[Limiting metabolic steps in the utilization of D-xylose by recombinant Ralstonia eutropha W50-EAB].

OBJECTIVE: To further improve the efficiency of xylose fermentation by modifying the pentose phosphate pathway (PPP) and the aldehyde reductase gene h16_A3186 in Ralstonia eutropha W50-EAB. METHODS: The transketolase (tktA, cbbT2) and transaldolase (tal) gene were cloned from R. eutropha chromosome by PCR and inserted into expressing vector pBBR1MCS-3. The resulting recombinant plasmids were transformed into W50-EAB to generate W50-KAB, W50-CAB and W50-TAB, respectively. The aldehyde reductase gene h16_A3186 was shortened from 834 bp to 135 bp by in-frame deletion from strain W50-E in which the xylE gene coding for xylose transporter was chromosomally integrated to construct recombinant strain W50'-E. Then the xylAB gene coding for xylose isomerase and xylulokinase from Escherichia coli were expressed in W50'-E to generate recombinant strain W50'-EAB. Recombinant plasmid pWL1-TAL was transformed into W50'-EAB to construct the strain W50'-TAB. The fermentation characteristics of the engineered strains were investigated. RESULTS: The expression of tktA, cbbT2 and tal genes in R. eutropha W50-EAB was confirmed by enzyme assay. The deletion of h16_A3186 gene was confirmed by PCR analysis and enzyme assay. Amplification of transketolase activity in R. eutropha W50-EAB showed negative effect on cell growth and D-xylose consumption. The recombinant strain W50-TAB and W50'-EAB exhibited a faster growth than W50-EAB with the maximum specific growth rate of 0.039 h(-1) and 0.040 h(-1), respectively, when cultivated on 0.1 mol/L D-xylose. And the PHB accumulation of W50-TAB and W50'-EAB reached 16.2 ± 1.01% and 19.8 ± 1.05% on the basis of cell dry weight, respectively. Furthermore, recombinant strain W50'-TAB exhibited better fermentation performance with the maximum specific growth rate of 0.042 h(-1) and PHB content of 27.9 ± 0.47%, respectively. Meanwhile, the recombinant strains W50-TAB, W50'-EAB and W50'-TAB showed higher biomass and more PHB accumulation when using glucose (0.01 mol/L) and D-xylose (0.09 mol/L) mixed sugars as fermentative substrate. CONCLUSION: Overexpression of the tal gene resulted in incressed D-xylose consumption. Deficiency of the aldehyde reductase relieved inhibition to D-xylose metabolism. Combination of the two strategies contributed to a higher efficiency of D-xylose utilisation and more PHB accumulation of the engineered R. eutropha strain.

[Effect of pps and aroGfbr overexpression on L-tryptophan production in Corynebacterium pekinense].

OBJECTIVE: In order to redirect carbon flows into aromatic amino acids biosynthesis pathway and further improve the production of L-tryptophan in Corynebacterium pekinense PD-67, two schemes were implemented. First, the supply of phosphoenolpyruvate (PEP), one of precursors of L-tryptophan biosynthesis, was increased. Second, the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS), a key enzyme in the aromatic amino acids biosynthesis, was relieved and the activity of DS was increased. METHODS: The phosphoenolpyruvate synthase gene (pps) was cloned from C. pekinense PD-67 chromosome by PCR and inserted into expression vector to construct a recombinant plasmid pXPPS; the aroG gene encoding DS isozymes was cloned from Escherichia coli chromosome by PCR and the mutation of Leu175Asp was introduced by site-directed mutagenesis using sequence-overlap extension PCR. The mutated gene named as aroGfbr was cloned to expression vector to construct a recombinant plasmid pXA; and the recombinant plasmid pXAPS co-expressing pps and aroGfbr was constructed. The three recombinant plasmids were transformed into PD-67 to generate the engineering strains PD-67/pXPS, PD-67/pXA and PD-67/pXAPS, respectively. The fermentation characteristics of the three engineering strains were investigated. RESULTS: The expression of pps and aroGfbr was confirmed by enzyme activity assays. The deregulation of feedback inhibition of AroGfbr was confirmed by determining DS activity in the presence of three aromatic amino acids. The overexpression of pps and aroGfbr resulted in an increase of L-tryptophan biosynthesis by 12.1% and 26.8%, respectively, while the co-expression of two genes increased the production of L-tryptophan by 35.9% in the engineering strain PD-67/pXAPS. CONCLUSION: Both of the overexpressions of the pps gene and aroGfbr gene can increase L-tryptophan biosynthesis, while the production was further improved by the co-expression of the two genes.

