05-Metabolic Engineering of Antibiotics

万古霉素代谢Erythromycin is a type of macrolide antibiotic that has been used for decades to treat a wide range of bacterial infections. Its broad spectrum of activity and low toxicity have made it a popular choice for healthcare providers. The mode of action of erythromycin involves inhibiting bacterial protein synthesis, which ultimately leads to bacterial cell death. The antibiotic is produced by the soil-dwelling bacterium Saccharopolyspora erythraea through a complex biosynthetic pathway. Understanding the metabolism of erythromycin is important for improving its production in industrial settings and for developing new derivatives with improved clinical efficacy. In this review, we will explore the metabolic pathway of erythromycin and the factors that influence its production.Biosynthesis of ErythromycinThe biosynthesis of erythromycin is a complex process that involves multiple enzymatic steps. The first step in the biosynthetic pathway is the condensation of malonyl-CoA and methylmalonyl-CoA to form a polyketide core, which serves as the precursor molecule for the synthesis of erythromycin. This reaction is catalyzed by the polyketide synthase (PKS) enzyme complex, which is composed of multiple subunits that work together to build the polyketide chain. The PKS enzymes are encoded by a cluster of genes known as the ery gene cluster, which is located on the chromosome of S. erythraea.Following the formation of the polyketide core, a series of enzymatic reactions are required to modify the structure of the polyketide chain to yield the final erythromycin molecule. These reactions include methylation, glycosylation, and oxidation steps, which are catalyzed by specific enzymes encoded by genes within the ery cluster. The final steps of the biosynthetic pathway involve the attachment of a macrolide ring and the formation of the deoxysugar moieties that are characteristic of erythromycin. These processes are mediated by a set of tailoring enzymes that further modify the structure of the polyketide core.Regulation of Erythromycin BiosynthesisThe production of erythromycin by S. erythraea is tightly regulated at the transcriptional and translational levels. Several regulatory proteins have been identified that control the expression of genes within the ery cluster in response to environmental cues and metabolic signals. One of the key regulators of erythromycin biosynthesis is the pathway-specific activator protein, which binds to specific DNA sequences within the ery gene cluster and stimulates the transcription of biosynthetic genes. In addition to activator proteins, global regulatory proteins that influence the overall metabolic state of the cell can also impact erythromycin production. For example, the availability of nutrients and the metabolic flux through central carbon metabolism are known to influence the synthesis of erythromycin. Improving Erythromycin ProductionEfforts to improve the production of erythromycin have focused on several strategies, including strain engineering, fermentation optimization, and bioprocess control. Strainengineering involves the manipulation of S. erythraea to enhance its ability to produce erythromycin. This can be achieved by overexpressing key biosynthetic genes, deleting negative regulators of the ery cluster, or introducing genes from related bacterial species to expand the metabolic capabilities of the host strain. Fermentation optimization aims to create an ideal growth environment for S. erythraea, including the selection of appropriate media, the optimization of fermentation conditions, and the addition of specific nutrients or inducers that stimulate erythromycin production. Bioprocess control involves monitoring and controlling the fermentation process to ensure optimal erythromycin yields, such as by regulating the pH, oxygen levels, and temperature within the fermentation vessel.Future PerspectivesThe metabolic pathway of erythromycin continues to be a topic of active research, as scientists seek to further elucidate the biosynthetic steps and regulatory mechanisms that underlie its production. In addition, efforts to engineer S. erythraea for improved erythromycin production are ongoing, with the goal of developing high-yielding strains that can be used in industrial settings. Furthermore, the development of new erythromycin derivatives with enhanced pharmacological properties, such as improved antimicrobial activity or reduced side effects, remains an important area of investigation. By gaining a deeper understanding of the metabolism of erythromycin and its biosynthetic pathway, researchers hope to advance the development and production of this important antibiotic for the treatment of bacterial infections.ConclusionIn conclusion, erythromycin is a valuable antibiotic that is produced by the bacterium S. erythraea through a complex biosynthetic pathway. Understanding the metabolic processes that govern erythromycin production is essential for improving its yield in industrial settings and for developing new derivatives with enhanced clinical efficacy. The regulatory mechanisms that control erythromycin biosynthesis and the strategies for improving its production are areas of active research. By advancing our knowledge of the metabolic pathway of erythromycin, researchers aim to enhance our ability to produce and utilize this important antibiotic for the treatment of bacterial infections.。

