Lauren B Pickens

University of California, Los Angeles, Los Angeles, CA, United States

Are you Lauren B Pickens?

Claim your profile

Publications (8)45.69 Total impact

  • Peng Wang, Woncheol Kim, Lauren B Pickens, Xue Gao, Yi Tang
    [Show abstract] [Hide abstract]
    ABSTRACT: A very accommodating host: Three tetracycline biosynthetic pathways were overexpressed and manipulated in the heterologous host Streptomyces lividans K4-114. Through the inactivation of various genes and characterization of the resulting biosynthetic intermediates, new tetracycline-modifying enzymes were identified.
    Angewandte Chemie International Edition 09/2012; 51(44):11136-40. · 11.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.
    Journal of Biological Chemistry 09/2011; 286(48):41539-51. · 4.65 Impact Factor
  • Lauren B Pickens, Yi Tang, Yit-Heng Chooi
    [Show abstract] [Hide abstract]
    ABSTRACT: Natural products and their derivatives play an important role in modern healthcare as frontline treatments for many diseases and as inspiration for chemically synthesized therapeutics. With advances in sequencing and recombinant DNA technology, many of the biosynthetic pathways responsible for the production of these chemically complex yet valuable compounds have been elucidated. With an ever-expanding toolkit of biosynthetic components, metabolic engineering is an increasingly powerful method to improve natural product titers and generate novel compounds. Heterologous production platforms have enabled access to pathways from difficult to culture strains, systems biology and metabolic modeling tools have resulted in increasing predictive and analytic capabilities, advances in expression systems and regulation have enabled the fine-tuning of pathways for increased efficiency, and characterization of individual pathway components has facilitated the construction of hybrid pathways for the production of new compounds. These advances in the many aspects of metabolic engineering not only have yielded fascinating scientific discoveries but also make it an increasingly viable approach for the optimization of natural product biosynthesis.
    Annual Review of Chemical and Biomolecular Engineering 01/2011; 2:211-36. · 7.51 Impact Factor
  • Lauren B Pickens, Yi Tang
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxytetracycline (OTC) is a broad-spectrum antibiotic that acts by inhibiting protein synthesis in bacteria. It is an important member of the bacterial aromatic polyketide family, which is a structurally diverse class of natural products. OTC is synthesized by a type II polyketide synthase that generates the poly-beta-ketone backbone through successive decarboxylative condensation of malonyl-CoA extender units, followed by modifications by cyclases, oxygenases, transferases, and additional tailoring enzymes. Genetic and biochemical studies have illuminated most of the steps involved in the biosynthesis of OTC, which is detailed here as a representative case study in type II polyketide biosynthesis.
    Journal of Biological Chemistry 09/2010; 285(36):27509-15. · 4.65 Impact Factor
  • Lauren B. Pickens, Yi Tang
    [Show abstract] [Hide abstract]
    ABSTRACT: In our previous work, we have sequenced and analyzed the gene cluster responsible for producing SF2575, a tetracycline family natural product with anticancer properties, from native host Streptomyces sp. SF2575. Chemically, tetracyclines share in common their characteristic linear four-ringed structure, heavily oxidized upper periphery and C-2 amide group. Commercially available tetracyclines such as doxycycline and recently tigecycline are produced semisynthetically due to the structural complexity of the tetracycline core which renders de novo synthesis difficult and makes biosynthesis an attractive option both for production and potential engineering of these compounds. In addition to the traditional tetracycline features, SF2575 has further tailoring which makes its structure even more complex including a salicylic acid substitution at C-4 and a C-glycosylation at C-9, which is itself acylated with an angelic acid moity. These features not only add new chemical diversity to the tetracycline family but are also vital to the bioactivity of SF2575 as a potent cytotoxic compound. Recently we have verified the early biosynthetic steps leading to common tetracycline intermediates including 6-methylpretetramid and have focused efforts on characterizing key tailoring enzymes. In this study, the enzymes responsible for the attachment of the salicylic acid moity to the A ring have been identified and characterized. These include an ATP dependent salicyl-CoA ligase SsfL1, and a GDSL family acyltransferase SsfX3. SsfX3 has shown broad substrate promiscuity and has been shown to accept a wide array of chemically diverse substrates leading to the isolation of novel tetracycline compounds. Interestingly, while SsfL1 has homology to enzymes from many natural product pathways which contain an aromatic acid ligand similar to salicylic acid, SsfX3 has only a single homolog from a natural product pathway found in the NCBI protein database, and the function of this homolog is yet to be identified. This study sheds light on these unusual tetracycline tailoring enzymes and is a promising step toward using this pathway to generate novel tetracycline analogs with potent anticancer activity.
    2009 AIChE Annual Meeting; 11/2009
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihydroxylase, were functionally reconstituted.
    Journal of the American Chemical Society 11/2009; 131(48):17677-89. · 10.68 Impact Factor
  • Lauren B. Pickens, Yi Tang
    [Show abstract] [Hide abstract]
    ABSTRACT: Tetracyclines are an important group of natural products that are synthesized by different Streptomyces species by way of a type II polyketide biosynthetic pathway. These compounds have played an important role in treating bacterial infections since their discovery in 1948. More recently, several semisynthetic analogs of tetracycline have been shown to have antitumor properties, which has sparked interest in their development as a possible cancer therapy. We have obtained a strain which has been identified as producing a novel tetracycline compound SF2575. This compound has a much more complicated structure than many naturally derived tetracyclines such as chlorotetracycline and oxytetracycline, and has also been shown to have potent antitumor activity. Due to the complexity of the molecule, biosynthetic methods likely provide the most tractable path to producing and manipulating this compound. To this end, we have sequenced the previously unidentified gene cluster responsible for the production of this compound. Through expression of the minimal PKS in a heterologous host, Streptomyces CH999, we were able to confirm the production of a decaketide backbone consistant with a tetracycline biosynthesis. We plan to use this new sequence information to systematically investigate the role of individual proteins in the biosynthetic pathway towards the goal of engineering this pathway to produce novel compounds.
    2008 AIChE Annual Meeting; 11/2008
  • Source
    Lauren B Pickens, Yi Tang
    [Show abstract] [Hide abstract]
    ABSTRACT: Tetracyclines have been important agents in combating infectious disease since their discovery in the mid-20th century. Following widespread use, tetracycline resistance mechanisms emerged and continue to create a need for new derivatives that are active against resistant bacterial strains. Semisynthesis has led to second and third generation tetracycline derivatives with enhanced antibiotic activity and pharmacological properties. Recent advancement in understanding of the tetracycline biosynthetic pathway may open the door to broaden the range of tetracycline derivatives and afford analogs that are difficult to access by synthetic chemistry.
    Metabolic Engineering 11/2008; 11(2):69-75. · 6.86 Impact Factor