De Novo Biosynthesis of Anisyl Alcohol and Anisyl Acetate in Engineered Escherichia coli.

Anisyl alcohol and its ester anisyl acetate are both important fragrance compounds and have a wide range of applications in the cosmetics, perfumery, and food industries. The currently commercially available anisyl alcohol and anisyl acetate are based on chemical synthesis. However, consumers increasingly prefer natural fragrance compounds. Therefore, it is of great significance to construct microbial cell factories to produce anisyl alcohol and anisyl acetate. In this study, we first established a biosynthetic pathway in engineered Escherichia coli MG1655 for the production of anisyl alcohol from simple carbon sources. We further increased the anisyl alcohol production to 355 mg/L by the increasing availability of erythrose-4-phosphate and phosphoenolpyruvate. Finally, we further demonstrated the production of anisyl acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent acetylation of anisyl alcohol to produce anisyl acetate. To our knowledge, this is the first report on the biosynthesis of anisyl alcohol and anisyl acetate directly from a renewable carbon source.

De novo biosynthesis of N-acetyltyramine in engineered Escherichia coli.

N-acetyltyramine as a tyramine alkaloid has drawn great attention because of its excellent anti-free radical, antithrombotic, and antitumour activity. Therefore, it is an attractive compound. In this study, we reported for the first time the construction a synthetic pathway of N-acetyltyramine in engineered Escherichia coli. First, the tyrosine decarboxylase tdc gene and arylalkylamine N-acyltransferase aanat gene were introduced into E. coli to generate a recombinant N-acetyltyramine producer with L-tyrosine as substrate. Subsequently, overexpressing aroGfbr and TyrAfbr enhance the availability of L-tyrosine to achieve de novo biosynthesis of N-acetyltyramine from glucose. Finally, overexpressing the transketolase I tktA and phosphoenolpyruvate synthase ppsA genes improved the N-acetyltyramine production to 854 mg/L.

De Novo Biosynthesis of Methyl Cinnamate in Engineered Escherichia coli.

Methyl cinnamate with a fruity balsamic odor is an important fragrance ingredient in perfumes and cosmetics. Chemical processes are currently the only means of producing methyl cinnamate. But consumers prefer natural flavors. Therefore, it is necessary to design and develop microbial cell factories for the production of methyl cinnamate. In this study, we established for the first time a biosynthetic pathway in engineered Escherichia coli for production of methyl cinnamate from glucose. We further increased the methyl cinnamate production to 302 mg/L by increasing the availability of the metabolic precursors. Finally, the titer was increased to 458 mg/L in a two-phase culture system.

De novo biosynthesis of τ-cadinol in engineered Escherichia coli.

τ-Cadinol is a sesquiterpene that is widely used in perfume, fine chemicals and medicines industry. In this study, we established a biosynthetic pathway for the first time in engineered Escherichia coli for production of τ-cadinol from simple carbon sources. Subsequently, we further improved the τ-cadinol production to 35.9 ± 4.3 mg/L by optimizing biosynthetic pathway and overproduction of rate-limiting enzyme IdI. Finally, the titer was increased to 133.5 ± 11.2 mg/L with a two-phase organic overlay-culture medium system. This study shows an efficient method for the biosynthesis of τ-cadinol in E. coli with the heterologous hybrid MVA pathway.

Accumulation of Astaxanthin Was Improved by the Nonmotile Cells of Haematococcus pluvialis.

The current commercial production of natural astaxanthin is mainly carried out using Haematococcus pluvialis vegetative cells in the "two-stage" batch mode. The motile vegetative cells are more sensitive to stress than nonmotile vegetative cells, thereby affecting the overall astaxanthin productivity in H. pluvialis cultures. In this study, we compared the differences between motile cells and nonmotile cells in astaxanthin productivity, morphological changes, the mortality rate, and the diameter of the formed cysts. The experimental design was achieved by two different types H. pluvialis cell under continuous light of 80 µmol photons m-2 s-1 for a 9-day induction period. The highest astaxanthin concentration of 48.42 ± 3.13 mg L-1 was obtained in the nonmotile cell cultures with the highest the productivity of 5.04 ± 0.15 mg L-1 day-1, which was significantly higher than that in the motile cell cultures. The microscopic examination of cell morphological showed a large number of photooxidative damaged cells occurring in the motile cell cultures, resulting in higher cell mortality rate (22.2 ± 3.97%) than nonmotile cell cultures (9.6 ± 0.63%). In addition, the analysis results of cell diameter statistics indicated that nonmotile cells were more conducive to the formation of large astaxanthin-rich cysts than motile cells. In conclusion, the works presented here suggest that the accumulation of astaxanthin was significantly improved by nonmotile cells of H. pluvialis, which provided a possibility of optimizing the existing H. pluvialis cultivation strategy for the industrial production.

Differences between Motile and Nonmotile Cells of Haematococcus pluvialis in the Production of Astaxanthin at Different Light Intensities.

