Hei Chan Lee

Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus

Enediyne antibiotics are known for their potent antitumor activities. One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non‐peptide chromophore (1a), and peptide apoprotein. The structurally diverse non‐peptide chromophore is responsible for its biological activity. One of its structural components, the naphthoic acid moiety (2,7‐dihydroxy‐5‐methyl‐1‐naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units. The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6‐methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin. Cultures of S. lividans TK24 and S. coelicolor YU105 containing plasmids with NNS were able to produce 2‐hydroxy‐5‐methyl‐1‐naphthoic acid (2a), a key intermediate of naphthoic acid moiety in NCS. In addition to 2a, a novel product, 2‐hydroxy‐5‐hydroxymethyl‐1‐naphthoic acid (2d) was isolated. This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds.

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.

Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2)

DNA-affinity capture assay (DACA) coupled with liquid chromatography–tandem mass spectrometry analysis was applied to identify the transcriptional regulators involved in the biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red) in Streptomyces coelicolor. The aim of this analysis was to determine the specific transcriptional regulators binding to the promoter region of actII-ORF4 or redD. The results of the DACA, as the first screening tool, identified eight proteins, including AdpA, as candidate regulators binding to those promoter regions. To show the direct physical relationship between the regulators and promoters, we purified four regulators over-expressed in soluble form in Escherichia coli and subjected these to an electrophoretic mobility shift assay (EMSA). The results of the EMSA appeared to be compatible with the DACA results for those regulators. A null mutant was also constructed for one of these regulators, SCO6008, which showed early Red production and quite delayed Act production in R5− medium. These observations suggest that DACA can be widely used to find new regulators and that the regulator SCO6008 may be involved in antibiotic production through its binding to the redD promoter.

Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

Genetic Engineering Approach for the Production of Rhamnosyl and Allosyl Flavonoids From Escherichia coli

The main functions of glycosylation are stabilization, detoxification and solubilization of substrates and products. To produce glycosylated products, Escherichia coli was engineered by overexpression of TDP‐L‐rhamnose and TDP‐6‐deoxy‐D‐allose biosynthetic gene clusters, and flavonoids were glycosylated by the overexpression of the glycosyltransferase gene from Arabidopsis thaliana. For the glycosylation, these flavonoids (quercetin and kaempferol) were exogenously fed to the host in a biotransformation system. The products were isolated, analyzed and confirmed by HPLC, LC/MS, and ESI‐MS/MS analyses. Several conditions (arabinose, IPTG concentration, OD600, substrate concentration, incubation time) were optimized to increase the production level. We successfully isolated approximately 24 mg/L 3‐O‐rhamnosyl quercetin and 12.9 mg/L 3‐O‐rhamnosyl kaempferol upon feeding of 0.2 mM of the respective flavonoids and were also able to isolate 3‐O‐allosyl quercetin. Thus, this study reveals a method that might be useful for the biosynthesis of rhamnosyl and allosyl flavonoids and for the glycosylation of related compounds. Biotechnol. Bioeng. 2010;107: 154–162.

Heterologous expression of metK1-sp and afsR-sp in Streptomyces venezuelae for the production of pikromycin

Two regulator genes, metK1-sp and afsR-sp, from Streptomyces peucetius ATCC 27952 were heterologously expressed in S. venezuelae ATCC 15439, to produce 14-membered pikromycin antibiotics. The production of pikromycin was increased by 1.6-fold and 2.6-fold by the expression of metK1-sp and afsR-sp, respectively. The overexpression of metK1-sp and afsR-sp in S. venezuelae stimulated the expression of the pathway-specific regulatory genes, pikD and ketosynthase, as demonstrated by RT-PCR. The elevated transcripts of the pikD and ketosynthase genes were consistent with the enhanced production of pikromycin.

Metabolic engineering of Escherichia coli for the biological synthesis of 7-O-xylosyl naringenin

Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physiological and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previously generated the E. coli BL21 (DE3) Δpgi host by deleting the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydrogenase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed with naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product was verified by HPLCLC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids.

Metabolic engineering of Streptomyces venezuelae for malonyl-CoA biosynthesis to enhance heterologous production of polyketides

Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.

Enhancement of pradimicin production in Actinomadura hibisca P157-2 by metabolic engineering

Actinomadura hibisca P157-2 produces potent antifungal antibiotic pradimicins. To enhance pradimicin production, ACCase from Streptomyces coelicolor and two regulatory genes metK1-sp and afsR-sp from Streptomyces peuticus were overexpressed into A. hibisca using an integration vector pSET152 under the control of the strong ermE* promoter. The constructed plasmids pACC152, pSAM152, pAFS152, pSA152 and pASA152 were transformed into A. hibisca by the conjugal method. The recombinant strains A. hibisca ACC, A. hibisca SAM, A. hibisca AFS, A. hibisca SA and A. hibisca ASA produced greater amounts of pradimicin than the parental strain with an increment of 3-fold, 2.1-fold, 2.8-fold, 3.4-fold, and 4.5-fold respectively. To increase the acetyl-coA pool, the strains were fed methyl oleate and acetate as carbon sources. The production was increased in wild-type A. hibisca, A. hibisca ACC and A. hibisca ASA by 2.2-fold, 4.12-fold and 5.98-fold respectively, with oleate and by 1.12-fold, 3.8-fold and 5.38-fold respectively with acetate. The strain A. hibisca ASA remained the best strain for the production of pradimicin. The higher transcriptional levels of structural genes in the strains harboring metK1-sp and afsR-sp compared to the wild-type strain were consistent with the enhanced production.

A novel low molecular weight phospholipase D from Streptomyces sp. CS684.

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.