The Gut MicrobiotaREVIEWInteractions Between the Microbiota and the Immune SystemLora V.Hooper,1*Dan R.Littman,2Andrew J.Macpherson 3The large numbers of microorganisms that inhabit mammalian body surfaces have a highly coevolved relationship with the immune system.Although many of these microbes carry out functions that are critical for host physiology,they nevertheless pose the threat of breach with ensuing pathologies.The mammalian immune system plays an essential role in maintaining homeostasis with resident microbial communities,thus ensuring that the mutualistic nature of the host-microbial relationship is maintained.At the same time,resident bacteria profoundly shape mammalian immunity.Here,we review advances in our understanding of the interactions between resident microbes and the immune system and the implications of these findings for human health.Complex communities of microorganisms,termed the “microbiota,”inhabit the body surfaces of virtually all vertebrates.In the lower intestine,these organisms reach extraordi-nary densities and have evolved to degrade a variety of plant polysaccharides and other dietary substances (1).This simultaneously enhances host digestive efficiency and ensures a steady nutrient supply for the microbes.Metabolic efficiency was likely a potent selective force that shaped the evolution of both sides of the host-microbiota lions of years of coevolution,however,have forged pervasive interconnections between the physiologies of microbial commu-nities and their hosts that extend beyond metabolic functions.These interconnections are particularly apparent in the relationship between the microbiota and the immune system.Despite the symbiotic nature of the intestinal host-microbial relationship,the close association of an abundant bacterial community with intesti-nal tissues poses immense health challenges.The dense communities of bacteria in the lower intes-tine (≥1012/cm 3intestinal contents)are separated from body tissues by the epithelial layer (10m m)over a large intestinal surface area (~200m 2in humans).Opportunistic invasion of host tissue by resident bacteria has serious health consequences,including inflammation and sepsis.The immune system has thus evolved adaptations that work to-gether to contain the microbiota and preserve the symbiotic relationship between host and microbiota.The evolution of the vertebrate immune system has therefore been driven by the need to protect thehost from pathogens and to foster complex micro-bial communities for their metabolic benefits (2).In this Review,we survey the state of our understanding of microbiota-immune system in-teractions.We also highlight key experimental challenges that must be confronted to advance our understanding in this area and consider how our knowledge of these interactions might be harnessed to improve public health.Tools for Analyzing the Microbiota –Immune System RelationshipMuch of our current understanding of microbiota –immune system interactions has been acquired from studies of germ-free animals.Such animals are reared in sterile isolators to control their exposure to microorganisms,including viruses,bacteria,and eukaryotic parasites.Germ-free animals can be studied in their microbiologically sterile state or can serve as living test tubes for the establishment of simplified microbial ecosystems composed of a single microbial species or defined species mixtures.The technology has thus come to be known as “gnotobiotics,”a term derived from Greek meaning “known life.”Gnotobiotic ani-mals,particularly rodents,have become critical experimental tools for determining which host immune functions are genetically encoded and which require interactions with microbes.The current impetus for gnotobiotic exper-imentation has been driven by several impor-tant technical advances.First,because any mouse strain can be derived to germ-free status (3),large numbers of genetically targeted and wild-type inbred isogenic mouse strains have become avail-able in the germ-free state.The contribution of different immune system constituents to host-microbial mutualism can thus be determined by comparing the effects of microbial colonization in genetically altered and wild-type mice (4,5).Second,next-generation sequencing tech-nologies have opened the black box of micro-biota complexity.Although advances in ex vivo culturability are still needed,the composition ofhuman and animal microbiotas can be opera-tionally defined from polymorphisms of bacterial genes,especially those encoding the 16S ribo-somal RNA sequences.Such analyses have made possible the construction of defined microbiotas,whose distinct effects on host immunity can now be examined (6).Moreover,these advances allow the study of experimental animals that are both isobiotic and,in a defined inbred host,isogenic.A dominant goal of these efforts is to benefit hu-man health [see Blumberg and Powie (7)].With the developing technology,the species differ-ences can be closed using mice with a defined humanized microbiota (8).On the horizon,there is even the prospect of humanized isobiotic mice that also have a humanized immune system (9).A third advance has been the development of experimental systems that allow the uncoupling of commensal effects on the immune system from microbial colonization.This cannot be achieved by antibiotic treatment alone because a small pro-portion of the targeted microbes will persist.Deletion strains of bacteria lacking the ability to synthesize prokaryotic-specific amino acids have been developed that can be grown in culture but do not persist in vivo,so the animals become germ-free again.This allows issues of mucosal immune induction,memory,and functional protection to be explored without permanent colonization (10).Finally,important insights about the impact of resident microbial communities on mammalian host biology have been acquired by using high-throughput transcriptomic and metabolomic tools to compare germ-free and colonized mice (11,12).These tools include DNA microarrays,which have led to a detailed understanding of how microbiota shape many aspects of host physiology,includ-ing immunity (13,14)and development (15),as well as mass spectrometry and nuclear magnetic resonance spectroscopy,which have provided im-portant insights into how microbiota influence metabolic signaling in mammalian hosts (12).The application of these new approaches to the older technology of gnotobiotics has revolutionized the study of interactions between the microbiota and the immune system.Looking Inside-Out:Immune System Control of the MicrobiotaA major driving force in the evolution of the mammalian immune system has been the need to maintain homeostatic relationships with the microbiota.This encompasses control of micro-bial interactions with host tissues as well as the composition of microbial consortia.Here,we dis-cuss recent insights into how the immune system exerts “inside-out ”control over microbiota local-ization and community composition (see Fig.1).Stratification and compartmentalization of the microbiota.The intestinal immune system faces unique challenges relative to other organs,as it must continuously confront an enormous micro-bial load.At the same time,it is necessary to avoid1The Howard Hughes Medical Institute and Department of Im-munology,The University of Texas Southwestern Medical Center at Dallas,Dallas,TX 75390,USA.2Howard Hughes Medical Institute and Molecular Pathogenesis Program,The Kimmel Center for Biology and Medicine of the Skirball Institute,New York University School of Medicine,New York,NY 10016,USA.3Maurice Müller Laboratories,University Clinic for Visceral Sur-gery and Medicine,University of Bern,Bern,Switzerland.*To whom correspondence should be addressed.E-mail:lora.hooper@8JUNE 2012VOL 336SCIENCE1268 o n M a y 20, 2015w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mpathologies arising from innate immune signaling or from microbiota alterations that disturb essential metabolic functions.An important function of the intestinal immune system is to control the expo-sure of bacteria to host tissues,thereby lessening the potential for pathologic outcomes.This oc-curs at two distinct levels:first,by minimizing direct contact between intestinal bacteria and the epithelial cell surface(stratification)and,second, by confining penetrant bacteria to intestinal sites and limiting their exposure to the systemic im-mune compartment(compartmentalization).Several immune effectors function together to stratify luminal microbes and to minimize bacterial-epithelial contact.Intestinal goblet cells secrete mucin glycoproteins that assemble into a~150-m m-thick viscous coating at the intestinal epithelial cell surface.In the colon,there are two structurally distinct mucus layers.Although the outer mucus layer contains large numbers of bacteria,the inner mucus layer is resistant to bacterial penetration (16).In contrast,the small intestine lacks clearly distinct inner and outer mucus layers(17).Here, compartmentalization depends in part on antibac-terial proteins that are secreted by the intestinal epithelium.RegIII g is an antibacterial lectin that is expressed in epithelial cells under the control of Toll-like receptors(TLRs)(18–20).RegIII g limits bacterial penetration of the small intestinal mucus layer,thus restricting the number of bacteria that contact the epithelial surface(5).Stratification of intestinal bacteria on the luminal side of the epithelial barrier also depends on secreted immunoglobulin A(IgA).IgA spe-cific for intestinal bacteria is produced with the help of intestinal dendritic cells that sample the small numbers of bacteria that penetrate the over-lying epithelium.These bacteria-laden dendritic cells interact with B and T cells in the Peyer’s patches,inducing B cells to produce IgA directed against intestinal bacteria(21).IgA+B cells home to the intestinal lamina propria and secrete IgA that is transcytosed across the epithelium and deposited on the apical surface.The transcytosed IgAs bind to luminal bacteria,preventing micro-bial translocation across the epithelial barrier(22).Mucosal compartmentalization functions to minimize exposure of resident bacteria to the sys-temic immune system(Fig.1B).Although bacteria are largely confined to the luminal side of the epithelial barrier,the sheer number of intestinal bacteria makes an occasional breach inevita-ble.Typically,commensal microorganisms that penetrate the intestinal epithelial cell barrier are phagocytosed and eliminated by lamina propria macrophages(23).However,the intestinal im-mune system samples some of the penetrant bac-teria,engendering specific immune responses that are distributed along the length of the intes-tine(21).Bacteria that penetrate the intestinal barrier are engulfed by dendritic cells(DCs)re-siding in the lamina propria and are carried alive to the mesenteric lymph nodes.However,these bacteria do not penetrate to systemic secondarylymphoid tissues.Rather,the commensal-bearingDCs induce protective secretory IgAs(21),whichare distributed throughout all mucosal surfacesby recirculation of activated B and T cells.Thus,distinctive anatomical adaptations in the mucosalimmune system allow immune responses directedagainst commensals to be distributed widely whilestill being confined to mucosal tissues.Other immune cell populations also promotethe containment of commensal bacteria to in-testinal sites.Innate lymphoid cells reside in thelamina propria and have effector cytokine pro-files resembling those of T helper(T H)cells(24).Innate lymphoid cells that produce interleukin(IL)–22are essential for containment of lymphoid-resident bacteria to the intestine,thus preventingtheir spread to systemic sites(25).The compartmentalization of mucosal andsystemic immune priming can be severely per-turbed in immune-deficient mice.For example,mice engineered to lack IgA show priming ofserum IgG responses against commensals,indi-cating that these bacteria have been exposed tothe systemic immune system(22).A similar out-come is observed when innate immune sensingisFig.1.Looking inside-out:immune system control of the microbiota.Several immune effectors function together to stratify luminal microbes and to minimize bacterial-epithelial contact.This includes the mucus layer,epithelial antibacterial proteins,and IgA secreted by lamina propria plasma partmen-talization is accomplished by unique anatomic adaptations that limit commensal bacterial exposure to the immune system.Some microbes are sampled by intestinal DCs.The loaded DCs traffic to the mesenteric lymph nodes through the intestinal lymphatics but do not penetrate further into the body.This compartmentalizes live bacteria and induction of immune responses to the mucosal immune system. There is recirculation of induced B cells and some T cell subsets through the lymphatics and the bloodstream to home back to mucosal sites,where B cells differentiate into IgA-secreting plasma cells. SCIENCE VOL3368JUNE20121269SPECIAL SECTIONThe Gut Microbiotadefective.Mice lacking MyD88or TRIF signal-ing adaptors for TLR-mediated sensing of bacteria also produce serum IgG responses against com-mensals(26).This probably results from the fact that in these settings,large numbers of commensals cross the epithelial barrier and phagocytic cells are less able to eliminate the penetrant organisms.Immune system control of microbiota com-position.The development of high-throughput sequencing technologies for microbiota analysis has provided insight into the many factors that determine microbiota composition.For example nutrients,whether derived from the host diet (27)or from endogenous host sources(28),are critically important in shaping the structure of host-associated microbial communities.Recent evidence suggests