Haematococcus pluvialis, as the best natural resource of astaxanthin, is widely used in nutraceuticals, aquaculture, and cosmetic industries. The purpose of this work was to compare the differences in astaxanthin accumulation between motile and nonmotile cells of H. pluvialis and to determine the relationship between the two cells and astaxanthin production. The experiment design was achieved by two different types of H. pluvialis cell and three different light intensities for an eight day induction period. The astaxanthin concentrations in nonmotile cell cultures were significantly increased compared to motile cell cultures. The increase of astaxanthin was closely associated with the enlargement of cell size, and the nonmotile cells were more conducive to the formation of large astaxanthin-rich cysts than motile cells. The cyst enlargement and astaxanthin accumulation of H. pluvialis were both affected by light intensity, and a general trend was that the higher the light intensity, the larger the cysts formed, and the larger the quantity of astaxanthin accumulated. In addition, the relatively low cell mortality rate in the nonmotile cell cultures indicated that the nonmotile cells have a stronger tolerance to photooxidative stress. We suggest that applying nonmotile cells as the major cell type of H. pluvialis to the induction period may help to enhance the content of astaxanthin and the stability of astaxanthin production.

[Study on immuno-effect with GRA4 or SAG2 gene recombinant BCG vaccine of Toxoplasma gondii].

OBJECTIVE: To compare the immuno-protection induced by the recombinant BCG vaccine of Toxoplasma gondii GRA4 gene (rBCG-GRA4) and SAG2 gene (rBCG-SAG2) in BALB/c mice. METHODS: 108 SPF BALB/c mice were divided into 6 groups: PBS, BCG, rBCG, rBCG-GRA4, rBCG-SAG2 and rBCG-GRA4+SAG2, each with 18 mice. Each mouse was injected by 100 microl corresponding materials for 2 times. Blood was taken from tail vein before inoculation. 4,6 and 8 weeks after inoculation, spleen was moved and blood was taken from orbit vein of 3 mice from each group for the detection of cytokines, IgG and IgM antibodies, T lymphocyte subgroups and transformation efficiency. 3 weeks after the last inoculation, 9 mice from each group were challenged intraperitoneally with 50 tachyzoites of T. gondii RH strain and their survival time was observed. RESULTS: rBCG vaccine of T. gondii induced immune response. The value of CD3+ CD4+/CD3+CD8+ of group BCG-GRA4+SAG2 was the highest (14.06+/-1.17) in the 4th week; the IgG titer in the BCG-GRA4+SAG2 group was the highest (0.18+/-0.02) in the 6th week and the IgM titer in the BCG-SAG2 group was the highest (0.82+/-0.05) in the 8th week. The average survival time of the mice in BCG-SAG2 group was about 8.61 days after challenged with tachyzoites, and that of the PBS control group, 7.33 days. The average survival time in the 3 immunized groups was one day longer than that of the control. CONCLUSION: The rBCG vaccine of T. gondii shows certain immuno-protection in mice.

The immunity induced by recombinant spike proteins of SARS coronavirus in Balb/c mice.

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

Toxoplasma gondii: expression pattern and detection of infection using full-length recombinant P35 antigen.

A complete P35 surface antigen of Toxoplasma gondii was sequenced (GenBank ). Immunoblot found that it reacted specially with T. gondii acute infected sera, and the recombinant P35 signal was specific for P35 antigen. The P35-GST protein was used as antigen to detect 125 sera samples by double-sandwich ELISA. P35-IgM positive rate in a chronic infected group, a persistent IgM positive chronic group, a recently seroconvered group and an acute infected group were 4% (1 out of 25), 16% (4 out of 25), 88% (22 out of 25), and 100% (25 out of 25), respectively. The sensitivity and specificity of the recombinant full-length P35 antigen were 100 and 96%, respectively. The detailed expression patterns of P35 antigen were studied in 36 IgM and IgG positive sequential samples from 10 recently seroconvered patients. Results showed that the P35-IgM positive rate decreased as the time after the first seroconversion increased. P35-IgM positive samples in the first, second, third, fourth, and fifth month after the first seroconversion test were 90, 78, 57, 50, and 33%, respectively. P35-IgG positive samples in the first, second, third, fourth, fifth, sixth, and seventh month after the first seroconversion test were 70, 100, 100, 100, 67, 100, and 100%, respectively. All samples were P35-IgM negative after the fifth month, and P35-IgG negative after the seventh month from seroconversion. P35-IgM existed the shortest time and was a more specific marker for T. gondii acute infection than P35-IgG, IgM, and IgG to whole tachyzoites antigens.

[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].

OBJECTIVE: To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. METHODS: The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. RESULTS: The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. CONCLUSION: The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.

[Expression and purification of Toxoplasma gondii GRA4 gene in prokaryotic system].

OBJECTIVE: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity. METHODS: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. RESULTS: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. CONCLUSION: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

[High efficiency expression and antigenicity analysis of the SAG2 gene from Toxoplasma gondii RH strain].

OBJECTIVE: To make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E. coli and study the antigenicity of the expressed product. METHODS: The SAG2 gene fragment of T. gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E. coli DH5alpha. After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E. coli DH5alpha. The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E. coli BL21 (DE3) and induced for expression. The expressed product was studied by SDS-PAGE and Western blot. RESULTS: 502 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about Mr 19,000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSION: The plasmid pET23a-SAG2 was constructed and a high efficiency expression of the SAG2 gene from T. gondii RH strain was made. The expressed product shows a specific antigenicity.

Expression and purification of SARS coronavirus membrane protein.

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.