Wenjun Zhang

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Name: Zhang, Wenjun

Organization: University of California , USA

Department: Department of Chemical and Biomolecular Engineering and Energy Biosciences Institute

Title: (PhD)

TOPICS

Organic Chemistry

Chemicals
References

Discovery of a Family of Desaturase-Like Enzymes for 1-Alkene Biosynthesis

Co-reporter:Zhe Rui, Nicholas C. Harris, Xuejun Zhu, Wei Huang, and Wenjun Zhang

ACS Catalysis 2015 Volume 5(Issue 12) pp:7091

Publication Date(Web):October 29, 2015

DOI:10.1021/acscatal.5b01842

1-Alkenes are important platform chemicals that are almost exclusively produced from fossil hydrocarbons. Bioproduction of 1-alkenes can mitigate our dependence on declining petrochemical resources, thereby representing an important step in the field of green chemistry. Here, we report the discovery of a new family of membrane-bound desaturase-like enzymes that convert medium-chain fatty acids (10–16 carbons) into the corresponding 1-alkenes through oxidative decarboxylation. We further show that these desaturase-like enzymes could be efficient in transforming lauric acid to 1-undecene in E. coli compared to the existing 1-alkene biosynthetic enzymes. This work expands the enzyme inventory for the transformation of fatty acid precursors to hydrocarbons and promotes the industrial production of medium-chain 1-alkenes through microbial fermentation.Keywords: 1-alkene; 1-undecene; biocatalysis; biosynthesis; desaturase

Identification of the Polyketide Biosynthetic Machinery for the Indolizidine Alkaloid Cyclizidine

Co-reporter:Wei Huang, Seong Jong Kim, Joyce Liu, and Wenjun Zhang

Organic Letters 2015 Volume 17(Issue 21) pp:5344-5347

Publication Date(Web):October 16, 2015

DOI:10.1021/acs.orglett.5b02707

The cyclizidine biosynthetic gene cluster was identified from Streptomyces NCIB 11649, which revealed the polyketide biosynthetic machinery for cyclizidine alkaloid biosynthesis. Both in vivo mutagenesis study and in vitro biochemical analysis provided insight into the timing and mechanism of the biosynthetic enzymes that produce cyclizidine-type indolizidine alkaloids.

Biosynthesis of Antimycins with a Reconstituted 3-Formamidosalicylate Pharmacophore in Escherichia coli

Co-reporter:Joyce Liu, Xuejun Zhu, Ryan F. Seipke, and Wenjun Zhang

ACS Synthetic Biology 2015 Volume 4(Issue 5) pp:559

Publication Date(Web):October 2, 2014

DOI:10.1021/sb5003136

Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.Keywords: 3-formamidosalicylate; antimycin biosynthesis; formyltransferase; heterologous expression; multicomponent oxygenase; nonribosomal peptide/polyketide hybrid;

Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase

Co-reporter:Zhe Rui, Wei Huang, Fei Xu, Mo Han, Xinyu Liu, Shuangjun Lin, and Wenjun Zhang

ACS Chemical Biology 2015 Volume 10(Issue 8) pp:1765

Publication Date(Web):June 5, 2015

DOI:10.1021/acschembio.5b00284

Sparsomycin is a model protein synthesis inhibitor that blocks peptide bond formation by binding to the large ribosome subunit. It is a unique dipeptidyl alcohol, consisting of a uracil acrylic acid moiety and a monooxo-dithioacetal group. To elucidate the biosynthetic logic of sparsomycin, a biosynthetic gene cluster for sparsomycin was identified from the producer Streptomyces sparsogenes by genome mining, targeted gene mutations, and heterologous expression. Both the genetic and enzymatic studies revealed a minimum set of non-ribosomal peptide synthetases needed for generating the dipeptidyl alcohol scaffold of sparsomycin, featuring unusual mechanisms in dipeptidyl assembly and off-loading.