that the immune system is also likely to be an important contributor to“inside-out”host control over microbiota composition.Certain secreted antibacterial proteins produced by epithelial cells can shape the composition of in-testinal microbial communities.a-defensins are small(2to3kD)antibacterial peptides secreted by Paneth cells of the small intestinal epithelium.Anal-ysis of the microbiota in mice that were either de-ficient in functional a-defensins or that overexpressed human a-defensin-5showed that although there was no impact on total numbers of colonizing bacte-ria,there were substantial a-defensin–dependent changes in community composition,with reciprocal differences observed in the two mouse strains(29).An interesting question is how far secreted in-nate immune effectors“reach”into the luminal microbial consortia.For example,the impact of hu-man a-defensin-5on luminal community composi-tion contrasts with the antibacterial lectin RegIII g, which limits penetration of bacteria to the epithelial surface but does not alter luminal communities(5). This suggests that some antimicrobial proteins,such as a-defensins,reach into the lumen to shape overall community composition,whereas others,such as RegIII g,have restricted effects on surface-associated bacteria and thus control microbiota location relative to host surface tissues.Questions remain as to ex-actly how a-defensin-5controls luminal community composition,however.In one scenario,these small antimicrobial peptides diffuse through the mucus layer and directly act on bacteria that inhabit the lu-men.Another possibility is that a-defensin-5exerts its antibacterial activity on bacteria that are trapped in the outer reaches of the mucus layer,with those bac-teria acting as reservoirs that seed luminal commu-nities and thus dictate their composition.Answering these questions will require improved tools for fine-mapping microbiota composition and consortia from the surface of the intestine to the interior of the lumen.The impact of the immune system on micro-biota composition is also suggested by several im-mune deficiencies that alter microbial communities in ways that predispose to disease.For example, Garrett et al.studied mice that lack the transcription factor T-bet(encoded by Tbx21),which governs inflammatory responses in cells of both the innate and the adaptive immune system(30).WhenTbx21–/–mice were crossed onto Rag2–/–mice,which lack adaptive immunity,the Tbx21–/–/Rag2–/–progeny developed ulcerative colitis in a microbiota-dependent manner(30).Remarkably,this colitisphenotype was transmissible to wild-type mice byadoptive transfer of the Tbx21–/–/Rag2–/–micro-biota.This demonstrated that altered microbiotawere sufficient to induce disease and could thus beconsidered“dysbiotic.”Similarly,mice lacking thebacterial flagellin receptor TLR5exhibit a syn-drome encompassing insulin resistance,hyper-lipidemia,and increased fat deposition associatedwith alterations in microbiota composition(31).These metabolic changes are transferable to wild-type mice that acquire the Tlr5–/–gut microbiota.A third example of immune-driven dysbiosis isseen in mice deficient for epithelial cell expres-sion of the inflammasome component NLRP6.These mice develop an altered microbiota withincreased abundance of members of the Bacte-roidetes phylum associated with increased intes-tinal inflammatory cell recruitment and susceptibilityto chemically induced colitis.Again,there is evi-dence that dysbiosis alone is sufficient to drive theintestinal inflammation,because conventionallyraised wild-type mice that acquire the dysbioticmicrobiota show similar immunopathology(32).Together,these findings suggest that the im-mune system affords mammalian hosts some con-trol over the composition of their resident microbialcommunities.It is also clear that these commu-nities can be perturbed by defects in the host im-mune system.This leads to the idea of the immunesystem as a form of ecosystem management thatexerts critical control over microbiota compo-sition,diversity,and location[see Costello et al.(33)].However,a number of questions remain.First,although it is apparent that the immune sys-tem shapes community composition at the specieslevel,it is not yet clear whether the immune sys-tem shapes the genetics and physiology of indi-vidual microbial species.Second,how much doesthe immune system combine with gastric acid andintestinal motility to control the longitudinal dis-tribution of microbial species in the gastrointes-tinal tract?Finally,it will be important to determinethe extent to which the immune system also con-trols microbial community composition and loca-tion in other organ systems,such as the respiratorytract,urogenital tract,and skin.Looking Outside-In:How MicrobiotaShape ImmunityThe earliest comparisons of germ-free and colonizedmice revealed a profound effect of microbial colo-nization on the formation of lymphoid tissues andsubsequent immune system development.It wasthus quickly apparent that the microbiota influ-ence the immune system from“outside-in.”Recentstudies have greatly amplified this understandingand have revealed some of the cellular and mo-lecular mediators of these interactions(see Fig.2).The impact of the microbiota on lymphoidstructure development and epithelial function.The tissues of the gastrointestinal tract are rich inmyeloid and lymphoid cells,many of whichreside in organized lymphoid tissues.It has longbeen appreciated that the gut microbiota have acritical role in the development of organized lym-phoid structures and in the function of immunesystem cells.For example,isolated lymphoid fol-licles in the small intestine do not develop ingerm-free mice,and such mice are also deficientin secretory IgA and CD8ab intraepithelial lym-phocytes.The specific microbial molecules en-dowed with this inductive function have not yetbeen described,however.Sensing of commensal microbiota through theTLR-MyD88signaling pathway triggers severalresponses that are critical for maintaining host-microbial homeostasis.The microbiota inducerepair of damaged intestinal epithelium through aMyD88-dependent process that can be rescued inmicrobe-depleted animals by gavage with bacteriallipopolysaccharide(LPS).The innate signals,con-veyed largely through myeloid cells,are required toenhance epithelial cell proliferation(34,35).Asdiscussed above,MyD88-dependent bacterial sig-nals are also required for the induction of epithelialantimicrobial proteins such as RegIII g(5,19).Thisexpression can be induced by LPS(19,20)or flagel-lin(36).The flagellin signals are relayed throughTLR5expressed by CD103+CD11b+dendritic cellsin the lamina propria,stimulating production of IL-23that,in turn,promotes the expression of IL-22by innate lymphoid cells(37).IL-22then stimu-lates production of RegIII g,which is also secretedupon direct activation of MyD88in epithelialcells(5,20).This is one clear example of theimportance of commensals in the induction of hostinnate responses,but it likely represents a tinyfraction of the multitude of effects of microbiota onthe host immune system.Microbiota shaping of T cell subsets.It hasrecently become evident that individual commensalspecies influence the makeup of lamina propria Tlymphocyte subsets that have distinct effector func-tions.Homeostasis in the gut mucosa is maintainedby a system of checks and balances between poten-tially proinflammatory cells,which include T H1cellsthat produce interferon-g;T H17cells that produceIL-17a,IL-17f,and IL-22;diverse innate lymphoidcells with cytokine effector features resemblingT H2and T H17cells;and anti-inflammatory Foxp3+regulatory T cells(T regs).Colonization of mice withsegmented filamentous bacteria(SFB)results inaccumulation of T H17cells and,to a lesser extent,inan increase in T H1cells(38,39).SFB appear able topenetrate the mucus layer overlying the intestinalepithelial cells in the terminal ileum,and they in-teract closely with the epithelial cells,inducing hostcell actin polymerization at the site of interactionand,presumably,signaling events that result in aT H17polarizing environment within the laminapropria.There is little known about host cell8JUNE2012VOL336SCIENCE 1270signaling pathways initiated by SFB.It is possible that SFB influence epithelial gene expression,re-sulting,for example,in expression of antimicro-bial proteins such as RegIII g and of molecules that participate in T H 17cell polarization.SFB may also act directly on cells of the immune sys-tem,either through interactions with myeloid cells that extend processes through the epithelium to the mucus layer or by production of metabolites that act on various receptors expressed by host cells.Other bacteria have been shown to enhance the anti-inflammatory branches of the adaptive immune system by directing the differentiation of T regs or by inducing IL-10expression.For example,coloniza-tion of gnotobiotic mice with a complex cocktail of 46mouse Clostridial strains,originally isolated from mouse feces and belonging mainly to cluster IVand XIV a of the Clostridium genus,results in the expansion of lamina propria and systemic T regs .These have a phenotype characteristic of T regs in-duced in the periphery in response to transforming growth factor (TGF)–b and retinoic acid [in contrast to thymic-derived natural (n)T regs (40)],and manyof these inducible T regs (iT regs )express IL-10.The exact Clostridial strains within the complex exper-imental mixture that drive this regulatory response remain to be defined.Furthermore,polysaccharide A (PSA)of Bacteroides fragilis induces an IL-10response in intestinal T cells,which prevents the expansion of T H 17cells and potential damage to the mucosal barrier (41).In contrast,mutant B.fragilis lacking PSA has a proinflammatory profile and fails to induce IL-10.Production of PSA by B.fragilis has been proposed to be instrumental for the bac-terium ’s success as a commensal.Within the intestine,the balance of effector lym-phoid cells and T reg cells can have a profound in-fluence on how the mucosa responds to stresses that elicit damage.The relative roles of commensal-regulated Tcells differ according to the models used to study inflammation.For example,in mice sub-jected to chemical or pathogen-induced damage to the mucosa,T H 17cells have a beneficial effect that promotes healing.In contrast,T H 1and T H 17cells,as well as IL-23–dependent innate lymphoid cells,promote colitis in models in which T reg cells aredepleted.It is likely that inflammatory bowel dis-eases in humans can be similarly triggered by commensal-influenced imbalance of lymphoid cell subsets.This is supported by numerous observations,including the strong linkage of IL23R polymor-phisms with Crohn ’s disease,a serious condition with relapsing intestinal inflammation and a risk of malignancy,and the severe enterocolitis associated with IL10and IL10R mutations (42,43).Microbiota effects on systemic immunity.The influence of commensal bacteria on the balance of T cell subsets is now known to extend well beyond the intestinal lamina propria.Homeostatic T cell proliferation itself is driven by the microbiota or their penetrant molecules (44).Systemic auto-immune diseases have long been suggested to have links to infections,but firm evidence for causality has been lacking.Recent studies in animal models,however,have reinforced the notion that commen-sal microbiota contribute to systemic autoimmune and allergic diseases at sites distal to the intestinal mucosa.Several mouse models for autoimmunity are dependent on colonization status.Thus,germ-free mice have marked attenuation of disease in models of arthritis and experimental autoimmune encephalomyelitis (EAE),as well as in various colitis models.In models of T H 17cell –dependent arthritis and EAE,monoassociation with SFB is sufficient to induce disease (42,45,46).In all of these models,induction of T H 17cells in the in-testine has a profound influence on systemic dis-ease.Exacerbation of arthritis and EAE is likely the consequence of an increase in the number of arthritogenic or encephalitogenic T H 17cells that traffic out of the lamina propria.The antigen spec-ificity of such cells remains to be examined.Induction of iT regs by the cluster IV and XIV a Clostridia also has a systemic effect on inflamma-tory processes.Colonization of germ-free mice with these bacteria not only results in attenuated disease after chemical damage of the gut epithelium but also reduces the serum IgE response after immuni-zation with antigen under conditions that favor a T H 2response (40).As with pathogenic T H 17cells,the antigen specificity of the commensal-induced iT regs that execute systemic anti-inflammatory func-tions is not yet known,although at least some of the T regs in the gut have Tcell receptors with specificity for distinct commensal bacteria (47).Finally,B.fragilis PSA affects the develop-ment of systemic T cell responses.Colonization of germ-free mice with PSA-producing B.fragilis results in higher numbers of circulating CD4+T cells compared to mice colonized with B.fragilis lacking PSA.PSA-producing B.fragilis also elicits higher T H 1cell frequencies in the circulation (48).Together,these findings show that commen-sal bacteria have a general impact on immunity that reaches well beyond mucosal tissues.Microbiota influences on invariant Tcells and innate lymphoid cells.A recent study extends the role of microbiota to the control of the function invariant natural killer T cells (iNKT cells),whichFig.2.Looking outside-in:how microbiota shape host immunity.Some of the many ways that intestinal microbiota shape host immunity are depicted.These include microbiota effects on mucosal as well as systemic immunity.ILFs,isolated lymphoid follicles.SCIENCEVOL 3368JUNE 20121271SPECIAL SECTION。

