Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline

A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.     

Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.

Proline 3-hydroxylase was purified from Streptomyces sp. strain TH1, and its structural gene was cloned. The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H. Mori, T. Shibasaki, Y. Uosaki, K. Ochiai, and A. Ozaki, Appl. Environ. Microbiol, 62:1903-1907, 1996). The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively. The K(m) values were 0.56 and 0.11 mM for L-proline and 2-oxoglutarate, respectively. The Kcat value of hydroxylation was 3.2 s-1. Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database. A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence. With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR. A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe. The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158. No sequence homolog was found in EMBL, GenBank, and DDBJ databases. ORF 3 was expressed in Escherichia coli DH1. Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp. strain TH1. It was concluded that the ORF 3 encodes functional proline 3-hydroxylase.     

脯氨酸-4-羟化酶在大肠杆菌中的密码子优化表达及对反式-4-羟脯氨酸生物合成的作用

为了使脯氨酸-4-羟化酶基因在重组大肠杆菌中得到高表达,通过调整大肠杆菌密码子偏好性以及mRNA二级结构,使得脯氨酸-4-羟化酶基因得到优化。将优化后的脯氨酸-4-羟化酶基因插入含有色氨酸串联启动子的p UC19质粒,构建重组质粒p UC19-ptrp2-Hyp,并导入大肠杆菌BL21(DE3)中。在摇瓶水平,重组菌以L-脯氨酸为底物发酵8 h,可积累(0.492±0.034)g/L的反式-4-羟脯氨酸。在发酵罐水平,通过补料分批发酵来提高反式-4-羟脯氨酸的产量,当补糖速率为18 g/h时,反式-4-羟脯氨酸的产量高达42.5 g/L,反式-4-羟脯氨酸产率为0.966 g/(L·h)。     

Cloning of an isozyme of proline 3-hydroxylase and its purification from recombinant Escherichia coli

An isozyme gene of proline 3-hydroxylase was cloned from Streptomyces sp. strain TH1 (Mori H, Shibasaki T, Yano K, Ozaki A, J. Bacteriol. 1997, 179: 5677–5683). The isozyme gene (870 bp) encodes a protein of molecular weight of 33,573. Both 3-hydroxylase genes are identical at 76.2% in amino acid sequence. His-motifs conserved in 2-oxoglutarate-dependent dioxygenases are conserved in both genes. Although characteristics of both recombinant 3-hydroxylases are similar, specific activities to l-proline and proline analogs are different.     

响应面法优化脯氨酸羟化酶转化反应工艺条件

通过优化脯氨酸羟化酶表达条件和转化反应条件,提高其转化反应效率。采用单因素法筛选脯氨酸羟化酶的最佳诱导温度、诱导剂浓度和转化反应条件,并采用响应面法预测影响转化反应各因素的最佳条件。结果显示,经过筛选和验证,蛋白表达最适诱导温度为28℃,IPTG浓度为0.2 mmol/L;转化反应最佳条件为:120 mmol/L 2-(N-吗啡啉)乙磺酸(pH 6.6)、1.5% Nonidet P-40、200 mmol/L L-脯氨酸、200 mmol/L α-酮戊二酸,6 mmol/L L-抗坏血酸、6.0 mmol/L 硫酸亚铁,最适反应温度为27℃,振荡速率为152 r/min。在最佳条件下,转化反应进行48 h后产物反式-4-羟基-L-脯氨酸的转化率可达100%,为合成反式-4-羟基-L-脯氨酸奠定了坚实的实验基础。     

Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.     

The aryldialkylphosphatase-encoding gene adpB from Nocardia sp. strain B-1: cloning, sequencing and expression in Escherichia coli.

Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-1, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene. Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-1 DNA for fragments containing adpB. A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined. Under control of the lac promoter of pUC19, adpB expression in E. coli cultures was approx. 15-fold higher than in strain B-1 under the native adpB promoter. Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels.     

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

The direct conversion of hydroxyproline to proline

Carrot slices convert added free trans-4-hydroxy-L-proline to L-proline by a path that brings about the loss of one half of the tritium from 5[³H]trans-4-hydroxy-L-proline. The first intermediate in this path is therefore most likely 4,5-dehydro-L-proline.     