[Engineering of a D-xylose metabolic pathway in eutropha W50].

OBJECTIVE: This study aimed to broaden the substrate spectrum of Ralstonia eutropha W50 to use D-xylose, which can produce poly-beta-hydroxybutyrates (PHB) at a high level. METHODS: The D-xylose transporter gene xylE from Escherichia coli K-12 W3110 was cloned by PCR technique and integrated into the R. eutropha W50 chromosome. The recombinant strain W50-E was obtained. The D-xylose catabolic genes xylAB from E. coli K-12 W3110 and the promotor of PHA synthase gene phaC1 from R. eutropha H16 were cloned into pBBR1MCS to construct a recombinant plasmid. The plasmid was transformed into R. eutropha W50 and W50-E to generate the recombinant strains W50-AB and W50-EAB respectively. The characteristics of D-xylose utilization by W50-AB and W50-EAB were investigated. RESULTS: The expression of xylA and xylB genes in R. eutropha W50 was confirmed by enzyme assay. The recombinant strain W50-AB could grow on 0.1 mol/L D-xylose with the maximum specific growth rate of 0.025 h(-1), but no growth and D-xylose consumption were observed when cultivated on 0.01 mol/L D-xylose. The recombinant strain W50-EAB exhibited a faster growth than W50-AB on 0.1 mol/L D-xylose, with the maximum specific growth rate of 0.035 h(-1). Furthermore, it exhibited a slow but defined growth and D-xylose consumption on 0.01 mol/L D-xylose. The PHB content assay showed that both recombinant strains accumulated a small amount of PHB, with a proportion of 15.07 +/- 1.01% and 15.07 +/- 1.64% on the basis of dry cell weight respectively, by using D-xylose (0.1 mol/L) as substrate. And their final D-xylose-PHB conversion rates were 0.0920 g x g(-1) and 0.0838 g x g(-1) respectively, which were much lower than their glucose-PHB conversion rates( > 0.22 g x g(-1)). However, the recombinant strains W50-AB and W50-EAB exhibited better fermentation performance and more PHB accumulation when using glucose(0.01 mol/L) and D-xylose (0.09 mol/L) mixed sugars as fermentative substrate. CONCLUSION: The recombinant strain W50-AB can metabolize D-xylose by the expression of xylAB genes, and the further expression of xylE gene is able to improve its D-xylose consumption rate. Meanwhile, the two recombinant strains can accumulate a small amount of PHB by using D-xylose as the sole carbon source.

[Engineering of an L-arabinose metabolic pathway in Ralstonia eutropha W50].

OBJECTIVE: To broaden the substrate spectrum including L-arabinose, Ralstonia eutropha W50, a mutant strain with high yield of poly-beta-hydroxybutyrate (PHB), was metabolically engineered by expressing the genes encoding L-arabinose catabolic enzymes and high-affinity L-arabinose transporter from Escherichia coli. METHODS: The promoter fragment of PHB synthase gene phaC1 (P(pha C1)) from R. eutropha H16 and the araBAD genes from E. coli W3110 were cloned by PCR and inserted into expression vector pBBR1 MCS. The resulting recombinant plasmid was transformed into W50 to generate W50-1. The araFGH gene from E. coli W3110 was introduced into W50-1 by plasmid system or homologous recombination, yielding W50-2 and W50-3 respectively. The fermentation characteristics of the three engineered strains were investigated. RESULTS: The flask fermentation experiments of the engineered strains show that W50-1 carrying the arabinose catabolic genes under the control of P(pha C1) could grow in the fermentation medium containing 0.1 mol/L arabinose as the sole carbon source, but could not utilize low concentration arabinose (0.01 mol/L). However, W50-2 and W50-3 containing the gene of high-affinity arabinose transporter were able to utilize low concentration arabinose. In the fermentation medium containing 0.1 mol/L arabinose, the biomass of W50-3 was 2.5 fold higher than that of W50-1, and the PHB accumulation amount of W50-3 accounted for 38.6% of the cell dry weight. CONCLUSION: R. eutropha W50 was able to metabolize L-arabinose by the expression of araBAD genes, and the simultaneous expression of araFGH genes could further improve its ability of L-arabinose utilization. By using L-arabinose as the sole carbon source, the recombinant strain W50-3 can accumulate a noticeable level of PHB.

