• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1924-1932
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• 2016

Mycophenolic acid (MPA) is an antibiotic produced by Penicillium brevicompactum. MPA has antifungal, antineoplastic, and immunosuppressive functions, among others. ${\beta}-Hydroxy-{\beta}-methylglutaryl-CoA$ (HMG-CoA) lyase is a key enzyme in the bypass metabolic pathway. The inhibitory activity of HMG-CoA lyase increases the MPA biosynthetic flux by reducing the generation of by-products. In this study, we cloned the P. brevicompactum HMG-CoA lyase gene using the thermal asymmetric interlaced polymerase chain reaction and gene walking technology. Agrobacterium tumefaciens-mediated transformation (ATMT) was used to insert a mutated HMG-CoA lyase gene into P. brevicompactum. Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening, PCR, Southern blot analysis, and enzyme content assay. The maximum MPA production by transformants was 2.94 g/l. This was 71% higher than wild-type ATCC 16024. Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P. brevicompactum.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04a
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• pp.69-75
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• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus It is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04b
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• pp.69-75
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• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd/dwf3 were Shown to be blocked in $D^4$ reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bri1/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRI1 could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.139-144
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• 2002

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd /dwf3 were shown to be blocked in D$^4$reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bril/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRIl could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.88-94
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• 2008

Two sugar biosynthetic cassette plasm ids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003-OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ $Na^+$] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12-membered ring aglycon (10-deoxymethynolide, 1) and a 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.155-161
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• 2005

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, in Vitis vinifera cell cultures. Therefore, four cell line suspensions of Vitis vinifera L. var. Gamay Freaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of $2.73\;\pm\;0.15,\;1.45\;\pm\;0.04,\;0.7\;\pm\;0.024\;and\;0.27\;\pm\;0.04$CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and $84\%$ for V. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be $9.7\%$, ranging from 4 to $17\%$. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities to L-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.372-376
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• 2006

Ferulic acid is a phenolic compound that serves as a major biosynthetic precursor of vanillin in higher plants. We investigated the ability of the 3 commercial enzymes - Ultraflo L, Viscozyme L, and ${\alpha}-Amylase$ - to induce the release ferulic acid from the Ipomoea batatas L. (sweet potato) stem. The rate of release for ferulic acid was optimal when Ultraflo L (1.0%) was used compared with the other enzymes, whereas Viscozyme L was most effective for the release of vanillic acid and vanillin. Thus, these enzymes may be useful for the large-scale production of ferulic acid and other phenolic compounds from sweet potato stem.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.193-199
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• 1991

The effects of pH on the cell growth, the elaboration of pullulan, and the morphology of Aureobasidium pullulans IFO 4464 were examined. A. pulluans grew in yeast-like form at constant pH7.5 and in mycelial form at constant pH2.5. At the both pH conditions, the elaboration of pullulan was very low, about 6.0~6.5g/l. The mixture of yeast-like form and mycelial form of cells was found at the constant pH4.5, at which condition, the elaboration of pullulan was high, about 24.5g/l. The pH shift experimemts showed that the specific production rates of pullulan were 0.048($hr^{-1}$)for the mycelial form and 0.058($hr^{-1}$)for the yeast like form, which indicated that the yeast-like form has the similar, only slightly higher, biosynthetic activity of pullulan to the mycelial form at pH4.5 and the pH of culture broth is more important factor for the elaboration of pullulan than the morphology of A. pullulans.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.635-638
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• 2004

An ATP-binding-cassette (ABC) transporter gene in the cephabacin biosynthetic gene cluster of Lysobacter lactamgenus was characterized. The amplified orf10 (cpbJ) gene was subcloned into pET-28a(+) vector and expressed in E. coli BL21(DE3) strain by 0.5 mM IPTG at $30^{\circ}C$. The membrane fraction of recombinant E. coli cells was separated by ultracentrifugation, and solubilized using 2.5% octyl-$\beta$-D-glucoside. Using the solubilized membrane fraction, the artificial proteoliposomes were reconstituted and analyzed for the biological activity of CpbJ protein. Upon measuring ATPase activity, the proteoliposome made from recombinant E. coli membrane proteins showed slightly higher activity than that from host E. coli membrane proteins. In the measurement of membrane transport activity, the reconstituted proteoliposome of recombinant E. coli membrane proteins exhibited higher activity when both substrates of cephalosporin C and L-Ala-L-Ser were applied, compared to the case of cephalosporin C or L-Ala-L-Ser only. It implies that the CpbJ protein is an ABC transporter secreting cephabacin antibiotics synthesized in cytoplasm.