Unexpected differential metabolic responses of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> to the abundant presence of glutamate and fucose

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.

     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

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.

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

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.

     

Change of the <Emphasis Type="Italic">N</Emphasis>-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase as active inclusion bodies

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.

     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

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.

     

Production of tartaric acid using immobilized recominant <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

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.

     

ClpXP affects the cell metabolism of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> partially in an RpoS-dependent manner

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.

     

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots to <Emphasis Type="SmallCaps">l</Emphasis>-glutamate

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.

     

Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Directed evolution and site-specific mutagenesis of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine dioxygenase derived from <Emphasis Type="Italic">Bacillus weihenstephanensis</Emphasis>

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 Ido^N126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with Ido^T130K showing a 170% increase in activity. Although the activity of the combined mutant Ido^N126H/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 Ido^N126H/T130K and Ido, 66.50?±?0.99 mM and 26.09?±?1.85 mM 4-HIL was synthesized, respectively, in 24 h.

Conclusion

Ido^N126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.

     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

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 k_cat/K_m 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.

     

Native promoters of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application in <Emphasis Type="SmallCaps">l</Emphasis>-lysine production

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.

     

Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from d-mannose by two-step fermentation pathway in Escherichia coli.

Results

E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using d-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation.

Conclusions

Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.

     

Characterization of a flavin-containing monooxygenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application to production of indigo and indirubin

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.

     

Expression,purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile <Emphasis Type="Italic">Laceyella sacchari</Emphasis>

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.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

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.

     

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the <Emphasis Type="Italic">N</Emphasis>-acetyltransferase from <Emphasis Type="Italic">Chryseobacterium</Emphasis> sp.

Objectives

To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA).

Results

A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme’s kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. ¹²⁶Leu, ¹³²Leu, and ¹³⁵Lys were implicated in the binding of phosphopantothenic acid, and ¹⁰⁰Tyr and ¹³¹Lys in that of adenosyl biphosphate. The data supported the participation of ⁸³Glu and ¹³³Tyr in catalyzing acetyl-group transfer to l-2-phenylglycine.

Conclusions

5-3B NatA catalyzes the enantioselective N-acetylation of l-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.