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum.

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 µM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 µM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.

Phenylacetic acid catabolism and its transcriptional regulation in Corynebacterium glutamicum.

The industrially important organism Corynebacterium glutamicum has been characterized in recent years for its robust ability to assimilate aromatic compounds. In this study, C. glutamicum strain AS 1.542 was investigated for its ability to catabolize phenylacetic acid (PAA). The paa genes were identified; they are organized as a continuous paa gene cluster. The type strain of C. glutamicum, ATCC 13032, is not able to catabolize PAA, but the recombinant strain ATCC 13032/pEC-K18mob2::paa gained the ability to grow on PAA. The paaR gene, encoding a TetR family transcription regulator, was studied in detail. Disruption of paaR in strain AS 1.542 resulted in transcriptional increases of all paa genes. Transcription start sites and putative promoter regions were determined. An imperfect palindromic motif (5'-ACTNACCGNNCGNNCGGTNAGT-3'; 22 bp) was identified in the upstream regions of paa genes. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of PaaR to this motif, and phenylacetyl coenzyme A (PA-CoA) blocked binding. It was concluded that PaaR is the negative regulator of PAA degradation and that PA-CoA is the PaaR effector. In addition, GlxR binding sites were found, and binding to GlxR was confirmed. Therefore, PAA catabolism in C. glutamicum is regulated by the pathway-specific repressor PaaR, and also likely by the global transcription regulator GlxR. By comparative genomic analysis, we reconstructed orthologous PaaR regulons in 57 species, including species of Actinobacteria, Proteobacteria, and Flavobacteria, that carry PAA utilization genes and operate by conserved binding motifs, suggesting that PaaR-like regulation might commonly exist in these bacteria.

[Effect of aromatic amino acid transport gene knock-out on L-tryptophan accumulation in Corynebacterium pekinense PD-67].

OBJECTIVE: A Corynebacterium pekinense PD-67 mutant with aromatic amino acid transport system gene (aroP) in-frame deletion was constructed to decrease the uptake of L-tryptophan and reduce the intracellular pool of L-tryptophan, further to deregulate the feedback regulation of L-tryptophan and increase the extracellular accumulation. The effects of aroP knock-out as well as anthranilate synthetase (EC4. 1. 3. 27; AS) gene overexpression on L-tryptophan accumulation of the mutant were investigated. METHODS: The aroP gene was cloned from C. pekinense PD-67 chromosome and ligated to integration vector, and then deleted about 600bp fragment by restriction endonuclease digestion. The mutant C. pekinense PD-67-deltaaroP was screened by homologous recombination. The mutant phenotype can be reversed by complementation with aroP gene from the expression vector. AS gene was cloned and ligated to expression vector to construct a recombinant plasmid. The plasmid was transformed into PD-67deltaaroP to generate the engineering strain PD-67deltaaroP/pXAS. The fermentation characteristics of the mutant and the engineering strain were investigated. RESULTS: The aroP gene in-frame deletion was screened and confirmed by PCR analysis and the AS gene expression was confirmed by determination of enzyme activity. The aroP knock-out resulted in increase of L-tryptophan accumulation by 65% compared with that of the parent strain, while the expression of AS gene resulted in increase of L-tryptophan yield on cell mass by 25.6% in engineered strain. CONCLUSION: The aroP gene knock-out of the strain PD-67 improved L-tryptophan accumulation. The expression of AS gene could further improve L-tryptophan yield on cell mass in engineered strain.

The ncgl1108 (PheP (Cg)) gene encodes a new L-Phe transporter in Corynebacterium glutamicum.

Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP(Cg), was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP (Cg), which is located upstream of the genetic cluster (ncgl1110 ~ ncgl1113) for resorcinol catabolism, was identified as a new L-Phe specific transporter from C. glutamicum RES167. The disruption of pheP (Cg) resulted in RES167∆ncgl1108, and this mutant showed decreased growth on L-Phe (as nitrogen source) but not on L-Tyr or L-Trp. Uptake assays with unlabeled and (14)C-labeled L-Phe and L-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in L-Phe uptake than RES167. Expression of pheP (Cg) in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 restored their ability to uptake for L-Phe and growth on L-Phe. The uptake of L-Phe was not inhibited by nine amino acids but by L-Tyr. The K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 for L-Phe were determined to be 10.4 ± 1.5 µM and 1.2 ± 0.1 nmol min(-1) (mg DW)(-1), respectively, which are different from K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg)) for L-Phe [4.0 ± 0.4 µM and 0.6 ± 0.1 nmol min(-1) (mg DW)(-1)]. In conclusion, this PheP(Cg) is a new L-Phe transporter in C. glutamicum.