幅度提高,系统的氨氮污泥负荷可达到0106～0173kgΠkg・d。

(4)当酒精消化液碳源不足时,可投加乙酸钠作为外加碳源进行生物脱氮。

当乙酸钠投加量相当于C OD Cr497mgΠL时,消化液的C OD CrΠT N为6161,T N的去除率可达63%。

参考文献[1]　李锡英,丁建南1处理酒精废醪的厌氧反应器类型1中国沼气,1997,15(3):35237.[2]　江鸿,郭仁惠,张盼月等.酒精糟废水的回收利用与净化研究.工业水处理,1998,18(3):43244.[3]　孙锦宜.含氮废水处理技术与应用.北京:化学工业出版社,2003.[4]　Hallin Sara,Pell M ikael.M etabolic properties of denitrifying bacteriaadapting to methanol and ethanol in activated sludge.W ater Res ources, 1998,32(1):13218.[5]　章非娟.生物脱氮技术.北京:中国环境科学出版社,1992.作者通讯处　杨健　200092　上海市四平路1239号　同济大学环境科学与工程学院电话　(021)65984275E2m ail　yishu@2005-04-19收稿水解酸化法预处理青霉素废水的试验3孙京敏1,2　王路光2　王世研2(11哈尔滨工业大学,黑龙江150090;21国家环境保护制药废水污染控制工程技术中心,石家庄050051)摘要　在系统分析青霉素废水水质的基础上,进行该废水不同稀释比的水解酸化试验。

结果表明:采用水解酸化工艺作为生物处理的预处理手段是有效的,选取HRT为8～10h,C ODCr的进水负荷取6～8kgΠm3・d,废水酸化率达到10%左右,C OD Cr去除率为20%左右。

关键词　青霉素废水　水解酸化　预处理0　引言抗生素原料药生产过程排出的废水有机污染物浓度高、组分复杂,含有大量发酵残余物,包括发酵代谢产物、残余的消沫剂、凝聚剂、破乳剂和残留的抗生素效价及其降解产物,可生化降解性差。

两种消除Klebsiella pneumoniae重组型质粒的方法王熙;萨娜;杨建国;田平芳【摘要】对于工业发酵菌种肺炎克雷伯氏菌(Klebsiella pneumoniae), 研究发现有两种消除其重组型质粒的有效方法,一种是连续传代培养,另一种是使用消除剂十二烷基硫酸钠(SDS).对K. pneumoniae重组菌连续20代传代培养后,发现其质粒具有较高的消除率;而以0.2% SDS复合Ca2+处理K. pneumoniae重组菌,也能有效消除其重组型质粒,且该方法省却了反复的传代培养,能快速得到质粒消除菌,更具简便易操作性.消除了质粒的K. pneumoniae能再次接纳新的质粒,有效避免了因质粒不相容性带来的转化不成功,进而可用作宿主菌积累更多的生理性状.【期刊名称】《北京化工大学学报（自然科学版）》【年(卷),期】2010(037)006【总页数】4页(P112-115)【关键词】质粒;消除;肺炎克雷伯氏菌;重组【作者】王熙;萨娜;杨建国;田平芳【作者单位】北京化工大学,北京市生物加工过程重点实验室,北京,100029;北京化工大学,北京市生物加工过程重点实验室,北京,100029;北京化工大学,北京市生物加工过程重点实验室,北京,100029;北京化工大学,北京市生物加工过程重点实验室,北京,100029【正文语种】中文【中图分类】Q933生理工程是新兴的工业生物技术研究方向,强调微生物的生理特性对发酵生产的重要作用[1]。

