Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.     

Non-enzymatic PLP-dependent oxidative deamination of amino acids induces higher alcohol synthesis

Pyridoxal phosphate (PLP) is an organic cofactor found in all transaminase enzymes. In this study PLP was used to replace the enzymatic deamination step in the Ehrlich pathway, for the oxidative conversion of amino acids into 2-keto acids. PLP functions in an enzymeindependent manner. It was further used in the synthesis of higher alcohols through a sequential enzymatic reduction in vitro and in vivo. PLP-dependent oxidation was investigated against five representative amino acids: valine, leucine, isoleucine, norvaline, and phenylalanine. In vitro amino acid oxidation resulted in approximately 45 ~ 75% [mole/mole] of each 2-keto acid conversion and in vitro ammonia formation was less than 2-keto acid formation, with 20% of conversion yields. Whole cell E. coli expressing reduction enzymes KivD/ADH with both single amino acid and amino acid mixture (4% yeast extract) gave the highest yield (30 ~ 55%) in the presence of the PLP-Cu complex and following enzymatic reactions.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.     

Enhancement of the direct antimicrobial activity of Lysep3 against <Emphasis Type="Italic">Escherichia coli</Emphasis> by inserting cationic peptides into its C terminus

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

Stability,oviposition attraction,and larvicidal activity of binary toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?相似文献   

<Emphasis Type="Italic">Escherichia coli</Emphasis> signal peptidase recognizes and cleaves archaeal signal sequence

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.     

Anaerobic biosynthesis of intermediates of reductive branch of tricarboxylic acids cycle by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with inactivated <Emphasis Type="Italic">frdAB</Emphasis> and <Emphasis Type="Italic">sdhAB</Emphasis> genes

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.     

Engineering the growth pattern and cell morphology for enhanced PHB production by <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli JM109?envC?nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109?minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109?envC?nlpD and E. coli JM109?minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109?minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109?envC?nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.     

Construction of a Synthetic Bypass for Improvement of Aerobic Synthesis of Succinic Acid through the Oxidative Branch of the Tricarboxylic Acid Cycle by Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains

The effect of the introduction of a synthetic bypass, providing 2-ketoglutarate to succinate conversion via the intermediate succinate semialdehyde formation, on aerobic biosynthesis of succinic acid from glucose through the oxidative branch of the tricarboxylic acid cycle in recombinant Escherichia coli strains has been studied. The strain lacking the key pathways of acetic, lactic acid and ethanol formation from pyruvate and acetyl-CoA and possessing modified system of glucose transport and phosphorylation was used as a chassis for the construction of the target recombinants. The operation of the glyoxylate shunt in the strains was precluded resulting from the deletion of the aceA, aceB, and glcB genes encoding isocitrate lyase and malate synthases A and G. The constitutive activity of isocitrate dehydrogenase was ensured due to deletion of isocitrate dehydrogenase kinase/phosphatase gene, aceK. Upon further inactivation of succinate dehydrogenase, the corresponding strain synthesized succinic acid from glucose with a molar yield of 24.9%. Activation of the synthetic bypass by the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase gene notably increased the yield of succinic acid. Functional activity of the synthetic bypass in the strain with the inactivated glyoxylate shunt and opened tricarboxylic acid cycle led to 2.7-fold increase in succinate yield from glucose. As the result, the substrate to the target product conversion reached 67.2%. The respective approach could be useful for the construction of the efficient microbial succinic acid producers.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

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.

     

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from <Emphasis Type="Italic">Bacillus coagulans</Emphasis> NL01

Objective

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid.

Results

The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7–8 but with a high temperature maximum of 80–85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni²⁺ and Co²⁺, respectively.

Conclusions

Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

     

Breadfruit (<Emphasis Type="Italic">Artocarpus altilis</Emphasis>): a source of high-quality protein for food security and novel food products

Protein deficiency has been observed as a leading cause of malnutrition and child death in the tropics. The current study evaluated the protein quality of 49 important breadfruit cultivars (41 Artocarpus altilis and 8 hybrids of A. altilis × A. mariannensis). While significant differences were found between cultivars, all varieties contained a full spectrum of the essential amino acids and are especially rich in phenylalanine, leucine, isoleucine, and valine. The cultivar Ma’afala contained significantly higher total essential amino acid content than other varieties and higher-quality protein than staples such as corn, wheat, rice, soybean, potato, and pea.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

First structure of archaeal branched-chain amino acid aminotransferase from <Emphasis Type="Italic">Thermoproteus uzoniensis</Emphasis> specific for <Emphasis Type="SmallCaps">l</Emphasis>-amino acids and <Emphasis Type="Italic">R</Emphasis>-amines

The gene TUZN1299 from the genome of the hyperthermophilic archaeon Thermoproteus uzoniensis encoding a new 32.8 kDa branched-chain amino acid aminotransferase (BCAT) was expressed in Escherichia coli. The recombinant protein TUZN1299 was purified to homogeneity in the PLP-bound form. TUZN1299 was active towards branched-chain amino acids (l-Val, l-Leu, l-Ile) and showed low but detectable activity toward (R)-alpha-methylbenzylamine. The enzyme exhibits high-temperature optimum, thermal stability, and tolerance to organic solvents. The structure of an archaeal BCAT called TUZN1299 was solved for the first time (at 2.0 Å resolution). TUZN1299 has a typical BCAT type IV fold, and the organization of its active site is similar to that of bacterial BCATs. However, there are some differences in the amino acid composition of the active site.