Effects of carbon source and metabolic engineering on butyrate production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Butyrate was produced in recombinant Escherichia coli strains by applying metabolic engineering strategies. The genes for producing butyrate were cloned from Clostridium acetobutylicum and then expressed in E. coli. To study important factors for improving the productivity of butyrate, we deleted pta and ptsG genes in E. coli and compared the effects of these gene deletions in E. coli B and K strains. The effect of carbon sources, glucose and glycerol, was also compared. A significant improvement of butyrate production was made when glycerol was used as a carbon source, resulting in 0.56 g/l of butyrate in LB medium with 1% (v/v) glycerol.     

Efficient Whole‐Cell Biocatalytic Synthesis of N‐Acetyl‐D‐neuraminic Acid

N‐Acetyl‐D ‐neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N‐acetyl‐D ‐glucosamine (GlcNAc) and N‐acetyl‐D ‐mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32±0.56 g/liter Neu5Ac were obtained from 65.61±2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4±0.4 % purity, 80.87±0.79 % recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes.     

Enhanced recombinant protein synthesis in batch and fed‐batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis

BACKGROUND: The formation of acetate as a metabolic by‐product in Escherichia coli fermentation is well known to have detrimental effects on cell growth and productivity. Various bioprocess and genetic approaches have previously been made to limit acetate accumulation, however, they tend to be conservative, limiting overall process productivity, or lead to other problems such as a decrease in maximum specific growth rate and decreased product yield on carbon. RESULTS: In this work, the utility of electrodialysis is examined as a potentially generic approach for in situ acetate removal and its impact on recombinant protein production. Using the induced synthesis of recombinant green fluorescent protein (GFP) in E. coli Tg1 (pGLO) as an example, it is shown that in situ removal of acetate to below inhibitory levels (～1 g L^?1) provides significant improvements in cell growth rate as well as specific biomass and recombinant protein yields. Experiments were performed in a 7.5 L stirred‐tank bioreactor using an external single cell‐pair electrodialysis module with an effective ion exchange membrane area of 0.01 m 2 . For this system increases in specific recombinant protein yield of up to 4‐fold have been observed dependent upon the time of induction, the mode of operation and the level to which acetate concentration is reduced in the fermentation broth. CONCLUSIONS: The implementation of ED can significantly increase the level of recombinant protein synthesis in batch and fed‐batch fermentation. The approach is considered to be generic, readily implemented and has wide application for the production of recombinant enzymes and proteins. Copyright © 2009 Society of Chemical Industry     

Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi‐Enzyme System

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D ‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L ‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xyl_A) was recombined with pRSETA vector, and the pRSETA::xyl_A obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L^?1 (20.6 U mg^?1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h^?1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h^?1 operation as 9.6 g L^?1 at t = 32 h. The highest oxygen uptake was 4.57 mol m^?3 s^?1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry     

Effects of Oxygen Supply Modes on the Production of Human Growth Hormone in Different Scale Bioreactors

Recombinant Escherichia coli has been studied as a main host for recombinant protein productions, but it is still difficult to cultivate E. coli in a large industrial‐scale process due to the oxygen supply limitation. In this study, E. coli BL(21) harboring a new constructed plasmid (pEHUb‐hGH) was used for producing recombinant human growth hormone (r‐hGH) in 5‐L and 30‐L scale fermentors by supplying air and high purity oxygen, respectively, where the high purity oxygen was produced from a vacuum pressure swing adsorption (PSA). The impact of oxygen supply modes, i.e., air and high purity oxygen, on cell growth and r‐hGH production was investigated in different scale fermentors. In the case of high purity oxygen supply, the final cell density and r‐hGH concentrations were 63.0 and 4.8 g/L in the 5‐L fermentor, 51.6 and 4.0 g/L in the 30‐L fermentor, respectively. In addition, the productivity of r‐hGH was doubled in the 5‐L fermentor, and increased 4‐fold in the 30‐L fermentor, compared to the results obtained in the case of the air supply. The supply of high purity oxygen eliminated the oxygen limitation and acetate formation effectively, and apparently, did not affect the degradation of r‐hGH. This shows that the recombinant E. coli cultivation with high purity oxygen produced from PSA may provide an effective method for large‐scale industrial production of recombinant proteins.     

