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Graphical Abstract Recent results obtained by Chindelevitch et al (2014) are challenged by Ebrahim et al, illustrating several issues in the reproducibility of computational biology methods.

Abstract Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re‐sequenced and fully re‐annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome‐scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost‐effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole‐cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.

Abstract Amino‐acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L‐threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed‐batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser–His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins. [ABSTRACT FROM AUTHOR]

Aromatic polyesters are widely used plastics currently produced from petroleum. Here we engineer Escherichia coli strains for the production of aromatic polyesters from glucose by one-step fermentation. When the Clostridium difficile isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (HadA) and evolved polyhydroxyalkanoate (PHA) synthase genes are overexpressed in a D-phenyllactate-producing strain, poly(52.3 mol% 3-hydroxybutyrate (3HB)-co-47.7 mol% D-phenyllactate) can be produced from glucose and sodium 3HB. Also, various poly(3HB-co-D-phenyllactate) polymers having 11.0, 15.8, 20.0, 70.8, and 84.5 mol% of D-phenyllactate are produced from glucose as a sole carbon source by additional expression of Ralstonia eutropha β-ketothiolase (phaA) and reductase (phaB) genes. Fed-batch culture of this engineered strain produces 13.9 g l-1 of poly(61.9 mol% 3HB-co-38.1 mol% Dphenyllactate). Furthermore, different aromatic polyesters containing D-mandelate and D-3- hydroxy-3-phenylpropionate are produced from glucose when feeding the corresponding monomers. The engineered bacterial system will be useful for one-step fermentative production of aromatic polyesters from renewable resources. [ABSTRACT FROM AUTHOR]

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody. [ABSTRACT FROM AUTHOR]

Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate- co-lactate) [P(3HB- co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB- co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB- co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and the molecular weights of P(3HB- co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca. 7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue. [ABSTRACT FROM AUTHOR]

The effects of three phosphoenolpyruvate (PEP)-dependent PTS carbon sources (glucose, mannose and maltose) and three non-PTS carbon sources (glycerol, galactose, and lactose) on the formation of four carotenoids with diverse structures and on the cell growth of the recombinant Escherichia coli were investigated. The biosynthetic pathways of four carotenoids, C diapolycopene, C diapotorulene, C lycopene, and C beta-carotene, were engineered in E. coli. The resulting E. coli cells were grown in a mineral medium supplemented with each of the six carbon sources. Among the six carbon sources, non-PTS glycerol showed the highest performance in production of all four carotenoid structures, whereas PTS glucose showed the lowest performance. Based on the conversion yield, carotenoid-producing capability, and the cell density, we found that there was no close correlation between PTS and non-PTS transport mechanism and carotenoid formations in E. coli. [ABSTRACT FROM AUTHOR]

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms ( Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass. [ABSTRACT FROM AUTHOR]

Extracellular proteins of filamentous fungi are important for biomedical and biotechnological applications. Aspergillus terreus not only comprises an important class of organisms that have significant commercial relevance to the biotechnology industry, but also is an emerging fungal pathogen. However, no information is available on the extracellular proteome of A. terreus. Thus, we analyzed the extracellular proteomes of A. terreus under different culture conditions using sucrose, glucose, or starch as a main carbon source. A total of 82 protein spots including 39 unique proteins was successfully identified by 2-DE and nano-LC-MS/MS. Of these, 12 proteins were detected in the presence of at least two different carbon sources, whereas 16 proteins were unique to sucrose-, 3 to glucose-, and 8 to starch-grown A. terreus. Most of the proteins with known functions are hydrolytic enzymes, such as hydrolases, glycosylases and proteases, some of which include potential allergens. Both oryzin and a predicted protein (ATEG_07481) were the most abundant in all three media. Particularly, oryzin was highly secreted in high concentration sucrose medium. These proteomic data will be useful for studying protein secretion in further detail, and finding fusion partners for the extracellular production of homologous or heterologous proteins in A. terreus. [ABSTRACT FROM AUTHOR]

Succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically grown in a glycerol-fed continuous bioreactor in order to investigate the physiological responses of the cell to different pH values (5.9, 6.2, or 6.5) and various dilution rates, D. In these experiments, A. succiniciproducens showed a pH-dependent glycerol consumption behavior. When pH was maintained at 5.9 or 6.5, glycerol started to accumulate even at a very low D of 0.027 h−1. Succinic acid yield was not significantly affected by the pH of the culture or the Ds. However, more acetic acid formation was observed when the growth rate of A. succiniciproducens was fast on glycerol at pH 6.2 (at D ≥ 0.15 h−1). The highest obtainable succinic acid/acetic acid ratio was 40:1, which was 10 times higher than that obtained by batch cultures grown on glucose. The maximum obtainable productivity of succinic acid was 2.1 g L−1 h−1), which was 14 times higher than that obtained by batch culture. [ABSTRACT FROM AUTHOR]