[Effect of gamma-glutamyl kinase gene knock-out on metabolism in L-arginine-producing strain Corynebacterium crenatum 8-193].

OBJECTIVE: In order to optimize precursor supply for L-arginine biosynthesis, we constructed a Corynebacterium crenatum 8-193 mutant with gamma-glutamyl kinase gene (proB) in-frame deletion. The effects of proB knock-out on physiological characteristics of the mutant were investigated. METHODS: The upstream and downstream fragments of proB were cloned from C. crenatum 8-193 chromosome and ligated to integration vector. The mutant C. crenatum 8-193-deltaproB was obtained by homologous recombination. The mutant phenotype can be reversed by complementation with proB gene from the expression vector. The physiological characteristics of the mutant were investigated by measurement of the activities of phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PYC). RESULTS: The proB gene in-frame deletion was screened and confirmed by PCR, gamma-glutamyl kinase determination and complementation. The mutant lost the ability of growth on minimal medium without proline addition. The proB knock-out mutant resulted a decrease of cell mass by 9.6% and an increase of L-arginine accumulation by 13.6% compared with that of the parent strain. The analysis of by-products of fermentation broth showed that the concentrations of glutamate-related and aspartate-related amino acids increased, and the concentrations of alpha-ketoglutaric acid, PEP and succinic acid decreased. The specific activities of PEPCx and PYC increased in 8-193-deltaproB. CONCLUSION: The proB gene knock-out of the strain 8-193 blocked branch catabolism of L-glutamate and improved efficiency of the glucose utilization and L-arginine accumulation.

[Corynebacterium pekinense transketolase: gene cloning, sequence analysis and expression].

OBJECTIVE: Transketolase (EC 2. 2. 1. 1; TK) is the key enzyme in non-oxidative phosphate pentose pathway. We cloned tkt gene from Corynebacterium pekinense AS 1.299 and its mutant PD-67 in order to investigate the effect of gene expression on physiological characteristics of C. pekinense. PD-67. METHODS: According to the homology between Corynebacterium glutamicum ATCC13032 and C. pekinense, we designed a pair of PCR primers to clone the tkt gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant tkt gene was expressed in C. pekinense PD-67 by subcloning the PCR fragment into plasmid pAK6. The physiological characteristics of the recombinant C. pekinense PD-67 was investigated by fermentation. RESULTS: Analysis of PCR fragments reveals that, besides the regulatory sequence, they contain the whole structure of tkt gene. There is no base change all over the structure genes and regulatory sequences between C. pekinense AS1. 299 and PD-67. Comparing with Corynebacterium glutamicum ATCC 13032, there exist 5 amino acids change in amino acid sequence. Four of them were located in the motifs involved in thiamine pyrophosphate binding sites. The tkt gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of TK in C. pekinense PD-67 (pTK3) is two times over that of the control strain C. pekinense PD-67 (pAK6). The recombinant C. pekinense PD-67 exhibits higher cell mass and accumulation of more tryptophan. CONCLUSION: The moderate amplification of TK activity resulted in increase of L-tryptophan production without affecting the cell growth.

[Effect of phosphoenolpyruvate carboxylase gene knock-out on metabolism in Corynebacterium pekinense PD-67].