一个新的茶树黄酮醇合酶基因的克隆和表达分析

Cs—COR113（GenBank登录号：FE942098）为一受冷诱导的茶树黄酮醇合酶基因的cDNA片段,采用RACE技术克隆了这一基因的全长cDNA,命名为CsFLS（GenBank登录号：FJ577509）。CsFLScDNA序列全长为1303bp,5＇-UTR和3’-UTR分别长91bp和175bp,包含一个编码336个氨基酸的完整开放阅读框。序列分析显示,CsFLS与烟草、矮牵牛、欧芹、拟南芥的黄酮醇合酶的同源性分别为74％、75％、75％和63％,含有2-酮戊二酸依赖性双加氧酶家族2个保守的基序,以及与黄酮醇合酶正确折叠有关的2个保守的甘氨酸残基。CsFLS的表达受低温诱导,但不受ABA诱导。     

Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2

Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.     

Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine.

We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA- and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.     

Novel proline hydroxylase activities in the pneumocandin-producing fungus Glarea lozoyensis responsible for the formation of trans 3- and trans 4-hydroxyproline

Novel proline 3-hydroxylase (P3H) and proline 4-hydroxylase (P4H) activities that convert free l-proline to both trans 3- and trans 4-hydroxy- l-proline were detected in protein extracts of the anamorphic fungus Glarea lozoyensis. The enzymatic conversion of l-proline to trans 3- and trans 4-hydroxy- l-proline was strictly dependent on alpha-ketoglutarate, ascorbate, and dithiothreitol. Ferrous iron was required for optimal P3H and P4H activity. These substrate and co-factor requirements indicate these enzyme activities belong to the class of 2-oxoglutarate-dependent dioxygenases. Both P3H and P4H were inhibited by zinc and other trace metals. The addition of proline to the fermentation medium resulted in an increase in the specific activity of P4H and a decrease in the synthesis of pneumocandin C(0). Additionally, the synthesis of trans 3- and trans 4-hydroxy- l-proline in vivo was affected differently by the proline concentration in the medium. This result suggested that two enzymes may be responsible for the regio- and stereospecific hydroxylation of l-proline.     

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.     

Pathways for lactose/galactose catabolism by Streptococcus salivarius

Using oligonucleotides derived from the N-terminal sequence of a triazine hydrolase from Nocardioides sp. strain C190, two DNA fragments containing trzN were cloned into Escherichia coli and their nucleotide sequences were determined. The 456-amino acid polypeptide predicted from the 1356-bp trzN ORF displayed significant similarity to triazine hydrolases from Pseudomonas and Rhodococcus isolates and belonged to the same amidohydrolase family. The trzN gene was flanked by two DNA sequences possessing 57 and 69% identity, respectively, at the protein level to Rhodococcus erythropolis sequences for a transposase and a transposase helper protein. Amplification primers specific to trzN were tested in soils inoculated with strain C190. The results demonstrated that the primers were specific to trzN, and could detect populations at 10(8) cfu g(-1) soil using 250-mg soil samples.     

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.     

Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1

Aims:  Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro-organism Enterobacter sp. FMB-1.
Methods and Results:  A cosmid clone containing c. 6 kbp region encoding an SIase gene was identified. The 5969-bp chromosomal DNA fragment covering the SIase ( esi ) gene in Enterobacter sp. FMB-1 was sequenced. Although this DNA fragment contained several open reading frames other than esi , only the presence of esi was sufficient to produce isomaltulose in recombinant Escherichia coli . The esi gene was expressed in E. coli , leading to the characterization of its SIase activity.
Conclusions:  The Enterobacter sp. FMB-1 esi gene was successfully cloned and expressed in E. coli . This gene encoded a functional SIase that produced isomaltulose from sucrose.
Significance and Impact of the Study:  This is the first molecular analysis of an SIase gene in an Enterobacter strain. The functional expression of the Enterobacter sp. FMB-1 esi gene in E. coli offers an alternative choice for the industrial production of isomaltulose.     

DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er

Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp. carotovora strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.