Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.     

Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

Functional characterization of orf6 and orf9 genes involved in the biosynthesis of L-oleandrose from Streptomyces antibioticus Tü99

Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and heterologously expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is in support of proving their functions. Furthermore biosynthetic pathway 5,5-gem-dimethyl-6-deoxyglucose (TDP-Lnoviose) has been proposed.     

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-d-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.     

Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> CCM 3239

An incomplete oligoketide (PK; ‘polyketide’) gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.     

Functional analyses of cytochrome P450 genes responsible for the early steps of brassicicene C biosynthesis

We previously revealed that Orf8 and Orf6, which were identified in the brassicicene C biosynthetic gene cluster in Alternaria brassicicola strain ATCC96836, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present Letter, the early biosynthetic steps after the formation of FD were investigated. Plasmids carrying the FD synthase gene, one (or two) of five cytochrome P450 genes (orf1, orf2, orf5, orf7, and orf11) identified in the cluster and a cytochrome P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Saccharomyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8β-hydroxylation enzyme that yields FD 8β-ol (4), followed by 16-hydroxylation by Orf7 to produce FD 8β16-diol (5).     

Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome

The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1^*–4^*). The function of the orf1^* product is not known. The product of orf2^* (tylM2) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3-N-methylation during mycaminose production, is encoded by orf3^* (tylM1). The product of orf4^* (ccr) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Cloning and expression of a full-length glutamate decarboxylase gene from a high-yielding γ-aminobutyric acid yeast strain MJ2

A yeast strain MJ2 that was found to produce a higher amount of γ-aminobutyric acid (GABA) was isolated from the surface of kiwi. Phylogenetic analysis based on the ITS sequence and morphological, biochemical studies indicated that it may belong to Saccharomyces cerevisiae. Under optimum conditions in Czapek’s broth medium with 0.5 % monosodium glutamate, it produced GABA at a concentration of 5.823 g/L after 48 h. A full-length glutamate decarboxylase gene (Scgad) was cloned by PCR amplification. The open reading frame (ORF) of the Scgad gene was composed of 1,755 nucleotides and encoded a protein (585 amino acids) with a predicted molecular weight of 65.897 kDa. The deduced amino acids sequence of Scgad shows 100 %, 65 % and 62 % similarity with S. cerevisiae, Candida glabrata and Kluyveromyces lactis GAD in the polypeptide level, respectively. The Scgad gene was expressed in Escherichia coli BL21 (DE3) cells, and the expression was confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The results suggested that the S. cerevisiae GAD (ScGAD) was successfully encoded in E. coli BL21 (DE3) cells. Furthermore, the enzyme activity of ScGAD encoded in E. coli BL21 (DE3) had been significantly enhanced using artificial neural network linked with genetic algorithm (ANN-GA) method.     

Optimizing cadmium and mercury specificity of CadR-based E. coli biosensors by redesign of CadR

The metalloprotein, CadR, was redesigned to optimize cadmium and mercury specificity of CadR-based E. coli biosensors. By truncating 10 and 21 amino acids from the C-terminal extension of CadR, CadR-TC10 and CadR-TC21 were obtained, respectively. The genes cadR, cadR-TC10 and cadR-TC21 were used as sensing elements to construct green fluorescent protein based E.coli biosensors. Induction at 30 °C for 4 h in supplemented M9 medium was the optimized condition for the biosensor. Compared with CadR-based biosensor, there was a clear decline in induction coefficient for CadR-TC21-based biosensor (decreased by 86 % in Zn(II), 44 % in Hg(II), and only 37 % in Cd(II)). While in CadR-TC10-based biosensor, the induction coefficient decreased by 95 % in Zn(II), 70 % in Hg(II), and 67 % in Cd(II). Improved performances of CadR mutants based E. coli biosensors indicated that truncating C-terminal extension of CadR could improve the specificity.     

PCR-Based Detection of Extended-Spectrum β-Lactamases (bla CTX-M-1 and bla TEM ) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae Isolated from Pigs in North Eastern India (Mizoram)

Cephalosporins are major antimicrobials used to treat serious infections. However, their effectiveness is being compromised by the emergence of extended-spectrum β-lactamases (ESBLs). A total of 138 enteric bacteria were isolated from 53 faecal samples of pigs collected from different districts of Mizoram, of which 102 (73.91 %) were Escherichia coli, 26 (18.84 %) were Salmonella spp. and 10 (7.25 %) were Klebsiella pneumoniae. Phenotypic confirmatory test (Double Discs Synergy Test) showed that 8 (5.80 %) E. coli isolates were ESBLs producer. PCR analysis confirmed that out of the eight isolate, 7 (5.07 %) harboured bla _CTX-M-1 gene and/or bla _TEM gene. Of the eight positive isolates, 7 (5.07 %) and 3 (2.17 %) were found to be positive for bla _CTX-M-1 gene and bla _TEM gene, respectively, of which 3 (2.17 %) isolates were positive for both the genes. Only 4 (2.90 %) E. coli isolates carried bla _CTX-M-1 gene alone. Agarose gel electrophoresis showed that all the isolates were carrying plasmids ranging between 0.9 and ~30 kb. Out of the seven isolates positive for bla _CTX-M-1 and/or bla _TEM , 2 (1.84 %) isolates were confirmed for bla _CTX-M-1 gene in their plasmid. Only one E. coli isolate was found to be positive for both the genes in its plasmid. The resistance plasmid could not be transferred to a recipient by in vitro horizontal gene transfer method.     

Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol

Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (?aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P < 0.05). The results showed that sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption.     

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml^?1) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.     

Gas Vesicle Genes Identified in Bacillus megaterium and Functional Expression in Escherichia coli

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E. coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-constrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In B. megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E. coli. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli

The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-β-d-maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 ± 2 Å for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids.     

Modification of glycolysis and its effect on the production of l-threonine in Escherichia coli

High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and l-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an l-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more l-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less l-threonine than THRD, THRDΔpfkB produced levels of l-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high l-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of l-threonine.     

Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases

Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3,7-O-bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3-O-glucoside-7-O-rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3-O-glucoside, were transformed into E. coli. Using this approach, 67 mg/L of quercetin 3-O-glucoside-7-O-rhamnoside was synthesized. For the synthesis of quercetin 3,7-O-bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1, AtUGT89C1, and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7-O-bisrhamnoside.