Control of the mechanical and thermal properties of semicrystalline polymers via a new processing route of the equal channel multiple angular extrusion

Deformation routes of equal‐channel multiple angular extrusion (ECMAE) are suggested that allow realization of new combinations of positions of planes and directions of simple shear. The routes are called F, B+C, D+C. Their effect on the structure and the properties of semicrystalline polymers is studied by the example of high density polyethylene, polyoxymethylene, polytetrafluorethylene. It is demonstrated that the enhanced strength and plasticity characteristics and the reduced anisotropy of microhardness in longitudinal and transversal directions are achieved in the series: route C → routes E,F → route B+C → route D+C. The methods of X‐ray structure analysis and dilatometry revealed differences in biaxial oriented fibril structures formed after these ECMAE routes have been applied and in the character of their distribution over preferred directions of orientation. Possibility of realization of bidirectional invar‐effect in rod‐shaped billets is presented. POLYM. ENG. SCI., 54:531–539, 2014. © 2013 Society of Plastics Engineers     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

Peracetic acid‐enhanced photocatalytic and sonophotocatalytic inactivation of E. coli in aqueous suspensions

BACKGROUND: Although chlorination is an effective and widely employed method of water disinfection, it suffers serious drawbacks such as the formation of toxic chlorinated by‐products. Therefore, other disinfection technologies have been researched and developed, including advanced oxidation. RESULTS: The efficacy of heterogeneous photocatalysis and sonophotocatalysis induced by UV‐A irradiation and low frequency (24–80 kHz) ultrasound irradiation in the presence of TiO₂ as the photocatalyst and peracetic acid (PAA) as an additional disinfectant to inactivate E. coli in sterile water was evaluated. PAA‐assisted UV‐A/TiO₂ photocatalysis generally leads to nearly complete E. coli inactivation in 10–20 min of contact time with the extent of inactivation depending on the photocatalyst type and loading (in the range 100–500 mg L^?1) and PAA concentration (in the range 0.5–2 mg L^?1). The simultaneous application of ultrasound and UV‐A irradiation in the presence of TiO₂ and PAA prompted further E. coli inactivation. CONCLUSIONS: The proposed advanced disinfection technology offers complete E. coli inactivation at short treatment times and low PAA doses. Copyright © 2010 Society of Chemical Industry     

Synthesis,Chiral Separation,Absolute Configuration Assignment,and Biological Activity of Enantiomers of Retro‐1 as Potent Inhibitors of Shiga Toxin

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro‐1, a compound active against Stx and other such protein toxins. Retro‐1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X‐ray diffraction data revealed (S)‐Retro‐1 to be slightly more active than (R)‐Retro‐1.     

A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

A pH‐based high‐throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active‐site‐modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3‐hydroxypropanal and 4‐hydroxybutanal for preparative synthesis of chiral deoxyketose‐type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH‐based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.     

Genome Mining of the Lipopeptide Biosynthesis of Paenibacillus polymyxa E681 in Combination with Mass Spectrometry: Discovery of the Lipoheptapeptide Paenilipoheptin

Paenibacillus polymyxa strains are qualified for agro‐biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A–D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet‐unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI‐LIFT‐TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open‐chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty‐acid part as well as in their peptide part.     

Revisiting the mechanism of the triosephosphate isomerase reaction: the role of the fully conserved glutamic acid 97 residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues—K12, H95, E97 and E165—are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three‐dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000‐fold reduction in k_cat is observed for E97Q, whereas the E97D mutant shows a 100‐fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000‐fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 Å resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans‐to‐gauche transition about the Cγ? Cδ (χ³) bond. In the E97D mutant, the K12 side chain maintains the wild‐type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.     