工业生物技术一直致力于打造具有最优性能的细胞工厂。

细胞工厂作为一个运营实体[2],其“内部建筑物”的“建造”过程具有时序性与累积性[3]。

因此,一个优良工业发酵菌株的性能是逐渐累积得来的。

质粒作为携带异源基因的主要工具,其存在与否会对宿主细胞的生理状态造成较大的影响。

所以,重组质粒的导入、消除与再导入将成为生理工程研究中一项常规实验操作。

质粒消除并非一个新的概念,临床上用于解决细菌耐药性蔓延的研究已逾20年[4]。

Mono and Diterpene Production in Escherichia coliK.Kinkead Reiling,1Yasuo Yoshikuni,2Vincent J.J.Martin,3Jack Newman,1Jo ¨rg Bohlmann,4Jay D.Keasling 1,2,31Department of Chemical Engineering,The University of California Berkeley,California 94720-1462;telephone:510-6424862;fax:510-6431228;e-mail:keasling @ 2UCSF/UCB Joint Graduate Program in Bioengineering,The University of California Berkeley,California 3Physical Biosciences Division,Lawrence Berkeley National Laboratory,1Cyclotron Road,Berkeley,California 4Biotechnology Laboratory,Department of Botany and Department of Forest Sciences,University of British Columbia,Vancouver V6T 1Z3,BC CanadaReceived 24October 2003;accepted 9March 2004Published online 18June 2004in Wiley InterScience ().DOI:10.1002/bit.20128Abstract:Mono-and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceu-ticals.Production of these compounds in microbial hosts,such as Escherichia coli,can be limited by intracellular levels of the polyprenyl diphosphate precursors,geranyl di-phosphate (GPP),and geranylgeranyl diphosphate (GGPP).To alleviate this limitation,we constructed synthetic oper-ons that express three key enzymes for biosynthesis of these precursors:(1)DxS,1-deoxy-D -xylulose-5-phosphate synthase;(2)IPIHp,IPP isomerase from Haematococcus pluvialis ;and (3)one of two variants of IspA,FPP syn-thase that produces either GPP or GGPP.The reporter plasmids pAC-LYC and pACYC-IB,which encode enzymes that convert either FPP or GGPP,respectively,to the pig-ment lycopene,were used to demonstrate that at full in-duction,the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene.To synthesize di-or monoterpenes in E.coli using the GGPP and GPP encoding operons either a diter-pene cyclase [casbene cyclase (Ricinus communis L )and ent -kaurene cyclase(Phaeosphaeria sp.L487)]oramonoter-pene cyclase [3-carene cyclase (Picea abies )]was coex-pressed with their respective precursor production operon.Analysis of culture extracts or headspace by gas chro-matography-mass spectrometry confirmed the in vivo production of the diterpenes casbene,kaur-15-ene,and kaur-16-ene and the monoterpenes a -pinene,myrcene,sabinene,3-carene,a -terpinene,limonene,h -phellandrene,a -terpinene,and terpinolene.Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di-and monoterpene cyclases and the overproduction of terpenes in bacteria.B 2004Wiley Periodicals,Inc.Keywords:isoprenoid;diterpene;monoterpene;cyclase;casbene;carene;kaureneINTRODUCTIONIsoprenoids are among the most diverse groups of com-pounds synthesized by biological systems;it has been estimated that there are over 50,000known isoprenoids,which include the terpenoids and carotenoids (McCaskill and Croteau,1997).The number of isoprene (C5)units of which they are comprised classifies terpenoids.Monoter-penes (C 10),such as menthol and camphor,and sesquiter-penes (C 15),such as zingiberene (ginger),are the major constituents of herbs and spices.Other sesquiterpenes and diterpenes (C 20)are pheromones,antimicrobial agents,or signal transducers (Fraga,1991;McGarvey and Croteau,1995).Higher molecular weight isoprenoids stabilize mem-branes (cholesterol and other C 30compounds)and serve as photoreceptive pigments (lycopene).The primary or ancillary biological activity of some terpenoids has been a benefit to the medical community.Several monoterpene derivatives are being investigated as anticancer agents.For example,the monoterpene limonene and related derivatives are believed to inhibit farnesylation of the growth-promoting protein RAS,and therefore inhibit malignant cell proliferation (Gelb et al.,1995;Gould,1997;Hohl,1996).Casbene,a plant diterpenoid,is an antifungal agent (Erkel and Anke,1994;Ferrandiz et al.,1994;Tan et al.,1999).Taxol,a diterpenoid isolated from the pacific yew tree (Taxus brevifolia ),and its derivatives are potent anti-cancer compounds used in the treatment of ovarian,breast,lung,neck,bladder,skin,and cervical cancers (Jennewein and Croteau,2001).Because the complexity of Taxol precludes commercial total chemical synthesis (Jennewein and Croteau,2001)nearly all of the TaxolB 2004Wiley Periodicals,Inc.Correspondence to:Jay D.KeaslingContract grant sponsors:ERC Program of the National Science Foundation;National Science Foundation;Office of Naval Research;University of California Discovery Grant Program;Maxygen (Redwood City,CA);American Cancer SocietyContract grant numbers:EEC-9731725;BES-9911463;N00014-00-1-0750;99-10044;PF-03-106-01-CDDbiosynthetic genes have been cloned,opening the way for in vivo recombinant production of a diterpene therapeutic. The building blocks for the biosynthesis of terpenes, isopentenyl diphosphate(IPP),and dimethylallyl diphos-phate(DMAPP),can be synthesized via two pathways:the mevalonate-dependent and the mevalonate-independent pathway(also referred to as the DXP pathway;Fig.1).In prokaryotes,isoprenoids are synthesized primarily via the DXP pathway(Boucher and Doolittle,2000),which in Escherichia coli produces farnesyl diphosphate(FPP). Farnesyl diphosphate is the precursor to trans-octaprenyl diphosphate,used in ubiquinone synthesis,and cis,trans-undecaprenyl diphosphate,used in bactoprenol biosyn-thesis(Apfel et al.,1999).Escherichia coli,however,does not naturally produce appreciable quantities of geranyl diphosphate(GPP)or geranylgeranyl diphosphate(GGPP), the precursors for mono-and diterpenes,respectively.In this study,we have developed strains of E.coli that harbor engineered operons encoding enzymes for the production of GPP and GGPP.The output of the GGPP biosynthetic pathway is assayed via the production of the pigment lycopene from these precursors.We report on the in vivo production of mono-and diterpenes from GPP or GGPP by three terpene cyclases:the monoterpene synthase 3-carene cyclase from Picea abies(Faldt et al.,2003),and the diterpene synthases casbene cyclase from Ricinus communis L(Mau and West,1994)and ent-kaurene cyclase from Phaeosphaeria sp.L487(Kawaide et al.,1997).The terpene-precursor platform host,described in this study,is designed to facilitate the transition of natural products from discovery to development and commercial-ization.Terpenes and their derivatives are often difficult to produce in significant yields either in higher plants or by synthetic means.Although the research on metabolic engineering of natural product biosynthesis is still at the gene discovery stage,the deluge of DNA-sequence data from genomics research provides an excellent opportu-nity for the design of microorganisms for the synthesis of natural bined with recent advances in enzyme directed evolution and combinatorial biocataly-sis,this approach may be a valuable alternative to traditional methods of high-throughput small-molecule synthesis and drug production.MATERIALS AND METHODSMedia and CultureLuria-Bertani(LB),M9,and2YT media were used in this study(Sambrook et al.,1989).Carbenicillin(50A g/mL), kanamycin(50A g/mL),ampicillin(100A g/mL),chloram-phenicol(50A g/mL),isopropyl-beta-D-thiogalactopyrano-side(IPTG),and arabinose were purchased from Fisher Scientific.Plasmids used in this study are listed in Table I. Construction and Expression of the SyntheticIsoprenoid