http://www.microbialcellfactories.com The latest research articles published by Microbial Cell Factories 2012-05-14T00:00:00Z Background: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes.Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. Results: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris. http://www.microbialcellfactories.com/content/11/1/61 Andrea Mellitzer Roland Weis Anton Glieder Karlheinz Flicker Microbial Cell Factories 2012, null:61 2012-05-14T00:00:00Z doi:10.1186/1475-2859-11-61 /content/figures/1475-2859-11-61-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 61 2012-05-14T00:00:00Z PDF Background: TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter. Results: Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens DeltatliA, a knock-out mutant of tliA. Conclusion: LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ. http://www.microbialcellfactories.com/content/11/1/60 Yeonwoo Park Yuseok Moon Jungmin Ryoo Nayeon Kim Hyounghoon Cho Jung Hoon Ahn Microbial Cell Factories 2012, null:60 2012-05-11T00:00:00Z doi:10.1186/1475-2859-11-60 /content/figures/1475-2859-11-60-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 60 2012-05-11T00:00:00Z PDF Background: The influence of carbon dioxide and oxygen on microbial secondary metabolite producers and the maintenance of these two parameters at optimal levels have been studied extensively. Nevertheless, most studies have focussed on their influence on specific product formation and condition optimization of established processes. Considerably less attention has been paid to the influence of reduced or elevated carbon dioxide and oxygen levels on the overall metabolite profiles of the investigated organisms. The synergistic action of both gases has garnered even less attention. Results: We show that the composition of the gas phase is highly important for the production of different metabolites and present a simple approach that enables the maintenance of defined concentrations of both O2 and CO2 during bioprocesses over broad concentration ranges with a minimal instrumental setup by using endogenously produced CO2. The metabolite profiles of a myxobacterium belonging to the genus Chondromyces grown under various concentrations of CO2 and O2 showed considerable differences. Production of two unknown, highly cytotoxic compounds and one antimicrobial substance was found to increase depending on the gas composition. In addition, the observation of CO2 and O2 in the exhaust gas allowed optimization and control of production processes. Conclusions: Myxobacteria are becoming increasingly important due to their potential for bioactive secondary metabolite production. Our studies show that the influence of different gas partial pressures should not be underestimated during screening processes for novel compounds and that our described method provides a simple tool to investigate this question. http://www.microbialcellfactories.com/content/11/1/59 Stephan Hüttel Rolf Müller Microbial Cell Factories 2012, null:59 2012-05-09T00:00:00Z doi:10.1186/1475-2859-11-59 /content/figures/1475-2859-11-59-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 59 2012-05-09T00:00:00Z PDF Background: The E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes. Results: The results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time. Conclusions: In the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon. http://www.microbialcellfactories.com/content/11/1/58 Alfred Fernández-Castané Glòria Caminal Josep López-Santín Microbial Cell Factories 2012, null:58 2012-05-09T00:00:00Z doi:10.1186/1475-2859-11-58 /content/figures/1475-2859-11-58-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 58 2012-05-09T00:00:00Z PDF Background: The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results: The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions: Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels. http://www.microbialcellfactories.com/content/11/1/57 Joel Jordà Paula Jouhten Elena Cámara Hannu Maaheimo Joan Albiol Pau Ferrer Microbial Cell Factories 2012, null:57 2012-05-08T00:00:00Z doi:10.1186/1475-2859-11-57 /content/figures/1475-2859-11-57-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 57 2012-05-08T00:00:00Z 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: The amyloid-beta peptide (Abeta42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Abeta42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Abeta42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides. http://www.microbialcellfactories.com/content/11/1/55 Anna Villar-Piqué Alba Espargaró Raimon Sabaté Natalia de Groot Salvador Ventura Microbial Cell Factories 2012, null:55 2012-05-03T00:00:00Z doi:10.1186/1475-2859-11-55 /content/figures/1475-2859-11-55-toc.gif Microbial Cell Factories 1475-2859 ${item.volume} 55 2012-05-03T00:00:00Z PDF 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 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