Strain isolation and optimization of process parameters for bioconversion of glycerol to lactic acid

BACKGROUND: The crude glycerol from biodiesel production represents an abundant and inexpensive source which can be used as raw material for lactic acid production. The first aim of this investigation was to select a strain suitable for producing lactic acid from glycerol with a high concentration and productivity. The second aim was to obtain the optimum fermentation conditions, as a basis for large‐scale lactate production in the future. RESULTS: Eight bacterial strains, which could aerobically convert glycerol to lactic acid, were screened from soil samples. One of the strains, AC‐521, which synthesized lactic acid with a higher concentration, was identified based on its 16S rDNA sequences and physiological characteristics. These results indicated that this strain was a member of Escherichia coli. The optimal fermentation conditions for Escherichia coli AC‐521 were 42 °C, pH 6.5, 0.85 min^?1 (K_La). CONCLUSION: Escherichia coli AC‐521 suitable for producing lactic acid from glycerol with high concentration and productivity was identified. After 88 h of fed‐batch fermentation, both the lactic acid concentration and glycerol consumption reached maximum, giving 85.8 g L^?1 of lactic acid with a productivity of 0.97 g L^?1 h^?1 and a yield of 0.9 mol mol^?1 glycerol. Copyright © 2009 Society of Chemical Industry     

Biochemical Characterization of the Lysine Acetylation of Tyrosyl‐tRNA Synthetase in Escherichia coli

Aminoacyl‐tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl‐tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic‐code‐expansion strategy to site‐specifically incorporate N^?‐acetyl‐l ‐lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5‐related N‐acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl‐CoA or acetyl‐phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.     

Escherichia coli Biofilm Characteristics on Polymeric Heat Exchanger Surfaces

Time‐dependent effects on the apparent roughness and surface free energy of different polymeric surfaces and stainless steel were studied during the biofouling process for Escherichia coli K12. The surface roughness increases during primary adhesion of E. coli on the surfaces and is later reduced as the surface between scattered bacteria is completely covered, forming a uniform biofilm. During the fouling process, the polar fraction of the surface free energy significantly increased, whereas the dispersive fraction decreased for all substrates. The attachment of E. coli and subsequent bacterial production of extracellular polymeric substances increased the polarity of the initially nonpolar polymeric surfaces to increase wettability.     

An unstructured model for the analysis of substrate consumption and product release in relation to biosynthesis and cell maintenance during batch cultures of Geotrichum candidum and Penicillium camembertii

An unstructured model has been developed to predict microbial growth based on carbon or nitrogen substrate consumption, ammonia or carbon dioxide production and proton transfer. The model has been validated for batch cultures of Geotrichum candidum and Penicillium camembertii growing on peptones and peptones + lactate based media. The contributions of the considered kinetics to biosynthesis and cellular maintenance can be deduced from this model. The nitrogen source (peptones) was mainly utilized in biosynthesis: for P camembertii growing on peptones, 86% of the metabolized peptones. G candidum metabolized peptones preferentially to lactate as a carbon source, resulting in lactate utilization by a maintenance mechanism during the stationary state. In contrast, P camembertii, which metabolized fewer amino acids as a carbon source, utilized lactate mainly for biosynthesis (83% of the consumed lactate). Most (up to 71%) of the ammonia released was produced by deamination of amino acids utilized as both carbon and nitrogen sources by growth‐associated metabolism. With peptones, proton transfer resulted from ammonia release, most likely as a result of the growth‐associated mechanism, as supported experimentally (55–58% of the released ammonia for both microorganisms). The contribution of lactate to proton transfer resulted in 76% of protons exchanged by a growth‐associated mechanism during P camembertii growth. For total carbon dioxide production, the contributions of the energy supplies for biosynthesis and cell maintenance were similar; except during P camembertii growth in the presence of lactate (65% of growth‐associated CO₂ production). © 2002 Society of Chemical Industry     

High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his‐tagged rhamnulose‐1‐phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm^?3 and 288 AU dm^?3 h^?1 respectively) compared with Luria–Bertoni (LB) medium (2292 AU dm^? 3 and 255 AU dm^?3 h^?1). The minimum quantity of (isopropyl‐β‐D ‐thiogalactoside) IPTG for optimal induction was estimated in 18–20 µmol IPTG gDCW^?1. The highest volumetric production of RhuA (8333 AU dm^?3) was obtained when IPTG was added in the late log‐phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37 °C. An effective two‐step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications. Copyright © 2003 Society of Chemical Industry