Cross-genome comparative transcriptome analyses were previously conducted using the sequenced Streptomyces coelicolor genome microarrays to understand the genetic nature of doxorubicin (DXR) and daunorubicin (DNR) overproducing industrial mutant (OIM) of Streptomyces peucetius. In this previous work, a whiB-like putative transcription factor ( wblA sco) was identified as a global antibiotic down-regulator in S. coelicolor (Kang et al., J Bacteriol 189:4315-4319, ). In this study, a total genomic DNA library of a DXR/DNR-overproducing S. peucetius OIM was constructed and screened using wblA sco as a probe, resulting in the isolation of a wblA ortholog ( wblA spe) that had 95% amino acid identity to wblA sco. Gene disruption of wblA spe from the S. peucetius OIM resulted in an approximately 70% increase in DXR/DNR productivity, implying that the DXR/DNR production in the S. peucetius OIM could be further improved via comparative transcriptomics-guided target gene manipulation. Furthermore, several putative wblA spe -dependent genes were also identified using S. coelicolor interspecies DNA microarray analysis between the S. peucetius OIM and wblA spe-disrupted S. peucetius OIM. Among the genes whose expressions were significantly stimulated in the absence of wblA spe, the overexpression of a conserved hypothetical protein (SCO4967) further stimulated the total production of DXR/DNR/akavinone by 1.3-fold in the wblA spe-disrupted S. peucetius OIM, implying that the sequential genetic manipulation of target genes identified from interspecies comparative microarray analysis could provide an efficient and rational strategy for Streptomyces strain improvement. [ABSTRACT FROM AUTHOR]

The branched chain amino acids (BCAAs), l-valine, l-leucine, and l-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems. Recent advances in the development of BCAA production strains are also reviewed with a particular focus on l-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering are suggested. [ABSTRACT FROM AUTHOR]

This study presents a novel methodology for the development of a chemically defined medium (CDM) using genome-scale metabolic network and flux balance analysis. The genome-based in silico analysis identified two amino acids and four vitamins as non-substitutable essential compounds to be supplemented to a minimal medium for the sustainable growth of Mannheimia succiniciproducens, while no substitutable essential compounds were identified. The in silico predictions were verified by cultivating the cells on a CDM containing the six non-substitutable essential compounds, and it was further demonstrated by observing no cell growth on the CDM lacking any one of the non-substitutable essentials. An optimal CDM for the enhancement of cell growth and succinic acid production, as a target product, was formulated with a single-addition technique. The fermentation on the optimal CDM increased the succinic acid productivity by 36%, the final succinic acid concentration by 17%, and the succinic acid yield on glucose by 15% compared to the cultivation using a complex medium. The optimal CDM also lowered the sum of the amounts of by-products (acetic, formic, and lactic acids) by 30%. The strategy reported in this paper should be generally applicable to the development of CDMs for other organisms, whose genome sequences are available. [ABSTRACT FROM AUTHOR]

An efficient system for the production of (R)-hydroxyalkanoic acids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced. [ABSTRACT FROM AUTHOR]

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxy-alkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications. [ABSTRACT FROM AUTHOR]

A new feeding strategy in fed-batch culture, exponential feeding combined with pH-stat is suggested to avoid the accumulation of substrate in culture broth. Exponential feeding was stopped whenever a predetermined amount of limiting substrate was supplied and then pH change was observed. When pH rose above an upper limit due to the depletion of substrate, feeding was restarted. With this feeding strategy, recombinant Escherichia coli could be grown to 101 g/l by controlling the specific growth rate at 0.1 h -1. [ABSTRACT FROM AUTHOR]

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l-1, 168 g l-1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l-1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l-1 h-1. [ABSTRACT FROM AUTHOR]

The article presents the author's comments on the research conducted by A. Pohlmann and colleagues on the genome sequence of Polyhydroxyalkanoates (PHA) producer, Ralstonia eutropha H16. In his view, the genome sequence provides a foundation for optimizing PHA biosynthesis and for developing metabolic-engineering strategies to generate bioplastics with improved properties.

Presents a correction to the article "The Genome Sequence of the Capnophilic Rumen Bacterium Mannheimia Succiniciproducens," that was previously published in the 2004 issue of the journal "Nature Biotechnology."