OperonsThe synthetic operons were constructed by splicing the genes using overlapping extensions(SOE).These oper-ons expressed a combination of three genes:the E.coli 1-deoxy-D-xylulose-5-phosphate synthase gene(dxs-AC# NP_414954;Blattner et al.,1997),the HaematococcusFigure1.The DXP pathway and downstream compounds addressed inthis study.In the upper box is depicted the metabolite flow leading to IPPthrough the DXP pathway of E.coli.Below is a flow of metabolitesleading to either terpenes or the pigment lycopene.Listed are the enzymesused in this study:in the SOE operons(DXP synthase,dxs;FPP synthase,ispA;IPP isomerase,IPIHp),in the pAC-LYC reporter plasmids(GGPPsynthase,crtE;phytoene synthase,crtB;and lycopene synthase,crtI)andin the pACYC-IB(crtB and crtI).REILING ET AL.:TERPENE PRODUCTION IN E.COLI201pluvialis isopentenyl diphosphate(IPP)isomerase gene (IPIHp-AC#AF082326;Sun et al.,1998),and the E.coli FPP synthase gene(isp A-AC#NP_414955;Blattner et al., 1997).dxs was obtained from the plasmid pDdxs(Kim and Keasling,2001).IPPHp was subcloned from the previously constructed plasmid,pAC-LYC04(Cunningham et al., 1994).IPPHp was spliced3V to dxs via PCR using overlap extensions and primers dxs-for and dxs-rev and IPPHp-for and IPPHp–rev(Table II).The dxs-for primer introduced a5V-Eco RI site upstream of the two-gene operon and the IPPHp–rev primer introduced a3V-Xba I site downstream. The overlapping dxs-rev and IPPHp-for primers introduced a Shine-Dalgarno site in place of the final two codons of the dxs gene changing the N-terminal sequence from Leu-Ala(GG CTGG CATAA)to Arg-Thr(GG AGGA CATAA).Thus,the10-base spacer between the Shine-Dalgarno site and the start codon of IPPHp required only a four base separation between the two genes.The variant isp A genes (isolated from the pCRispA plasmids,described below) were spliced to the3V end of the dxs-IPPHp operon by the introduction of a3V-Nco I site flanking the dxs-IPPHp fragment via PCR using the primers dxs-for2and IPPHp-rev2.The dxs-for2primer introduced a5V-Nhe I site and a Shine-Dalgarno site into the dxs-IPPHp fragment.The IPPHp-rev2primer mutated the two C-terminal residues of IPPHp from Glu-Ala(GCGTGA)to Gly-Gly(GGAGGA) and thus introduced a Shine-Dalgarno site seven base pairs 5V to the ATG of the Nco I site.This dxs-IPPHp PCR product was ligated into the Nhe I-Nco I sites of the pBAD24 plasmid to produce pBADSOE1,which was subsequentlyTable I.Plasmids used in this study.Vector Parent plasmid Antibiotic marker Expression control Inserted gene Reference pTRcCas pTrc99a Carbenicillin LacIq Casbene Cyclase This studypTRcKau pTrc99a Carbenicillin LacIq ent-Kaurene Cyclase This studypTRcCar pTrc99a Carbenicillin LacIq Carene Cyclase This studypBADSOE1pBAD24Carbenicillin AraC dxs,IPIHp This studypBADSOE4pBAD24Carbenicillin AraC dxs,IPIHp,ispA(WT)This studypBBRSOE4pBBR1MCS-2Kanamycin AraC dxs,IPIHp,ispA(WT)This studypBBRSOE5pBBR1MCS-3Kanamycin AraC dxs,IPIHp,ispA(Y80D)This studypBBRSOE6pBBR1MCS-4Kanamycin AraC dxs,IPIHp,ispA(S81F)This studypAC-LYC pACYC154Chloramphenicol Constitutive crtE,crtB,crtI Cunningham et al.,1994 pACYC-IB pACYC154Tetracycline Constitutive crtB,crtI Ohnuma et al.,1996Table II.PCR primers used in this study.Primer Sequence aS81F-for5V-GTGTATCCACGCTTACTTTTTAATTCATGATGATT-3VS81F-rev5V-AATCATCATGAATTAAAAAGTAAGCGTGGATACAC-3VY80D-for5V-TGAGTGTATCCACGCTGACTCATTAATTCATGATG-3VY80D-rev5V-CATCATGAATTAATGAGTCAGCGTGGATACACTCA-3VEcoR Idxs-for5V-AGGAGGA ATTCACC ATG AGTTTTGATATTGCCAAATAC-3Vdxs-rev5V-TCTGAGCAACGAACGAAG CAT ATAT TTA TG TCCTCC AGGCCTTG ATTTTG-3V IPPHp-for5V-CAAAATCAAGGCCT GGAGGA CA TAA ATAT ATG CTTCGTTCGTTG CTCAGA-3V IPPHp-rev5V-GCTCTAGA TCA CGCTTCGTTGATGTGATGC-3VXba INhe I EcoR Idxs-for25V-TTGGGCTAGCA GGAGGA ATTC-3VIPPHp-rev25V-GCATC CAT GGTA TCA TCCTCC GTTGATGTGATG-3VNco INco IispA-fo r5V-TGATACC ATG GACTTTCCGCAGCAACTCG-3VispA-rev5V-GCTCTAGA TTA TTTATTACGCTGGATGATG-3VXba IXba IKaurene-for5V-GCTCTAGA ATG TTTGCCAAATTCGATATGC-3VKaurene-rev5V-CCCAAGCTT TTA CGTGCCGACGTGTTTCAAAG-3VHind IIINcoI3CNcoF5V-TAGCCATGGCTGTTATTTCCATTTTGCCGTTG-3V3CXbaR5V-GCTCTAGATTACATAGGCACAGGTTCAAGAACGG-3Va Italics represent the Shine-Dalgarno(SD)sequences,underlined text denotes the restriction site sequences,and bold type indicates the start/stop codons. 202BIOTECHNOLOGY AND BIOENGINEERING,VOL.87,NO.2,JULY20,2004sequenced.The isp A gene or its variants were amplified from the pCRispA vectors described below using the PCR primers isp A-for and isp A-rev,which introduced an Nco I site in frame with the start codon and an Xba I site immediately after the stop codon.The produced fragments were ligated into the multiple cloning site of the pBAD24 vector,which contains the arabinose-inducible ara BAD promoter,to yield pBADSOE4,pBADSOE5,or pBAD-SOE6(Fig.2).The ispA region of each of the pBADSOE operons were subsequently sequenced in the context of the pET30a expression vectors(described below)to confirm the integrity of the gene.The operon fragments generated by a Cla I-Xba I digest were ligated into the broad-host-range vector pBBR1MCS-2to produce pBBRSOE4,pBBRSOE5, or pBBRSOE6.The fragments generated by this digest contained the prenyl diphosphate biosynthetic operon,the arabinose-inducible P BAD and associated regulator(araC). All the PCR reactions were run for30cycles at95j C for30s, 55j C for30s,and72j C for1.5min and used Expand High Fidelity PCR System(Roche).Cloning of Mono-and Diterpene CyclasesThe terpene cyclases used in this study were the3-carene cyclase from Picea abies(AC#AF461460),the ent-kaurene cyclase from Phaeosphaeria sp.L487(AC#AB003395), and the casbene cyclase from Ricinus communis L(AC# L32134).The casbene cyclase cDNA,initially provided in a pET21d plasmid,was ligated into pTrc99A at the Nco I and Sal I restriction sites to generate pTrcCAS.The ent-kaurene cyclase gene was amplified from plasmid DNA and5V-Xba I and3V-Hind III restriction sites were introduced by PCR using the primers kaurene-for and kaurene-rev,and the Expand HF PCR system(Roche).This PCR fragment was digested and ligated into the Xba I-Hind III sites of pTrc99A to produce pTrcKau.Carene cyclase was ampli-fied from plasmid DNA,and a5V-Nco I site and a3V-Xba I site were introduced via PCR using the primers3CNcoF and3CXbaR(Table II).The resulting PCR fragment was ligated into the like restriction sites of pTrc99a to yield pTrcCar.The sequence of the cyclase genes amplified by PCR was confirmed by DNA sequencing.Each of the se-quenced clones of carene exhibited the silent mutation C!A at base pair561.The cyclase plasmids were cotransformed into either E.coli DH5a(pTrcKau)or DH10b(pTrcCas and pTrcCar)with the appropriate iso-prenoid operon for in vivo terpene production. Production and Purification of Wild-Type and Mutant IspA PrenyltransferasesWild-type and mutant prenyltransferase genes were ex-pressed from a pET30a vector and the6-His-tagged re-combinant enzymes purified by immobilized-metal affin-ity chromatography(IMAC).The isp A genes were subcl-oned into a pET30a vector from the SOE operons using PCR to introduce5V-Nco I-Eco RI-3V restriction sites(Table II),sequenced,and transformed into E.coli BL21(DE3) (Novagen)for protein production.Overnight cultures were grown from a single colony and used to inoculate(1%v/v) 500mL of LB medium.The cultures were grown at37j C to an optical density measured at a wavelength of600nm (OD600)of0.6at which time they were induced with 