Bacterial Genome Mining of Enzymatic Tools for Alkyne Biosynthesis

Co-reporter:Xuejun Zhu

ACS Chemical Biology 2015 10(12) pp: 2785-2793

Publication Date(Web):October 6, 2015

DOI:10.1021/acschembio.5b00641

The alkyne is an important functionality widely used in material science, pharmaceutical science, and chemical biology, but the importance of this functionality is contrasted by the very limited number of enzymes known to be involved in alkyne biosynthesis. We recently reported the first known carrier protein-dependent pathway for terminal alkyne formation, and in silico analysis suggested that this mechanism could be widespread in bacteria. In this paper, we screened additional homologous gene cassettes presumed to be involved in alkyne biosynthesis using both in vitro biochemical study and an E. coli-polyketide synthase (PKS) reporting system for in vivo analysis. We discovered and characterized a new terminal alkyne biosynthetic pathway comprised of TtuA, -B, and -C from Teredinibacter turnerae T7901. While the acyl-CoA ligase homologue (TtuA) demonstrated promiscuity in the activation and loading of medium-chain fatty acids onto the carrier protein (TtuC), the desaturase homologue (TtuB) showed stringent substrate specificity toward C10 fatty acyl moieties. In addition, TtuB was demonstrated to be a bifunctional desaturase/acetylenase that efficiently catalyzed two sequential O2-dependent dehydrogenation reactions. A novel terminal-alkyne bearing polyketide was further produced upon coexpression of ttuABC and a PKS gene in E. coli. The discovery and characterization of TtuA, -B, and -C provides us with a new bifunctional desaturase/acetylenase for mechanistic and structural study and expands the scarce enzyme inventory for the biosynthesis of the alkyne functionality, which has important applications in synthetic and chemical biology.

Identifying the Minimal Enzymes Required for Biosynthesis of Epoxyketone Proteasome Inhibitors

Co-reporter:Joyce Liu;Xuejun Zhu;Dr. Wenjun Zhang

ChemBioChem 2015 Volume 16( Issue 18) pp:2585-2589

Publication Date(Web):

DOI:10.1002/cbic.201500496

Abstract

Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anticancer drugs. Although the biosynthetic gene clusters for several peptidyl epoxyketone natural products have recently been identified, the enzymatic logic involved in the formation of the terminal epoxyketone pharmacophore has been relatively unexplored. Here, we report the identification of the minimal set of enzymes from the eponemycin gene cluster necessary for the biosynthesis of novel metabolites containing a terminal epoxyketone pharmacophore in Escherichia coli, a versatile and fast-growing heterologous host. This set of enzymes includes a non-ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), and an acyl-CoA dehydrogenase (ACAD) homologue. In addition to the in vivo functional reconstitution of these enzymes in E. coli, in vitro studies of the eponemycin NRPS and ¹³C-labeled precursor feeding experiments were performed to advance the mechanistic understanding of terminal epoxyketone formation.

Unusual Acetylation-Dependent Reaction Cascade in the Biosynthesis of the Pyrroloindole Drug Physostigmine

Co-reporter:Joyce Liu;Tailun Ng;Dr. Zhe Rui;Omer Ad ;Dr. Wenjun Zhang

Angewandte Chemie 2014 Volume 126( Issue 1) pp:140-143

Publication Date(Web):

DOI:10.1002/ange.201308069

Abstract

Physostigmine is a parasympathomimetic drug used to treat a variety of neurological disorders, including Alzheimer’s disease and glaucoma. Because of its potent biological activity and unique pyrroloindole skeleton, physostigmine has been the target of many organic syntheses. However, the biosynthesis of physostigmine has been relatively understudied. In this study, we identified a biosynthetic gene cluster for physostigmine by genome mining. The 8.5 kb gene cluster encodes eight proteins (PsmA–H), seven of which are required for the synthesis of physostigmine from 5-hydroxytryptophan, as shown by in vitro total reconstitution. Further genetic and enzymatic studies enabled us to delineate the biosynthetic pathway for physostigmine. The pathway features an unusual reaction cascade consisting of highly coordinated methylation and acetylation/deacetylation reactions.

Unusual Acetylation-Dependent Reaction Cascade in the Biosynthesis of the Pyrroloindole Drug Physostigmine

Co-reporter:Joyce Liu;Tailun Ng;Dr. Zhe Rui;Omer Ad ;Dr. Wenjun Zhang

Angewandte Chemie International Edition 2014 Volume 53( Issue 1) pp:136-139

Publication Date(Web):

DOI:10.1002/anie.201308069

Abstract

Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase

Co-reporter:Zhe Rui;Xin Li;Xuejun Zhu;Joyce Liu;Bonnie Domigan;Jamie H. D. Cate;Ian Barr

PNAS 2014 Volume 111 (Issue 51 ) pp:18237-18242

Publication Date(Web):2014-12-23

DOI:10.1073/pnas.1419701112

Aliphatic medium-chain 1-alkenes (MCAEs, ∼10 carbons) are “drop-in” compatible next-generation fuels and precursors to commodity chemicals. Mass production of MCAEs from renewable resources holds promise for mitigating dependence on fossil hydrocarbons. An MCAE, such as 1-undecene, is naturally produced by Pseudomonas as a semivolatile metabolite through an unknown biosynthetic pathway. We describe here the discovery of a single gene conserved in Pseudomonas responsible for 1-undecene biosynthesis. The encoded enzyme is able to convert medium-chain fatty acids (C10–C14) into their corresponding terminal olefins using an oxygen-activating, nonheme iron-dependent mechanism. Both biochemical and X-ray crystal structural analyses suggest an unusual mechanism of β-hydrogen abstraction during fatty acid substrate activation. Our discovery unveils previously unidentified chemistry in the nonheme Fe(II) enzyme family, provides an opportunity to explore the biology of 1-undecene in Pseudomonas, and paves the way for tailored bioconversion of renewable raw materials to MCAE-based biofuels and chemical commodities.

