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Xiuling Shang Xin Chai Xuemei Lu Yuan Li Yun Zhang Guoqiang Wang Chen Zhang Shuwen Liu Yu Zhang Jiyin Ma Tingyi Wen 《Biotechnology letters》2018,40(2):383-391
Objective
To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering.Results
Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using β-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R 2 = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction.Conclusions
The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.3.
Nicole Werner Miriam Dreyer Wenke Wagner Nicolas Papon Steffen Rupp Susanne Zibek 《Biotechnology letters》2017,39(3):429-438
Objectives
To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.Results
We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.Conclusion
C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.4.
Thijs Welle Anna T. Hoekstra Ineke A. J. J. M. Daemen Celia R. Berkers Matheus O. Costa 《Metabolomics : Official journal of the Metabolomic Society》2017,13(7):83
Introduction
Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.Objective
The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.Methods
Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.Results
Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.Conclusions
The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.5.
Hyun-Soo Kim 《Biotechnology letters》2016,38(11):1955-1960
Objective
To identify a novel gene responsible for organic solvent-tolerance by screening a transposon-mediated deletion mutant library based on Saccharomyces cerevisiae L3262.Results
One strain tolerant of up to 0.5 % (v/v) n-hexane and cyclohexane was isolated. The determination of transposon insertion site identified one gene, YLR162W, and revealed disruption of the ORF of this gene, indicating that organic solvent tolerance can be conferred. Such a tolerant phenotype reverted to the sensitive phenotype on the autologous or overexpression of this gene. This transposon mutant grew faster than the control strain when cultured at 30 °C in YPD medium containing 0.5 % (v/v) n-hexane and cyclohexane respectively.Conclusion
Disruption of YLR162W in S. cerevisiae results in increased tolerance to organic solvents.6.
Gbekeloluwa B. Oguntimein Miguel RodriguezJr. Alexandru Dumitrache Todd Shollenberger Stephen R. Decker Brian H. Davison Steven D. Brown 《Biotechnology letters》2018,40(2):303-308
Objective
To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential.Results
Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain.Conclusion
A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.7.
Objectives
To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.Results
Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.Conclusions
Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.8.
Gianfranco Picone Francesco Savorani Alessia Trimigno Bruno Mezzetti Francesco Capozzi Søren Balling Engelsen 《Metabolomics : Official journal of the Metabolomic Society》2016,12(10):150
Introduction
The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch.Objectives
This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome.Methods
The effects of the genetic modification upon the grape metabolome were evaluated through 1H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one.Results
The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration.Conclusion
NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.9.
Shuanghong Zhang Dingyu Liu Zhitao Mao Yufeng Mao Hongwu Ma Tao Chen Xueming Zhao Zhiwen Wang 《Biotechnology letters》2018,40(5):819-827
Objective
To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum.Results
A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the ??10 and ??35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks.Conclusions
To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.10.
Phan Nguyen Thuy An Masamitsu Yamaguchi Eiichiro Fukusaki 《Metabolomics : Official journal of the Metabolomic Society》2017,13(3):29
Introduction
Metamorphosis is a complicated process in which cell proliferation, differentiation, and death are orchestrated to form the mature structures of insects. In Drosophila, this process is controlled by ecdysone, a steroid hormone responsible for tissue remodeling and organogenesis that gives rise to the adult fly.Objective
By using a metabolomics approach, this study aimed to elucidate global changes in the central metabolic pathways in Drosophila throughout metamorphosis and then further examine the effects of temperature and origin on metabolic profiles.Methods
Targeted and non-targeted metabolic profiling of time-course samples from Drosophila were constructed to cover a wide range of cellular metabolites during metamorphosis.Results
This was the first wide-scale metabolomics study of Drosophila metamorphosis focusing on central metabolism. The abundance of detected metabolites changed drastically and correlated strongly with the development of Drosophila pupae. In non-stress conditions, temperature affected the developmental time, but the metabolic state at a certain stage of metamorphosis remained stable. Between D. melanogaster Canton S and Oregon R, similar metabolic profiles throughout metamorphosis was observed. However, there were still differences in purine and pyrimidine metabolism at an early stage in the pupal period, which was matched by differences in ecdysteroid levels.Conclusion
This study supported the strength of metabolomics in the field of developmental biology. The results provided a general view on the metabolic profile of Drosophila during metamorphosis, which provides basic 3 knowledge for future metabolomics studies using Drosophila.11.
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Justin J. J. van der Hooft Wejdan Alghefari Eleanor Watson Paul Everest Fraser R. Morton Karl E. V. Burgess David G. E. Smith 《Metabolomics : Official journal of the Metabolomic Society》2018,14(11):144
Introduction
Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity.Objectives
For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium.Methods
To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h.Results
This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy.Conclusions
Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.14.
Wenyi Sun Xiaobing Yang Xueying Wang Xinping Lin Yanan Wang Sufang Zhang Yushi Luan Zongbao K. Zhao 《Biotechnology letters》2017,39(7):1001-1007
Objectives
To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.Results
The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.Conclusions
Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.15.
Pei Han Yong Huang Yumin Xie Wu Yang Wenying Xiang Peter J. Hylands Cristina Legido-Quigley 《Metabolomics : Official journal of the Metabolomic Society》2018,14(7):91
Introduction
In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.Objective
The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.Methods
Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).Results
The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.Conclusion
Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.16.
Tianzhen Li Wei Zhou Huiping Bi Yibin Zhuang Tongcun Zhang Tao Liu 《Biotechnology letters》2018,40(7):1057-1065
Objectives
To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.Results
We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.Conclusions
Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.17.
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Wenyi Sun Xiaobing Yang Xueying Wang Xiang Jiao Sufang Zhang Yushi Luan Zongbao K. Zhao 《Biotechnology letters》2018,40(6):933-940
Objectives
To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides.Results
Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium.Conclusions
We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.19.
Yalan Zou Tao Chen Lili Feng Shuanghong Zhang Dongxu Xing Zhiwen Wang 《Biotechnology letters》2017,39(9):1369-1374
Objective
To construct a strain of Corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ALA) via the C4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.Results
The recombinant C. glutamicum strain AP2 harboring a codon-optimized hemA gene from Rhodobacter sphaeroides was used as host strain for 5-ALA production. A plasmid harboring the serine operon, which contained serB, serC and the site-specific mutant serA Δ197 , was constructed and introduced into C. glutamicumAP2, leading to an increase of 70% in 5-ALA production. Further overexpression of the glyA gene increased production of 5-ALA by 150% over the control. 5-ALA production was thus significantly enhanced by engineering the glycine biosynthetic pathway. C.glutamicum AG3 produced 3.4 ± 0.2 g 5-ALA/l in shake-flask cultures in CGIIIM medium with the addition of 7.5 g glycine/l.Conclusion
This is the first report of remodeling the serine and glycine biosynthetic pathway to improve the production of 5-ALA in C. glutamicum.20.
Ye Mun Low Ivan Kok Seng Yap Kartini Abdul Jabar Mohd Yasim Md Yusof Chun Wie Chong Cindy Shuan Ju Teh 《Metabolomics : Official journal of the Metabolomic Society》2017,13(5):65