0.1m M IPTG.Cells were harvested4h post-induction and the pellets were frozen atÀ80j C.Cells harvested from 500mL of culture were suspended in5mL of50m M Tris-HCl at pH7.5,10m M MgCl2,1m M PMSF,DNaseI,10m M CaCl2,and400m M NaCl(buffer A)and lysed on ice by three successive sonications using a50%duty cycle at 25%power for1min.Lysates were cleared by centrifuga-tion at31,400RCF and4j C for30min and supernatants were filtered through a0.2A m syringe filter.The cleared lysates were loaded at1mL/min onto a1mL fast flow chelating sepharose resin(Pharmacia)previously charged with100m M NiSO4and equilibrated with buffer A sup-plemented with10m M ing a step gradient,the column was first washed with10column volumes of buffer A supplemented with100m M imidazole and the prenyl-transferases were eluted with5column volumes of buffer A supplemented with500m M imidazole.The resulting enzyme preparations were dialyzed against50m M Tris-HCl pH7.5,10m M MgCl2,and10%glycerol,twice.After dialysis,glycerol was added to bring the solution to50%, and then the solution was frozen atÀ80j C.Site-Directed Mutagenesis of isp A and In Vitro Prenyltransferase AssayWild-type isp A was isolated from the genomic DNA of a single E.coli colony by PCR using the Roche HF-PCR system and TA-cloned into pCR4(Invitrogen)resulting in the plasmid pCRispA.PCR-mediated site-directed muta-tions were carried out using the Quick-Change kit(Strat-agene)and the primers S81F-for,S81F-rev,Y80D-for,and Y80D-rev(Table II)with the plasmid pCRispA as a tem-plate.The mutations were confirmed by DNA sequencing. The E.coli wild-type and mutant prenyltransferases were assayed in vitro to determine their product profiles and enzymatic activity levels.Five microliters of purified protein were diluted into45A L of warmed reaction buffer (50m M Tris-HCL pH7.5,30m M MgCl2,37j C)to finalFigure2.Diagram of the engineered SOE operons.For each gene thestart and stop codons are indicated along with the position of introducedrestriction and Shine-Dalgarno sites.REILING ET AL.:TERPENE PRODUCTION IN E.COLI203substrate and enzyme concentrations as follows:400A M of IPP and DMAPP to6.7A M IspA(Ser81Phe),540A M of IPP and270A M of DMAPP to1.2A M IspA(WT),and 600A M of IPP and200A M of DMAPP to6.5A M IspA (Tyr80Asp).The reaction proceeded at37j C overnight. To quench the reaction and increase the sensitivity of HPLC analysis,10A L of100m M EDTA was added to the reac-tion.Forty microliters of the quenched reaction were loaded onto a C18reverse-phase high-performance liquid chromatography(HPLC)column previously equilibrated with buffer I(25m M NH3HCO2).Buffer I was run for an additional30s after sample loading followed first by a4-min gradient from100%buffer I to100%buffer II(100% acetonitrile)and finally by a1-min wash with buffer II.A standard for each polyprenyl diphosphate(FPP,GPP, GGPP)was run using this HPLC protocol at three different concentrations to derive the relationship between HPLC peak area and polyprenyl diphosphate concentration.In Vivo Lycopene AssayTo analyze the flux to GGPP,we utilized a previously described assay for lycopene production(Cunningham et al.,1994;Kim and Keasling,2001).Briefly,the plasmid pAC-LYC consists of the genes encoding GGPP synthase, phytoene synthase,and lycopene synthase genes from Erwinia herbicola(Fig.1).DH5a cells,cotransformed with either the FPP or the GGPP biosynthetic operon and the constitutive lycopene operon,were inoculated into 2YT medium and grown to an OD600of2.5.To induce gene expression,aliquots of cultures were diluted to a final OD600of0.1into fresh2YT medium containing arabinose. These cultures were shaken in a29j C water bath for24h, at which time cells from1mL of culture medium were harvested by centrifugation for2min at18,188RCF and room temperature.The supernatant was discarded and 1mL of acetone was added,followed by incubation in a 55j C-water bath for5min.The acetone-extracted lycopene from each sample was assayed by measurement of the absorbance at470nm.Expression of Diterpene Cyclases and GC-MS Analysis of ProductsDiterpene production was confirmed for each cyclase using gas chromatography-mass spectrometry(GC-MS)analysis of ethyl acetate-extracted cultures.For the diterpene cyclases,overnight cultures were grown from a single colony and used to inoculate(1%v/v)50mL of medium. The cultures were grown at37j C until an OD600of0.4at which time they were induced with1m M IPTG and13m M arabinose.Cells were harvested after4h of post-induction growth,and pellets suspended in1mL of phosphate-buffered saline.For full-scan GC-MS analysis,diterpenes from0.7mL of suspended cells were extracted with an equal volume of ethyl acetate.Extractions were carried out in glass vials to avoid contaminants that may be extracted from plastic tubes.Initial studies on diterpenes explored the products of a pBBRSOE5/pTrcKau E.coli in a DH5a background.To minimize background and maximize signal,these studies where performed in M9medium and the prepared ethyl acetate extracts were concentrated up to 20-fold by a gentle flow of nitrogen gas prior to GC-MS analysis.For later studies with casbene,the greater signal due to the activity of the cyclase and the more robust growth of E.coli in LB warranted a switch to this media. To identify the diterpenes,total ion scans were run on the GC-MS monitoring the40to275(m/z)ions.For time course analysis with pBBRSOE5/pTrcCas,0.7mL of growing culture was extracted with an equal part of ethyl acetate.To increase the sensitivity and selectivity of detection,the MS was operated in selected ion-monitoring (SIM)mode using ions of121,136,257,and272m/z, which represent the molecular ion and three abundant ions of casbene.The diterpene cembrene was used as an internal standard for chromatographic runs and to calibrate casbene production.All the diterpene GC-MS analyses utilized a Hewlett Packard HP6890gas chromatograph equipped with a Hewlett Packard5973mass selective detector and a HP-5MS capillary column(30MÂ250A m IDÂ0.25A m film thickness).Diterpenes from splitless1-A L injections were separated using a GC oven temperature program of 80j C for2min.followed by a10j C/min ramp to300j C. Injector and MS quadrupole detector temperatures were 250j C and150j C,respectively.In Vivo and In Vitro GC-MS Analysis of Monoterpene Cyclase ProductsFor detection of monoterpenes,a solid-phase microextrac-tion(SPME;Supelco)filament was exposed to the head-space of samples.Each100-A m polydimethylsiloxane filament was conditioned before use by exposing it to the 250j C injection chamber for2h with the split set to divert flow away from the column.A filament was considered ready for use when two consecutive blank GC-MS runs exhibited overlapping baseline traces.Each filament was used for less than100samplings with blanks run between experiments to confirm the integrity of the filament.For headspace sampling,terpenes were eluted from the im-pregnated filament by a2-min exposure to the220j C GC injection chamber.For the analysis of monoterpenes,the GC-MS was equipped with a cyclo-sil B chiral column (30MÂ250A m idÂ0.25A m film thickness,Agilent Technologies).The GC oven program used for all SPME runs was as follows:a column loading at60j C for 4minutes,followed by a ramp at10j C/minute to140j C and a second ramp at80j C/minute to200j C where the temperature was held for2min.For full scans,an ion range of40to140(m/z)was