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1.
Jiwei Mao Quanli Liu Xiaofei Song Hesuiyuan Wang Hui Feng Haijin Xu Mingqiang Qiao 《Biotechnology letters》2017,39(7):977-982
Objective
To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.Result
When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.Conclusion
Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.2.
Karin Förster-Fromme Sarah Schneider Georg A. Sprenger Christoph Albermann 《Biotechnology letters》2017,39(2):219-226
Objectives
To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.Results
The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3H-GDP-l-fucose with a Vmax of 8 pmol/min mg with a Km of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.Conclusions
The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.3.
Ying Wang Guo-Si Li Pei Qiao Ling Lin Hai-Long Xue Li Zhu Mian-Bin Wu Jian-Ping Lin Li-Rong Yang 《Biotechnology letters》2018,40(11-12):1551-1559
Objective
To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).Results
l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.Conclusions
By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.4.
Deqiang Zhu Jianrong Wu Xiaobei Zhan Li Zhu Zhiyong Zheng Minjie Gao 《Biotechnology letters》2017,39(2):227-234
Objectives
N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).Results
PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l?1, compared with 3.6 ± 0.15 g l?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l?1 in a 1 l fermenter.Conclusions
The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.5.
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.6.
Hongchao Wang Chen Zhang Haiqin Chen Qin Yang Xin Zhou Zhennan Gu Hao Zhang Wei Chen Yong Q. Chen 《Biotechnology letters》2016,38(10):1761-1768
7.
Ziqiang Wang Munan Su Yanliang Li Yunshan Wang Zhiguo Su 《Biotechnology letters》2017,39(12):1859-1863
Objective
To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid.Results
E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg?1. The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U?1 h?1 during the continuous enzymatic production process.Conclusion
ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.8.
Objectives
l-isoleucine dioxygenase (IDO) specifically transforms l-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.Results
The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with IdoN126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with IdoT130K showing a 170% increase in activity. Although the activity of the combined mutant IdoN126H/T130K (0.95?±?0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing IdoN126H/T130K and Ido, 66.50?±?0.99 mM and 26.09?±?1.85 mM 4-HIL was synthesized, respectively, in 24 h.Conclusion
IdoN126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.9.
Ryosuke Fujiwara Shuhei Noda Yoshifumi Kawai Tsutomu Tanaka Akihiko Kondo 《Biotechnology letters》2016,38(9):1543-1549
Objectives
To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.Results
The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s kcat/Km value was 0.54 mM ?1 s?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l?1 from 10 g glucose l?1.Conclusion
A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.10.
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.11.
Jesús Salvador López-Bucio Javier Raya-González Gustavo Ravelo-Ortega León Francisco Ruiz-Herrera Maricela Ramos-Vega Patricia León José López-Bucio Ángel Arturo Guevara-García 《Plant molecular biology》2018,96(4-5):339-351
Key message
The function and components of l-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid.Abstract
Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. l-Glutamate (l-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of l-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine–threonine–tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to l-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to l-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an l-Glu pathway linking the auxin response, and cell division for primary root growth.12.
Sisi Patricia Lolita Ameria Hye Sook Jung Hee Sook Kim Sang Soo Han Hak Sung Kim Jin Ho Lee 《Biotechnology letters》2015,37(8):1637-1644
Objective
To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments.Results
The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu2+ and recovered by adding 1–10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l?1 and 103 mg indirubin l?1 from 2.5 g l-tryptophan l?1.Conclusion
The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.13.
Objectives
To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.Results
Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l?1). Treatments with 100 µM AgNO3 significantly increased intracellular plumbagin production by up to 7.6 mg g?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g?1 DW). In contrast, treatments with 150 mg chitosan l?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g?1 DW, respectively.Conclusions
Chitosan (150 mg l?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.14.
Objective
To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.Results
Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l?1, 100 g wet cells l?1, and 25 g LA l?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l?1.Conclusion
Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.15.
Evelyn Balsano Maranda Esterhuizen-Londt Enamul Hoque Stephan Pflugmacher Lima 《Biotechnology letters》2017,39(8):1201-1209
Objectives
To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.Results
Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l?1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l?1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.Conclusions
M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.16.
Weiyu Wang Jiaqi Sun Wenjun Xiao Li Jiang Ruyue Wang Jun Fan 《Biotechnology letters》2017,39(11):1733-1740
Objectives
To optimize the production of active inclusion bodies (IBs) containing human d-amino acid oxidase (hDAAO) in Escherichia coli.Results
The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing d-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing d-alanine, d-serine and d-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the l-serine racemization activity of the bacterially expressed maize serine racemase.Conclusions
Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.17.
Objective
A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.Results
Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.Conclusion
It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.18.
Background
Corn stover, as one important lignocellulosic material, has characteristics of low price, abundant output and easy availability. Using corn stover as carbon source in the fermentation of valuable organic chemicals contributes to reducing the negative environmental problems and the cost of production. In ethanol fermentation based on the hydrolysate of corn stover, the conversion rate of fermentable sugars is at a low level because the native S. cerevisiae does not utilize xylose. In order to increase the conversion rate of fermentable sugars deriving from corn stover, an effective and energy saving biochemical process was developed in this study and the residual xylose after ethanol fermentation was further converted to l-lactic acid.Results
In the hybrid process based on the hydrolysate of corn stover, the ethanol concentration and productivity reached 50.50 g L?1 and 1.84 g L?1 h?1, respectively, and the yield of ethanol was 0.46 g g?1. The following fermentation of l-lactic acid provided a product titer of 21.50 g L?1 with a productivity of 2.08 g L?1 h?1, and the yield of l-lactic acid was 0.76 g g?1. By adopting a blank aeration before the inoculation of B. coagulans LA1507 and reducing the final cell density, the l-lactic acid titer and yield reached 24.25 g L?1 and 0.86 g g?1, respectively, with a productivity of 1.96 g L?1 h?1.Conclusions
In this work, the air pumped into the fermentor was used as both the carrier gas for single-pass gas stripping of ethanol and the oxygen provider for the aerobic growth of B. coagulans LA1507. Ethanol was effectively separated from the fermentation broth, while the residual medium containing xylose was reused for l-lactic acid production. As an energy-saving and environmental-friendly process, it introduced a potential way to produce bioproducts under the concept of biorefinery, while making full use of the hydrolysate of corn stover.19.
Tian Tang Qun Gao Hua Lin Francis Biville Jingyuan Xiong Xiaofang Pei Bo Zheng Xiaoli Zou Chuan Wang 《Metabolomics : Official journal of the Metabolomic Society》2017,13(12):157
Introduction
ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium (S. typhimurium). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated.Objectives
The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism.Method
We constructed a clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (ΔrpoS TT-1), clpP deletion mutant TT-16 (ΔclpP TT-1), and clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS).Results
Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l-5-oxoproline, l-glutamic acid, and l-tryptophan) and intracellular concentrations of 10 compounds (l-isoleucine, glycine, serine, l-methionine, l-phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted.Conclusion
ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.20.
Shinji Takenaka Takahiro Ozeki Kosei Tanaka Ken-ichi Yoshida 《Biotechnology letters》2017,39(11):1699-1707