwf2-overexpression enhanced FK506 concentration up to 398.9 mg/L, compared with 143.5 mg/L of the parent strain D852. Finally, fed-batch fermentations of HT-DeltaGDH-DAZ were carried out, which led to the FK506 production of 435.9 mg/L, 1.47-fold higher than the parent strain D852 (158.7 mg/L). Conclusions: Results confirmed that the promising targets led to an increase in FK506 titer. The present work is the first attempt to engineer the primary precursor pathways to improve FK506 production in S. tsukubaensis with genome-scale metabolic network guided metabolic engineering. The relationship between model prediction and experimental results demonstrates the rationality and validity of this approach for target identification. This strategy can also be applied to the improvement of other important secondary metabolites. http://www.microbialcellfactories.com/content/12/1/52 Di Huang Shanshan Li Menglei Xia Jianping Wen Xiaoqiang Jia Microbial Cell Factories 2013, null:52 2013-05-24T00:00:00Z doi:10.1186/1475-2859-12-52 /content/figures/1475-2859-12-52-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 52 2013-05-24T00:00:00Z PDF Background: Novel analytical tools, which shorten the long and costly development cycles of biopharmaceuticals are essential. Metabolic flux analysis (MFA) shows great promise in improving our understanding of the metabolism of cell factories in bioreactors, but currently only provides information post-process using conventional off-line methods. MFA combined with real time multianalyte process monitoring techniques provides a valuable platform technology allowing real time insights into metabolic responses of cell factories in bioreactors. This could have a major impact in the bioprocessing industry, ultimately improving product consistency, productivity and shortening development cycles. Results: This is the first investigation using Near Infrared Spectroscopy (NIRS) in situ combined with metabolic flux modelling which is both a significant challenge and considerable extension of these techniques. We investigated the feasibility of our approach using the industrial workhorse Pichia pastoris in a simplified model system. A parental P. pastoris strain (i.e. which does not synthesize recombinant protein) was used to allow definition of distinct metabolic states focusing solely upon the prediction of intracellular fluxes in central carbon metabolism. Extracellular fluxes were determined using off-line conventional reference methods and on-line NIR predictions (calculated by multivariate analysis using the partial least squares algorithm, PLS). The results showed that the PLS-NIRS models for biomass and glycerol were accurate: correlation coefficients, R2, above 0.90 and the root mean square error of prediction, RMSEP, of 1.17 and 2.90 g/L, respectively. The analytical quality of the NIR models was demonstrated by direct comparison with the standard error of the laboratory (SEL), which showed that performance of the NIR models was suitable for quantifying biomass and glycerol for calculating extracellular metabolite rates and used as independent inputs for the MFA (RMSEP lower than 1.5 x SEL). Furthermore, the results for the MFA from both datasets passed consistency tests performed for each steady state, showing that the precision of on-line NIRS is equivalent to that obtained by the off-line measurements. Conclusions: The findings of this study show for the first time the potential of NIRS as an input generating for MFA models, contributing to the optimization of cell factory metabolism in real-time. http://www.microbialcellfactories.com/content/12/1/51 Mariana Fazenda Joao Dias Linda Harvey Alison Nordon Ruan Edrada-Ebel David LittleJohn Brian McNeil Microbial Cell Factories 2013, null:51 2013-05-21T00:00:00Z doi:10.1186/1475-2859-12-51 /content/figures/1475-2859-12-51-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 51 2013-05-21T00:00:00Z PDF Background: Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations. Results: Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of beta-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a PrpoS [rightwards arrow] rpoS'-'lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria. Conclusions: While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and biochemical networks that ultimately enable P. putida to endure environmental stresses. Instead, the main value of polyP could be ensuring a reservoire of energy during prolonged starvation. This is perhaps one of the reasons for polyP persistence in live systems despite its apparent lack of essentiality. http://www.microbialcellfactories.com/content/12/1/50 Pablo Nikel Max Chavarría Esteban Martínez-García Anne Taylor Víctor de Lorenzo Microbial Cell Factories 2013, null:50 2013-05-20T00:00:00Z doi:10.1186/1475-2859-12-50 /content/figures/1475-2859-12-50-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 50 2013-05-20T00:00:00Z PDF Background: In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results: Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag(R)II and Twin-Strep-tag(R) fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion: The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. http://www.microbialcellfactories.com/content/12/1/49 Thorben Dammeyer Kenneth Timmis Philip Tinnefeld Microbial Cell Factories 2013, null:49 2013-05-20T00:00:00Z doi:10.1186/1475-2859-12-49 /content/figures/1475-2859-12-49-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 49 2013-05-20T00:00:00Z PDF Metabolic flexibility may be generally defined as "the capacity for the organism to adapt fuel oxidation to fuel availability". The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus. http://www.microbialcellfactories.com/content/12/1/48 Michelle O¿Donnell Paul O¿Toole Reynolds Ross Microbial Cell Factories 2013, null:48 2013-05-16T00:00:00Z doi:10.1186/1475-2859-12-48 /content/figures/1475-2859-12-48-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 48 2013-05-16T00:00:00Z PDF Background: In vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae. Results: To minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional. Conclusion: The presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitates the construction of any plasmid carrying two or more genes. http://www.microbialcellfactories.com/content/12/1/47 Niels Kuijpers Daniel Solis-Escalante Lizanne Bosman Marcel van den Broek Jack Pronk Jean-Marc Daran Pascale Daran-Lapujade Microbial Cell Factories 2013, null:47 2013-05-10T00:00:00Z doi:10.1186/1475-2859-12-47 /content/figures/1475-2859-12-47-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 47 2013-05-10T00:00:00Z PDF 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[bullet operator]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. Conclusions: The present study describes the identification of a gene target and the application a of successful metabolic engineering strategy to the unconventional host E.coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations. http://www.microbialcellfactories.com/content/12/1/46 Donatella Cimini Mario De Rosa Elisabetta Carlino Alessandro Ruggiero Chiara Schiraldi Microbial Cell Factories 2013, null:46 2013-05-09T00:00:00Z doi:10.1186/1475-2859-12-46 /content/figures/1475-2859-12-46-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 46 2013-05-09T00:00:00Z PDF Background: p-Hydroxycinnamic acid (pHCA) is an aromatic compound that serves as a starting material for the production of many commercially valuable chemicals, such as fragrances and pharmaceuticals, and is also used in the synthesis of thermostable polymers. However, chemical synthesis of pHCA is both costly and harmful to the environment. Although pHCA production using microbes has been widely studied, there remains a need for more cost-effective methods, such as the use of biomass as a carbon source. In this study, we produced pHCA using tyrosine ammonia lyase-expressing Streptomyces lividans. In order to improve pHCA productivity from cellulose, we constructed a tyrosine ammonia lyase- and endoglucanase (EG)-expressing S. lividans transformant and used it to produce pHCA from cellulose. Results: A Streptomyces lividans transformant was constructed to express tyrosine ammonia lyase derived from Rhodobacter sphaeroides (RsTAL). The transformant produced 786 or 736 mg/L of pHCA after 7 days of cultivation in medium containing 1% glucose or cellobiose as the carbon source, respectively. To enhance pHCA production from phosphoric acid swollen cellulose (PASC), we introduced the gene encoding EG into RsTAL-expressing S. lividans. After 7 days of cultivation, this transformant produced 753, 743, or 500 mg/L of pHCA from 1% glucose, cellobiose, or PASC, respectively. Conclusions: RsTAL-expressing S. lividans can produce pHCA from glucose and cellobiose. Similarly, RsTAL- and EG-expressing S. lividans can produce pHCA from glucose and cellobiose with excess EG activity remaining in the supernatant. This transformant demonstrated improved pHCA production from cellulose. Further enhancements in the cellulose degradation capability of the transformant will be necessary in order to achieve further improvements in pHCA production from cellulose. http://www.microbialcellfactories.com/content/12/1/45 Yoshifumi Kawai Shuhei Noda Chiaki Ogino Yasunobu Takeshima Naoko Okai Tsutomu Tanaka Akihiko Kondo Microbial Cell Factories 2013, null:45 2013-05-07T00:00:00Z doi:10.1186/1475-2859-12-45 /content/figures/1475-2859-12-45-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 45 2013-05-07T00:00:00Z PDF Background: Extracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation. Results: Echinacea purpurea suspension (5%, wt/vol) in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-[unknown][unknown]gene. Conclusions: E. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations. http://www.microbialcellfactories.com/content/12/1/44 Carlo Rizzello Rossana Coda Davinia Macías Daniela Pinto Barbara Marzani Pasquale Filannino Giammaria Giuliani Vito Paradiso Raffaella Di Cagno Marco Gobbetti Microbial Cell Factories 2013, null:44 2013-05-04T00:00:00Z doi:10.1186/1475-2859-12-44 /content/figures/1475-2859-12-44-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 44 2013-05-04T00:00:00Z PDF Background: Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results: We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion: A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. http://www.microbialcellfactories.com/content/12/1/43 Neeraj Pandey Annapurna Sachan Qi Chen Kristin Ruebling-Jass Ritu Bhalla Kiran Kumar Panguluri Pierre Rouviere Qiong Cheng Microbial Cell Factories 2013, null:43 2013-05-02T00:00:00Z doi:10.1186/1475-2859-12-43 /content/figures/1475-2859-12-43-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 43 2013-05-02T00:00:00Z PDF