OBJECTIVE: In order to optimize precursor supply for L-tryptophan biosynthesis, a Corynebacterium pekinense PD-67 mutant with phosphoenolpyruvate carboxylase gene (ppc) in-frame deletion was constructed. The effect of ppc knock-out on physiological characteristics of the mutant was investigated. METHODS: The upstream and downstream fragments of ppc were cloned from C. pekinense PD-67 chromosome and ligated to integration vector. The mutant C. pekinense PD-67-deltappc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and measurement of pyruvate carboxylase (PCx) and pyruvate kinase (PK). RESULTS: The mutant with ppc gene in-frame deletion was screened and confirmed by PCR check and phosphoenolpyruvate carboxylase determination. The mutant exhibited slow growth and less cell mass, 80% as much as the parent strain. The ppc knock-out resulted in decrease of L-tryptophan accumulation and overproduction of pyruvate-related amino acids, which accompanied by increase of PK activity and the decrease of PCx activity, in C. pekinense PD-67. CONCLUSION: The knock-out of ppc gene affected the metabolism of the strain to some extent. Only by blocking the anaplerotic pathway PEPCx participated was insufficient to increase the accumulation of L-tryptophan in C. pekinense PD-67.

[Cloning and analysis of promoter-active fragments from Corynebacterium glutamicum 10147].

OBJECTIVE: To clone promoter-active fragments from Corynebacterium glutamicum for further construction of expression vectors. METHODS: Random Sau3A I digested fragments of C. glutamicum 10147 chromosome were shot-gun cloned into the promoter-probe vector pAKC6 and promoter activity of the inserted fragments was selected by chloramphenicol resistance of transformed C. glutamicum cells. RESULTS: Thirty promoter-carrying fragments were isolated. Three C. glutamicum clones harboring pAKC6 with promoter fragments displayed chloramphenicol acetyltransferase (CAT) activity of more than 24 U/mg. The fragment F57 led to the highest CAT activity of 32.50 U/mg, even more than that produced by the promoter Ptrc, 26.33 U/mg. CONCLUSION: The strength of promoter on fragments F21, F54 and F57 is as strong as promoter Ptrc in C. glutamicum. These fragments can be used to construct expression vector.

[Cloning, expression and sequence analysis of DS I gene in Corynebacterium pekinense AS1.299 and PD-67].

OBJECTIVE: 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54;DS) is the key enzyme in tryptophan synthesis pathway. Cloning DS I gene from Corynebacterium pekinense and expression of DS I gene might facilitate testing the existence and function of DS I in Corynebacterium pekinense. METHODS: According to the homology between Corynebacterium glutamicum ATCC13032 and Corynebacterium pekinense, we designed a pair of PCR primers to clone the DS I gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant DS I gene was expressed in C. pekinense PD-67 by subcloning the the PCR fragment into plasmid pAK6. RESULTS: Analysis of PCR fragments revealed that they contained the whole DS I gene. There was no base change all over the structure genes and regulatory sequences between C. pekinense AS1.299 and PD-67. An internal promoter was found in the upstream of the DS I gene from C. pekinense and it functioned in E. coli 3257. The DS I gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of DS I in C. pekinense PD-67 (pAD1) was much higher than that of the control strain C. pekinense PD-67(pAK6). CONCLUSION: This is the first report that DS I gene existed in Corynebaterium Pekinense, The amplification of the specific activity of DS I is expected to increase L-tryptophan accumulation of C. pekinense PD-67.

[Construction of Corynebacterium glutamicum/E. coli shuttle promoter-probe vector].

Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin- and ampicillin-resistance-encoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum . This vector carried the promoterless chloramphenicol acetyltranstersae (CAT) gene as a reporter downstream from useful cloning site. For testing this promoter-probe vector, C. glutamicum genomic DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique Bgl II. Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed. After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.

[Cloning, sequence analysis and expression of anthranilate synthetase gene in Corynebacterium pekinense].

Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67. The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.

[Cloning, sequence analysis and expression of alanine racemase gene in Pseudomonas putida].

Two distinct alanine racemase genes from Pseudomonas putida 200 were cloned and sequenced. DadX encodes a peptide of 357 amino acids with a calculated molecular weight of 38.82kDa. The putative product of alr gene is a peptide of 409 amino acids with molecular weight of 44.182kDa. A homology comparison revealed identities of 96.64%, 71.99%, 44.88% and 47.37% of the DadX alanine racemase to those from P. putida KT2440, Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli, respectively. The amino acids sequence deduced from alr gene showed the homologies of 94.38%, 22.89%, 25.72% and 26.44% to those from the microorganisms above, respectively. Two motifs believed essential to the enzyme activity are found both in DadX and Alr, such as pyridoxal-5'-phosphate binding site. Both dadX and alr were expressed in E. coli TG1. Neither alanine racemase activity or serine racemase activity was detected in the host strain. Only alanine racemase activity was found in E. coli TG1/pCTD. But both E. coli TG1/pCTA and TG1/pCBA exhibit activity toward L-alanine and L-serine. Transcription of alr gene in E. coli is independent from extraneous promoter, a result confirmed by the significant enzyme activity observed in the E. coli TG1/pCBA, which indicates the presence of a possible promoter upstream the structure gene.