Characterization of AntB, a Promiscuous Acyltransferase Involved in Antimycin Biosynthesis

Co-reporter:Moriah Sandy, Xuejun Zhu, Zhe Rui, and Wenjun Zhang

Organic Letters 2013 Volume 15(Issue 13) pp:3396-3399

Publication Date(Web):June 17, 2013

DOI:10.1021/ol4014365

The in vivo and in vitro characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl carrier and produces more antimycin analogues with varying C-8 acyloxy moieties.

Tandem Enzymatic Oxygenations in Biosynthesis of Epoxyquinone Pharmacophore of Manumycin-type Metabolites

Co-reporter:Zhe Rui, Moriah Sandy, Brian Jung, Wenjun Zhang

Chemistry & Biology 2013 Volume 20(Issue 7) pp:879-887

Publication Date(Web):25 July 2013

DOI:10.1016/j.chembiol.2013.05.006

•Epoxyquinone pharmacophore was biosynthesized through tandem enzymatic oxygenations•Single-component monooxygenase (AsuE1) de-aromatizes 3,4-AHB moiety by hydroxylation•When flavin is scarce, flavin reductase (AsuE2) specifically elicits AsuE1 activity•FMN-dependent monooxygenase (AsuE3) catalyzes the formation of the epoxide moietyMany natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites. Our biochemical studies reveal that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA) precursor is activated and loaded on aryl carrier protein (AsuC12) by ATP-dependent adenylase (AsuA2). AsuE1 and AsuE3, both single-component flavin-dependent monooxygenases, catalyze the exquisite regio- and enantiospecific postpolyketide synthase (PKS) assembly oxygenations. AsuE1 installs a hydroxyl group on the 3,4-AHB ring to form a 4-hydroxyquinone moiety, which is epoxidized by AsuE3 to yield the epoxyquinone functionality. Despite being a single-component monooxygenase, AsuE1 activity is elicited by AsuE2, a pathway-specific flavin reductase. We further demonstrate that the epoxyquinone moiety is critical for anti-MRSA activity by analyzing the bioactivity of various manumycin-type metabolites produced through mutasynthesis.Figure optionsDownload full-size imageDownload high-quality image (79 K)Download as PowerPoint slide

Enzymatic Synthesis of Dilactone Scaffold of Antimycins

Co-reporter:Moriah Sandy, Zhe Rui, Joe Gallagher, and Wenjun Zhang

ACS Chemical Biology 2012 Volume 7(Issue 12) pp:1956

Publication Date(Web):September 12, 2012

DOI:10.1021/cb300416w

Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. The antimycin structural skeleton is built on a nine-membered dilactone ring containing one alkyl, one acyloxy, two methyl moieties, and an amide linkage connecting to a 3-formamidosalicylic acid. Although a biosynthetic gene cluster for antimycins was recently identified, the enzymatic logic that governs the synthesis of antimycins has not yet been revealed. In this work, the biosynthetic pathway for antimycins was dissected by both genetic and enzymatic studies for the first time. A minimum set of enzymes needed for generation of the antimycin dilactone scaffold were identified, featuring a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line containing both cis- and trans-acting components. Several antimycin analogues were further produced using in vitro enzymatic total synthesis based on the substrate promiscuity of this NRPS-PKS machinery.

Chemical Logic and Enzymatic Machinery for Biological Assembly of Peptidyl Nucleoside Antibiotics

Co-reporter:Christopher T. Walsh and Wenjun Zhang

ACS Chemical Biology 2011 Volume 6(Issue 10) pp:1000

Publication Date(Web):August 18, 2011

DOI:10.1021/cb200284p

Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization.

Enzymes for fatty acid-based hydrocarbon biosynthesis

Co-reporter:Nicolaus A Herman, Wenjun Zhang

Current Opinion in Chemical Biology (December 2016) Volume 35() pp:22-28

Publication Date(Web):December 2016

DOI:10.1016/j.cbpa.2016.08.009

A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool

Co-reporter:Xuejun Zhu, Peyton Shieh, Michael Su, Carolyn R. Bertozzi and Wenjun Zhang

Chemical Communications 2016 - vol. 52(Issue 75) pp:NaN11242-11242

Publication Date(Web):2016/08/19

DOI:10.1039/C6CC05990B

Directed evolution was used to improve the activity of JamB, a membrane-bound bifunctional desaturase/acetylenase. To quickly assess the protein engineering outcomes, we developed a new platform for quantifying extracellular alkyne-tagged metabolites through a fluorogenic click reaction. Random mutagenesis yielded the best JamB variant with ∼20-fold increased activity in E. coli.