Recombinant whole-cell mediated baeyer-villiger oxidation of perhydropyran-type ketones

Recombinant Escherichia coli cells expressing eight Baeyer-Villiger monooxygenases of bacterial origin have been utilized to oxidize prochiral heterocyclic ketones containing a pyran ring system. Within the biotransformation, two stereogenic centers were introduced with high control of enantioselectivity. The chemoselectivity of the enzymatic reaction was found to be high in favor of the Baeyer-Villiger process when using substituted ketone precursors incorporating functional groups labile to oxidation. A significantly different behavior was observed for two groups of monooxygenases with respect to substrate acceptance, which is consistent with our previous classification into two enzyme clusters.     

Microbiological transformations 52.: Biocatalysed Baeyer–Villiger oxidation of 1-indanone derivatives

The microbiological Baeyer–Villiger oxidation of various substituted 1-indanones is described. Three bacterial strains have been explored: an E. coli TOP10 [pQR 239] constructed to overexpress the cyclohexanone monoxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871, an E. coli TOP10 [hapE] strain recently constructed to overexpress 4-hydroxyacetophenone monoxygenase (HAPMO) of Pseudomonas fluorescens ACB and the wild type Pseudomonas sp. NCIMB 9872 strain known to metabolise cyclopentanone. This last strain oxidised some of the proposed substrates, leading to the corresponding lactones with good to excellent yields depending on the aromatic ring substituents.     

Biocatalyst assessment of recombinant whole-cells expressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5

An Escherichia coli-based expression system for the Baeyer-Villiger monooxygenase (BVMO) from Xanthobacter sp. ZL5 was screened for whole-cell-mediated biotransformations. Biooxidation studies included kinetic resolutions and regiodivergent conversions of structurally diverse cycloketones. An extended phylogenetic analysis of the BVMOs currently available as recombinant systems with experimentally determined Baeyer-Villigerase activity showed that the enzyme originating from Xanthobacter sp. ZL5 clusters together with the sequences of bacterial CHMO-type BVMOs. The regio- and enantiopreferences experimentally observed for this enzyme are clearly similar to the biocatalytic performance of cyclohexanone monooxygenase from Acinetobacter as prototype for this group of BVMOs and support our previously reported family grouping.     

Modular and scalable biocatalytic tools for practical safety, health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.     

The structure of the neuraminic acid-containing capsular polysaccharide of Escherichia coli serotype K9

The acidic capsular polysaccharide isolated from Escherichia coli O9:K9:H12 was investigated by using n.m.r. spectroscopy, methylation analysis, periodate oxidation, and bacteriophage-borne enzyme degradation. The polysaccharide, the structure of which is shown below, is the third E. coli capsular polysaccharide reported to contain neuraminic acid, the others being the K1 and K92 polysaccharides, and it is the first in the E. coli series shown to contain a 4-linked neuraminic acid unit.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5′-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3′ end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Synthesis and antibacterial activity of pyranmycin derivatives with N-1 and O-6 modifications

Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1 positions. The revealed SAR results demonstrated that the antibacterial activity of pyranmycin can be modulated by different acylic substituents at O-6. Among these results, the 6-O-aminoethyl derivative, JT050, showed effective activity against resistant strain Escherichia coli (pTZ19U-3) and E. coli (pSF815), which provides insight into further structural modifications.     

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from -threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using -glutamate as the amino donor.     

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.     

基于转录物组学的大肠杆菌血红素过氧化物酶EfeB的生理功能解析

大肠杆菌血红素过氧化物酶EfeB属于染料脱色过氧化物酶超家族。该家族的酶类一般具有良好的合成染料降解能力,但是其在生物体内的功能尚不清楚。为了深入研究EfeB的生理功能,本文通过同源重组的方法构建了efeB敲除菌株Eco△efeB,比较了亲本菌株E.coli BL21和Eco△efeB在基因组转录水平上、不同条件下的细胞生长以及对铁离子应答上的差异。结果表明:efeB基因的缺失引起了菌体1 765个基因的差异表达,这些基因主要与菌体的细胞代谢途径、细胞膜合成和鞭毛运动有关;在pH为7.0时,BL21和EcoΔefeB的生长无显著差异,但在pH4.5时BL21的生长明显优于EcoΔefeB,efeB基因的功能性表达可以支持大肠杆菌在低pH下的生存;当培养环境中有Fe²⁺存在时,efeB显著上调。以上结果为EfeB生理功能的认知和利用提供了一定的理论依据。     

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).     

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.     

Comparison of cyclohexanone monooxygenase as an isolated enzyme and whole cell biocatalyst for the enantioselective oxidation of 1,3-dithiane

Both whole cells of recombinant Escherichia coli TOP10, overexpressing cyclohexanone monooxygenase (CHMO) and isolated cyclohexanone monooxygenase, were used to carry out the enantioselective oxidation of 1,3-dithiane (1) to (R)-1,3-dithiane-1-oxide (2). The two biocatalysts were evaluated under various experimental conditions (e.g., shaken flask or bioreactor; non-bound or resin-adsorbed substrate; different substrate concentrations) in terms of volumetric productivity and enantioselectivity. While productivity was similar in the two cases (up to 0.58 g L⁻¹ h⁻¹), the optical purity of the product was much higher with the isolated enzyme (up to 98% e.e.) than with the whole cell biocatalyst (up to 85% e.e.).     

Development of cloning vehicles from the Streptomyces plasmid pFJ103

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies     

Cloning of a cDNA that encodes an invertebrate glutamate receptor subunit.

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terininal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.     

Effect of the uvrD3 mutation on ultraviolet radiation-induced DNA-repair replication in Escherichia coli K12

Ultraviolet-radiation-induced DNA-repair replication was measured in wild-type, polA1, uvrD3, and uvrD3 strains of Escherichia coli K12. A large stimulation of repair replication was observed in the uvrD3 strain, compaired to the wild-type and polA1 strains. This enhanced repair replication was reduced in the polA1 uvrD3 strain. Therefore, a uvrD3 mutation appears to affect the amount of repair replication performed by DNA polymerase I. In the polA1 strain, there also appears to be an effect of the uvrD3 mutation on the amount of repair replication performed by DNA polymerase III (and/or II). The enhanced repair replication observed for the uvrD3 strains appears to be in response to the enhanced DNA degradation observed for these strains.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.