- Email: [email protected]
New directions in metabolic engineering
133
New directions in metabolic engineering John R Jacobsen*
Metabolic term
engineering
typically
biochemistry ones.
refers
is a rapidly to the
to introduce
Recent
microbiology
progress and
new
field.
modification
properties
The
advances
in this field. results
are constantly
being
developed.
Addresses *Department
of Chemical
Engineering,
CA 94305.5025, USA ‘Departments of Chemical
Engineering,
Stanford
Unlverslty,
Chemistry
Stanford University, Stanford, CA 94305.5025, e-mail: [email protected] Current
Opinion
in Chemical
Biology
1998,
Stanford,
and Biochemlstty,
USA
2:133-l
37
http://biomednet.com/elecref/1367593100200133 0 Current
Biology
Ltd ISSN
1367-5931
Abbreviations cytd cyto DAHP E4P 6-dEB DEBS HV PllA PIIB PEP PHA PHB PTS VHb
cytochrome d cytochrome o 3-deoxy-o-arabino-heptulosonate-7-phosphate erythrose-4-phosphate 6-deoxyerythronolide 6-deoxyerythronolide hydroxyvalerate
lvill emphasize new approaches Lvhich have helped to broaden
to metabolic engineering the scope of this field.
existing
biology,
to yield exciting
and technologies
Khoslat
of cellular
or modify
molecular
are driving
areas continue
and new problems
evolving
genetic
in genetics,
chemistry
Many well-studied
and Chaitan
B B synthase
pristinamycin IlA pristinamycin 11~ phosphoenolpyruvate polyhydroxyalkanoate polyhydroxybutyrate phosphotransferase system Vitreoscilla hemoglobin
Introduction The redirection of cellular metabolism to create or enhance desirable attributes has been accomplished using a variety of techniques and applied co an even greater variety of goals. The challenges associated with metabolic engineering vary with the goal. For the production of relatively inexpensive chemicals such as solvents, economic competition demands emphasis on product yields, utilization of inexpensive feedstocks, and separation costs. Xlost products of interest in this category are derived from primary metabolism. In contrast, for most structurally complex (bio)pharmaceuticals, the identification of the end product of a biosynthetic pathway is of the greatest Optimizing the pathway itself, though not importance. irrelevant, is typically not the primary determinant of success. Products in this category are typically secondary metabolites (or heterologous proteins, which may be considered as a special category of secondary metabolites). We will focus on advances of the past two years, and
Primary metabolites I\ major challenge for metabolic
engineering is to provide nc\j’ routes to produce commodity chemicals that arc economically competitive with established methods. hlthough the industrial production of butanol and acetone by Clos~t.inj~m cl~efobgylinlm is currently unable to compete with the economics of synthesis from petroleum, environmental concerns are generating renewed interest in fermentation-based production. :\dvances in metabolic engineering of solvent production have followed from the development of important tools for the genetic manipulation of L’. crretohrrr)~/imm [1,2]. Cloning of the genes responsible for acetone formation in this organism allowed overexpression of the rate-limiting enzymes and a corresponding rise in solvent production [3]. hlore recently. integrational plasmid technology has been used to disrupt the pathways leading to (undesirable) carboxylic acid production redirecting butyryl-CoA toward butanol production
[q].
Another option for process improvement via metabolic engineering is to produce a desired chemical from less expensive feedstocks. The conversion of low-cost lignocellulosic feedstocks to ethanol has been targeted by pathway engineering in Zynomonns mobil’ir. Although Z. mobilis efficiently ferments the glucose component of these feedstocks to ethanol, it lacks the pathways for pentose metabolism necessary for the efficient utilization of the feedstock. In separate experiments, Picataggio and co-workers introduced genes for the metabolism of xylose [j] and arabinose (6’1 permitting efficient utilization of these
pentoses.
Polyhydroxyalkanoates (PH‘4s) have received considerable attention as biodegradable polymers with useful material properties (reviewed in [7]). PHAs are produced as energy reserves in microorganisms such as Alm/&er~es eutr-oph. As with solvent production, cheaper, more efficient
economic methods
considerations demand of PHA production. In
addition, the material properties of PHAs need to be controlled and optimized for industrial applications. ?ittempts to increase PHA yields have focused on the expression of the three-enzyme pathway in the natural host as well as E&e?%& roli [8] and plants [9,10]. Recently, the pathway has been polyhydroxybutyrate (PHB) synthesis expressed in cotton fiber cells and the resulting transgenic fibers were shown to contain low levels (0.34% by weight) of polyhydroxybutyrate [ll’]. Even at this level, the PHB conferred measurably improved insulating properties to the fiber.