[Cloning, sequence analysis and expression of N-acetylglutamate kinase gene in Corynebacterium crenatum].

N-Acetylglutamate kinase (EC 2.7.2.8;NAGK) genes from wild-type Corynebacterium crenatum AS 1.542 and a L-arginine-producing mutant C. crenatum 971.1 were cloned and sequenced. Analysis of argB sequences revealed that only one ORF existed, which used ATG as the initiation codon and coded a peptide of 317 amino acids with a calculated molecular weight of 33.6kDa. Only one nucleotide difference was found in the structure gene and the difference did not cause a change of amino acid by comparison of the gene sequences between the wild type C. crenatum AS 1.542 and the mutant 971.1. The ORF sequence of argB from C. crenatum AS 1.542 showed homologies of 99.89%, 76.62%, 37.94% to those from Corynebacterium glutamicum ATCC 13032, Corynebacterium efficient YS-314 and Escherichia coli k12. And the amino acid sequence deduced from ORF displayed homologies of 100%, 78.55%, 25.25% to those from microorganisms above, respectively. An internal promoter was found in the upstream of the argB gene from C. crenatum. The argB gene from C. crenatum AS 1.542 was expressed both in C. crenatum AS 1.542 and 971.1. The NAGK activity of transformed C. crenatum AS 1.542 was greatly increased by the induction of IPTG. The NAGK activity of transformed C. crenatum 971.1 was almost twice as much as that of C. crenatum 971.1 under the same induction. The amplification of the NAGK activity yielded 25% increase of L-arginine production in C. crenatum 971.1.

[Expression of feedback-resistant aspartate kinase gene in Corynebacterium crenatum].

The AEC-resistant aspartate kinase gene from C. crenatum CD945 was cloned into vector pJC1. Its expression was investigated both in the wild type C. crenatum AS1.542 and its mutant C. crenatum CD945. The result showed that C. crenatum AS1.542 harboring AK(fbr) gene could grow on the defined medium with the co-existence of 12 mg/mL both of AEC and L-threonine respectively. Overexpression of AK(fbr) gene in C. crenatum CD945 results in a 4-fold increase of specific enzyme activity than the parental strain. The amplification of the activity of aspartate kinase yields 22% increase of L-lysine production and 23% increase of L-lysine productivity without affecting the growth rate.

Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose.

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41-29.1 mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0 mol% in the biopolymer, indicating promising potentials of commercialization in the future.

[Cloming, sequence analysis of imidase gene from Alcaligenes eatrophus and its expression in E. coli].

A hydantoin-cleaving microorganism 112R4 is screened and identified to be Alcaligenes eutrophus. The resting cell of Alcaligenes eutrophus 112R4 can catalyze the hydrolysis of hydantoin, dihydropyrimidine and succinimide effectively, but not function to 5-monosubstituted hydantoins or 5,5'-disubstituted hydantoins. The microorganism can utilize succinimide as a sole carbon source and nitrogen source, which indicates the presence of a complete transformation pathway of succinimide, and a hydantoin-cleaving enzyme, imidase, is suggested to be contained in this metabolic pathway. A 6 kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R4 is shown to be correlative with the transformation of succinimide. A 2 kb DNA fragment containing the gene of imidase is subcloned and sequenced. Deletion analysis verifies that one open reading frame of 876 nucleotides, which encodes a peptide of 291 amino acids, with a calculated molecular weight of 33688, is responsible for the encoding of imidase. This is the first report of the nucleotide and amino acid sequences of imidase (GenBank accession number: AF373287). A homology search performed in protein database reveals an identity of 14% with polysaccharide deacetylase conserved domain, an identity of 60% with N-terminal 20 amino acids of Blastobacter sp. A17p-4, but no apparent similarity with all known cyclic-amide-cleaving enzymes. This result suggested that the imidase should be classified as a new member of cyclic amidases. Under the control of lac promoter and IPTG induction, the imidase activity of transformed E. coli reached 3200 U/L, which is about 7-fold higher than that of gene donor strain.