ngsheng Qi

Cyclodextrin glucanotransferase: from gene to applications

Cyclodextrin glucanotransferase (CGTase) is an important industrial enzyme which is used to produce cyclodextrins. CGTase genes from more than 30 bacteria have been isolated and several of the enzymes have been identified and biochemically characterized. For a better understanding of the reaction mechanism and function of CGTase, the enzyme has been analyzed at gene level and protein level with regard to its structure and the similarity of different CGTase subgroups. The biological role of the enzyme is proposed based on the genetic and enzymatic analyses. Methods to enhance the production of active CGTase by bacteria are compared. The enzyme can be applied in biotechnology for the production of cyclodextrins and oligosaccharides with novel properties.

In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

Abstract For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography. In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg−1 and 0.035 U mg−1 protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg−1 when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a Ki of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 104 g mol−1, and which occurred independently of pre-existing PHA granules.

Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase

Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography-mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5.5 x 10(5) g mol(-1) and a polydispersity of 1.6. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C. crescentus genomic sequence. This putative C. crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB(-)4 and recombinant Escherichia coli JM109(pBHR69), which produced the beta-ketothiolase and acetoacetyl-CoA reductase from R. eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity.

Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli

In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70-80% pure PHA synthase, then dissolved and denatured by 6 M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni(2+)-nitrilotriacetate-agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8 m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% alpha-helix, 50% beta-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69 kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128 kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.

A novel-designed Escherichia coli for the production of various polyhydroxyalkanoates from inexpensive substrate mixture

To efficiently produce polyhydroxyalkanoates (PHAs) from substrate mixture containing various carbon sources, Escherichia coli phosphotransferase system (PTS) mutants were constructed, in which the carbon catabolite repression in the presence of glucose was released. When cultivated in the medium containing glucose and xylose, E. coli LR1010, harboring phaCRe and phaABRe genes from Ralstronia eutropha, could simultaneously consume glucose and xylose and accumulate short-chain-length PHA, whereas E. coli LR1120 and LR1110, harboring phaC1 gene from Pseudomonas aeruginosa, could accumulate medium-chain-length PHA. At the same time, LR1110 could simultaneously utilize the mixture of glucose and fatty acids. Semiquantitative reverse transcription polymerase chain reaction analysis indicated that disrupting phosphoenolpyruvate: sugar PTS in this strain released the repression on fad genes.

Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12

δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B12, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B12, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B12 with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized.

Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production

In the application of engineered Escherichia coli in industrial polyhydroxybutyrate production process, one of the major concerns is the induction of the metabolic pathway. In this study, we developed a stress-induced system by which the PHB biosynthesis pathways can be induced under stress conditions. Fermentation results showed that recombinant E. coli DH5α (pQKZ103) harboring this system was able to accumulate polyhydroxybutyrate up to 85.8% of cell dry weight in minimal glucose medium without adding any inducer. Growth experiment with GFP as a reporter indicated that the induction of this system happened at the late exponential phase and was sensitive to stressed environment. This system can also be applied in many other biotechnological processes.

A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

BackgroundFor metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. In this study, we developed a strategy for obtaining gene overexpression by engineering promoter clusters consisted of multiple core-tac- promoters (MCPtac s) in tandem.ResultsThrough a uniquely designed in vitro assembling process, a series of promoter clusters were constructed. The transcription strength of these promoter clusters showed a stepwise enhancement with the increase of tandem repeats number until it reached the critical value of five. Application of the MCPtac s promoter clusters in polyhydroxybutyrate (PHB) production proved that it was efficient. Integration of the phaCAB genes with the 5CPtac s promoter cluster resulted in an engineered E.coli that can accumulate 23.7% PHB of the cell dry weight in batch cultivation.ConclusionsThe transcription strength of the MCPtac s promoter cluster can be greatly improved by increasing the tandem repeats number of the core-tac-promoter. By integrating the desired gene together with the MCPtac s promoter cluster into the chromosome of E. coli, we can achieve high and stale overexpression with only a small size. This strategy has an application potential in many fields and can be extended to other bacteria.

Production in Escherichia coli of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources

ABSTRACT The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose.

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA(M) from S. arizona on ALA production (2052mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.