monitored.In SIM mode,only the 136,121,and93ions were monitored.For in vitro monoterpene production,overnight cultures of a pBBRSOE6/pTrcCar cotransformed strain were grown from a single colony and used to inoculate(1%v/v)three204BIOTECHNOLOGY AND BIOENGINEERING,VOL.87,NO.2,JULY20,200430-mL cultures of LB medium.Cultures were grown to an OD600of0.4,induced with1m M IPTG and0.13m M arabinose,and grown for an additional6h in250-mL, baffled,shake flasks.These cultures were pelleted and frozen atÀ80j C.Cells from frozen pellets were suspended in1mL of assay buffer(25m M Hepes pH7.2,100m M KCl,10m M MnCl2,10%glycerol,and5m M DTT)and lysed on ice by sonication via three iterations at50%duty cycle for1min.Lysates were clarified by centrifugation at 17,530RCF for20min at4j C.A100-A L aliquot of the supernatant was diluted into900A L of assay buffer in a 4.5-mL serum vial.A one-A L aliquot of1mg/mL solution of GPP in water was added to the serum vial bringing the substrate concentration to2A M GPP,and then the vial was sealed.The reactions were allowed to proceed for30min at room temperature.The SPME filament was exposed to the headspace of the in vitro reactions for30s.The adsorbed volatiles were desorbed directly into the220j C GC-MS injection port for2min.For in vivo monoterpene production,overnight cultures cotransformed with pTrcCar and pBBRSOE6were grown from a single colony and used to inoculate(1%v/v)three 200-mL cultures of LB medium in baffled,500-mL flasks. The start of each culture was staggered by20min to allow time for SPME sampling between replicate measurements. Cultures were grown to an OD600of0.4,induced with1m M IPTG and0.13m M arabinose and shaken for an additional 5min to ensure thorough mixing of the inducer.Thirty-mL aliquots of the induced culture were placed into250-mL serum vials and capped with Teflon septa.A new vial was used for each time point measurement.In sampling,each Teflon septum was pre-puncture with a21-gauge needle taking care to cover the end of the needle to prevent outgassing from the culture.The SPME filament was exposed to the headspace of a serum vial in a32j C water bath for8min.As the interval between removing the fila-ment from the headspace and inserting the SPME into the GC-MS affected the total yields,the sampling set-up and the GC-MS equipment were colocalized.Calibration curves for 3-carene were determined using an8-min exposure of the SPME filament to the headspace of LB medium warmed to 32j C and spiked with each standard(Fluka)to a final concentration of1,10,100,and1000A g/L.RESULTSConversion of the E.coli FPP Cyclase(IspA)to Either a GGPP or GPP SynthasePreliminary experiments showed that the synthase from Archaeoglobus fulgidus(Gps)performed poorly when ex-pressed in E.coli(data not shown).We suspected that poor expression of the gps gene was due to an abundance of rare Arg codon.To circumvent this and other potential pro-Figure3.Purification and characterization of the polyprenyl diphosphatesynthase(IspA)variants.IspA wild-type and Ser81Phe and Phe80Aspvariants were purified and their product profiles characterized.(a)SDS-PAGE gel loaded with the samples as marked and indicating the progressionto purified enzymes.(b)Stacked HPLC traces of the IspA variant activityassays along with the analytical standards.REILING ET AL.:TERPENE PRODUCTION IN E.COLI205blems associated with heterologous protein expression,a GGPP synthase and a GPP synthase were constructed by introducing mutations into the active site of the native E.coli FPP synthase gene,isp A.Based on previous studies, which demonstrated that GPP or GGPP could be produced by a mutant form of FPP synthase(Ohnuma et al.,1996a), two amino acids in the E.coli FPP synthase(IspA)were targeted for mutation.For the first mutation,Tyr80of IspA was mutated to an Asp residue producing a putative GGPP synthase.For the second mutation,the codon specifying Ser81of IspA was changed to a Phe codon to produce a putative GPP synthase.Before in vivo expression studies,the shift in product specificity for the two mutant prenyltransferases was con-firmed in vitro with purified enzymes.The purified his-tagged wild-type,Tyr80Asp,and Ser81Phe IspA variant enzyme preparations were more than95%pure,as assayed by Coomassie-stained SDS-PAGE protein gel electropho-resis(Fig.3a).Purified synthases were provided IPP and DMAPP as substrates and the products were analyzed using HPLC-UV.The comparison of the retention times of the prenyl diphosphates produced by the enzymes to those of analytical standards confirmed the production of GGPP and GPP by the Tyr80Asp and Ser81Phe variant,respectively (Fig.3b).Although detailed enzyme kinetics studies were not performed,in vitro analysis confirmed that these mutations in IspA had the predicted effect in changing product specificity.When the enzymatic reactions were allowed to go to completion,the Tyr80Asp and Ser81Phe formed GGPP and GGP as in excess of80%and90%of the final product mixture,respectively.As might be expected from the results of the Bacillus-engineered synthases,the relative reaction rates of the engineered synthases compared to the wild-type enzyme also showed a reduction in overall activity.Although the product specificities of the IspA enzymes may have been influenced by the substrate concentrations and ratios and by the incubation time,these results demonstrate that the product distribution shifts in the mutant enzymes in the predicted fashion. Engineered Isoprenoid Biosynthetic Operons Increase Lycopene Production In VivoTo augment the intracellular pools of GPP and GGPP in E.coli,we constructed three operons containing combina-tions of the following genes:DXP synthase(dxs),IPP isomerase(IPPHp),and wild-type or variants of the E.coli FPP synthase(ispA).The production of GGPP and FPP was measured using lycopene biosynthesis as a reporter.The arabinose-induced lycopene production by cells containing the SOE4(FPP)operon was sixfold higher than that by cells without any metabolic augmentation(E.coli strain DH5a)(Fig.4)indicating that the SOE4operon increased flux through the isoprenoid biosynthetic pathway.To assess the biosynthesis of GGPP in vivo,lycopene pro-duction was measured in strains cotransformed with pBBRSOE5and pACYC-IB.The pACYC-IB plasmid differs from the previously described pAC-LYC plasmid by the deletion of the crtE gene,which encodes an FPP to GGPP synthase(Ohnuma et al.,1994).Therefore,ly-copene production by this strain was dependent almost entirely upon the capacity of the SOE5operon to produce GGPP.In vitro studies on the E.coli enzyme IspB indicate that some GGPP may be present in the cell as an inter-mediate in the production of octaprenyl diphosphate and thus may support a minimal lycopene background(Asai et al.,1994;Kainou et al.,2001).Arabinose-inducible ly-copene production was observed for the strains cotrans-formed with pBBRSOE5and pACYC-IB in contrast to an absence of lycopene in the control strains cotransformed with pBBRSOE4and pACYC-IB(Fig.4).The peak le-vels of lycopene production achieved in withthe Figure4.Lycopene production using the SOE4(FPP)and SOE5(GGPP)operons in the vectors pBAD24(A)or pBBRMCS2(B)at various arabinose concentrations.The insert is a picture of concentrated arabinose-induced,lycopene-producing cells,which exhibit the characteristic redpigmentation of lycopene.206BIOTECHNOLOGY AND BIOENGINEERING,VOL.87,NO.2,JULY20,2004。