Quaternary organization of subunits in the L-leucine dehydrogenase from Bacillus cereus

L-Leucine dehydrogenase from Bacillus cereus was examined in the electron microscope. The quaternary structure reveals a molecule that is built up from 8 subunits, identical in mass, arranged in 2 layers which are oriented mainly in a staggered form. In each layer subunits are positioned at the vertices of a square, leaving free a central protein-deficient region of 2.6 nm in diameter. The enzyme measures 11.1 nm in diagonal and 9.0 nm in edge length. Mean subunit diameter is 4.0 nm. The overall shape is a cube, slightly compressed, with 90% edge length in height.     

蜡样芽孢杆菌亮氨酸脱氢酶基因在大肠杆菌中的表达及重组酶性质

本研究采用PCR技术从蜡样芽孢杆菌Bacillus cereus基因组DNA中克隆出亮氨酸脱氢酶基因,构建重组表达质粒p ET28α(+)-ldh,实现在大肠杆菌中的高效表达,并分析重组亮氨酸脱氢酶的酶学性质。结果表明,从Bacillus cereus成功克隆的亮氨酸脱氢酶编码基因约为1 000 bp,表达的重组亮氨酸脱氢酶相对分子质量约为40 k Da。酶学研究结果表明:该酶的最适反应温度为37℃,其热稳定性好,30℃的半衰期长达330 h;最适反应p H为9.5;在p H 7.0~8.0的缓冲液中保存24 h后仍保持原有酶活力的80%以上;金属离子Fe2+对该酶具有明显的促进作用,而EDTA强烈抑制亮氨酸脱氢酶的活性。动力学分析结果表明该酶对底物NADH催化的Km和Vmax分别为0.635 mmol/L和1.54μmol/(L·min)。亮氨酸脱氢酶基因在大肠杆菌中的成功表达为手性氨基酸的生物合成提供了可能。     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Purification of recombinant Bacillus cereus ResD-ResE proteins expressed in Escherichia coli strains

Recombinant E. coli strains expressing the Bacillus cereus ATCC 14579^T resD and resE genes fused with the ubiquitin gene were constructed, and purification of the ResD and ResE proteins was performed. The approach used in the study allowed us to increase the protein yield of the electrophoretic homogeneous ResD and ResE proteins without denaturation steps up to 150 mg per gram of wet cell weight.     

Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD⁺-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L⁻¹ d⁻¹, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.     

Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli

The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLλ, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from pRA96. Expression of L-leucine dehydrogenase was controlled by its constitutive B. cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition. After cell cultivation at 30 °C and shifting to 41 °C to induce plasmid replication, E. coli BL21[pIET98] yielded 200 U LeuDH/mg protein, which corresponds to >50% of the soluble cell protein. Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure. Received: 19 July 1999 / Received revision: 4 November 1999 / Accepted: 5 November 1999     

Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis

The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Purification of fusion ferritin from recombinant E. coli using two-step sonications

Fusion ferritin, combined by heavy chain ferritin (21 kDa) and light chain ferritin (19 kDa), was expressed in recombinant E. coli. The fusion ferritin was easily purified by two-step sonications as well as gel filtration chromatography. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 Fe³⁺ mg mg^–1 of protein and 66%. Those values are larger than the previous ones of 0.2 Fe³⁺ mg mg^–1 (Kim et al. 2001).     

Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system.

DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli. A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR. The fused product was then inserted into the novel FLAG vector by restriction and ligation. This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III. The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha. Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography. The purified material was cleaved with enterokinase to remove the FLAG peptide. Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.     

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.     

Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli

Streptococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60°C with activation energy of 51 KJ mole^–. The apparent K_m values for NADP and D-G3P or DL-G3P were estimated to be 0.385 ± 0.05 and 0.666 ± 0.1 mM, respectively and the V_max of the purified protein was estimated to be 162.5 U mg^–1. The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.     

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

Background  

(+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli.     

Production of free fatty acids from switchgrass using recombinant Escherichia coli

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake‐flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass‐based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91–98, 2018     

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with P_BAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.     

Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.