http://www.microbialcellfactories.com The most accessed research articles published by Microbial Cell Factories 2012-05-08T00:00:00Z This paper gives an overview of the recent advances in engineering the central carbon metabolism of the industrially important bacteria Escherichia coli, Bacillus subtilis, Corynobacterium glutamicum, Streptomyces spp., Lactococcus lactis and other lactic acid bacteria. All of them are established producers of important classes of products, e.g. proteins, amino acids, organic acids, antibiotics, high-value metabolites for the food industry and also, promising producers of a large number of industrially or therapeutically important chemicals. Optimization of existing or introduction of new cellular processes in these microorganisms is often achieved through manipulation of targets that reside at major points of central metabolic pathways, such as glycolysis, gluconeogenesis, the pentose phosphate pathway and the tricarboxylic acid cycle with the glyoxylate shunt. Based on the huge progress made in recent years in biochemical, genetic and regulatory studies, new fascinating engineering approaches aim at ensuring an optimal carbon and energy flow within central metabolism in order to achieve optimized metabolite production. http://www.microbialcellfactories.com/content/11/1/50 Maria Papagianni Microbial Cell Factories 2012, null:50 2012-04-30T00:00:00Z doi:10.1186/1475-2859-11-50 /content/figures/1475-2859-11-50-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 50 2012-04-30T00:00:00Z PDF Background: Glycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes. Results: Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose. Conclusions: The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools. http://www.microbialcellfactories.com/content/11/1/46 Karla Martínez-Gómez Noemí Flores Héctor Castañeda Gabriel Martínez-Batallar Georgina Hernández-Chávez Octavio Ramírez Guillermo Gosset Sergio Encarnación Francisco Bolivar Microbial Cell Factories 2012, null:46 2012-04-18T00:00:00Z doi:10.1186/1475-2859-11-46 /content/figures/1475-2859-11-46-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 46 2012-04-18T00:00:00Z PDF Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way. http://www.microbialcellfactories.com/content/8/1/17 Neus Ferrer-Miralles Joan Domingo-Espin Jose Luis Corchero Esther Vazquez Antonio Villaverde Microbial Cell Factories 2009, null:17 2009-03-24T00:00:00Z doi:10.1186/1475-2859-8-17 /content/figures/1475-2859-8-17-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 17 2009-03-24T00:00:00Z XML Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags. http://www.microbialcellfactories.com/content/4/1/1 Hans Sorensen Kim Mortensen Microbial Cell Factories 2005, null:1 2005-01-04T00:00:00Z doi:10.1186/1475-2859-4-1 /content/figures/1475-2859-4-1-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 1 2005-01-04T00:00:00Z XML Background: Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and produced by such bacteria as Ralstonia eutropha H16 and Bacillus megaterium. In contrast to currently utilized plastics and most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this bioplastic interesting for various industrial applications. Results: In this study, we report on introducing the bacterial PHB pathway of R. eutropha H16 into the diatom Phaeodactylum tricornutum, thereby demonstrating for the first time that PHB production is feasible in a microalgal system. Expression of the bacterial enzymes was sufficient to result in PHB levels of up to 10.6% of algal dry weight. The bioplastic accumulated in granule-like structures in the cytosol of the cells, as shown by light and electron microscopy. Conclusions: Our studies demonstrate the great potential of microalgae like the diatom P. tricornutum to serve as solar-powered expression factories and reveal great advantages compared to plant based production systems. http://www.microbialcellfactories.com/content/10/1/81 Franziska Hempel Andrew Bozarth Nicole Lindenkamp Andreas Klingl Stefan Zauner Uwe Linne Alexander Steinbuchel Uwe Maier Microbial Cell Factories 2011, null:81 2011-10-17T00:00:00Z doi:10.1186/1475-2859-10-81 Plastic fantastic - the future of biodegradables Solar-powered polyester production is proved feasible by causing microalgae to express enzymes borrowed from specialized bacteria, to result in biosynthesis of poly-3-hydroxybutyrate. /content/figures/1475-2859-10-81-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 81 2011-10-17T00:00:00Z XML Background: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). Results: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. Conclusions: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development. http://www.microbialcellfactories.com/content/11/1/54 Xiao-Yun Zhou Xiao-Xi Yuan Zhen-Yu Shi De-Chuan Meng Wen-Jun Jiang Lin-Ping Wu Jin-Chun Chen Guo-Qiang Chen Microbial Cell Factories 2012, null:54 2012-05-02T00:00:00Z doi:10.1186/1475-2859-11-54 /content/figures/1475-2859-11-54-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 54 2012-05-02T00:00:00Z PDF Background: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi alpha-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the alpha-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained alpha-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. Conclusions: We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins. http://www.microbialcellfactories.com/content/11/1/53 Karen De Pourcq Wouter Vervecken Isabelle Dewerte Albena Valevska Annelies Van hecke Nico Callewaert Microbial Cell Factories 2012, null:53 2012-05-01T00:00:00Z doi:10.1186/1475-2859-11-53 /content/figures/1475-2859-11-53-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 53 2012-05-01T00:00:00Z PDF Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains. http://www.microbialcellfactories.com/content/11/1/36 Jurgen Nijkamp Marcel van den Broek Erwin Datema Stefan de Kok Lizanne Bosman Marijke Luttik Pascale Daran-Lapujade Wanwipa Vongsangnak Jens Nielsen Wilbert Heijne Paul Klaassen Chris Paddon Darren Platt Peter Kotter Roeland van Ham Marcel Reinders Jack Pronk Dick Ridder Jean-Marc Daran Microbial Cell Factories 2012, null:36 2012-03-26T00:00:00Z doi:10.1186/1475-2859-11-36 /content/figures/1475-2859-11-36-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 36 2012-03-26T00:00:00Z PDF Background: Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results: We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions: This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains. http://www.microbialcellfactories.com/content/11/1/56 Julie Lobstein Charlie Emrich Chris Jeans Melinda Faulkner Paul Riggs Mehmet Berkmen Microbial Cell Factories 2012, null:56 2012-05-08T00:00:00Z doi:10.1186/1475-2859-11-56 /content/figures/1475-2859-11-56-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 56 2012-05-08T00:00:00Z PDF Background: D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT. Results: Intracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold. Conclusions: This is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems. http://www.microbialcellfactories.com/content/11/1/47 Kanidtha Jariyachawalid Poramaet Laowanapiban Vithaya Meevootisom Suthep Wiyakrutta Microbial Cell Factories 2012, null:47 2012-04-19T00:00:00Z doi:10.1186/1475-2859-11-47 /content/figures/1475-2859-11-47-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 47 2012-04-19T00:00:00Z PDF