134
Biocatalysis
The
material
and biotransformation
properties
of
polymers
are
strongly
in-
fluenccd by their composition as well as by the ;1verage molecular weight and molecular weight distribution. For example, co-polymers of hydroxybut)-rate and hydroxyvalerdte (H\‘) exhibit superior material properties to PHB. It \vas recently demonstrated that poly-(3hydroxybutyrate-ro-3-hydroxyvalerate is produced by a recombinant E. co/i strain harboring a plasmid-encoded PHB synthesis pathway without further genetic modifications [12]. The induction of appropriate enzymes in the host organism lvith acetate, propionate or oleate allowed the production of HV from propionate, resulting in co-polymers with an Hi7 content as high as 33%. Polymer size and polydispersity are affected by the expression level of polyhydroxyalkanoate synthase, the enzyme responsible for the polymerization of the monomers. Overexpression of the synthase under the control of an inducible promoter resulted in a threefold to fivefold lower average molecular weight and greater polydispersity, although the overall level of PHA produced was not significantly affected [ 13’1. ‘I-his suggests a model in which each enzyme unit is responsible for the synthesis of a single polymer chain.
An area that has received considerable attention due to its utility as well as its complexity is the biosythcsis of aromaric metabolites. ‘l’he large number of branch points that must be controlled in order to direct the maximal amount of metabolic flux to the product or products of interest make this an extraordinarily challenging problem. A central focus of this field is increasing the flux through 3deox~-I)-arabino-hepculosonate-7-phosI~h~te (DMIP). the first committed intermediate in the synthesis of aromatic amino acids. DAI~IP is derived from cr~throse-4-phosphate (E4P) and phosphoenolpyrurate (PEP) in a reaction catalyzed by Dr\HP synthase. Overexpression of DAHP synthase shifted the focus to increasing available UP and PEP as well as control of the downstream steps [lq,lS]. Overexpression of transketolase [16] or PEP synthase [ 171 also increases DAHP production. presumably b) increasing the available supply of E4P and PEP. hlore recent studies have focused on the role of transaldolase. which also products E3P. Transaldolase overexpression increases obtained
DAHP production: however. no advantage is in cells that overexpress translketolase. suggest-
ing that transketolase overexpression saturating levels of EIP [ 181.
To further
increase
the available
is adequate
supply
to reach
of PEI’, \‘alle
and
co-workers [ 19’1 examined the glucose uptake pathnay. Internalization is driven by the glucose phosphotransferase system (P’I’S). lvhich converts glucose to glucose-6-phosphate using PEP 3s a phosphate donor. By inacti\.ation of the PTS and selection for growth on ,glucose. mutant by a strains \vere obtained that internalize glucose non-PTS system while maintaining wild-type growth rata [ 19’1. Higher levels of D.4llP production are observed, presumably due to 21larger available supply of PEP.
Secondary metabolites The challenges associated lvith engineering the biosynthesis of secondary metabolites are qualitatively different from those associated with bulk chemicals. Products of interest are often complex molecules that are necessarily derived from biological sources. In this arena. pathlvay engineering may increase the efficiency of existing production methods but may also lead to the development of new products. The relatively high vdur of these products shifts the emphasis from economics and efficiency to innovation. The antibiotic pristinamyin. produced by J’tqptorqres p/-istimNe.spir-,l/is, consists of a mixture of compounds includin,. pristinamycins 11,~ and 1113(PII,., and PIIll), Ivhich arc normally present in the ratio SO:20 (Pll,~:Pll~~). PII. is derived from the oxidation of PI113 in a reaction catalyzed by Pll,~ synthase. A new semisynthetic derivative of pristinamycin with improved properties utilizes PII, as a starting material. and it nas therefore desirable to increase the yield of PII. by engineering the complete conversion of Pll~+ The .wnA and snnB genes (which code for the two components of the heterodimcric PII. synthase) were cloned behind the strong. constituitive em/E* promoter and intcgratcd into the chromosome using the pSAhl2 integrative vector [20’]. Chromosomal integration has the advantages of high stability and fixed single-copy status, which arc desirable traits for industrial application. The engineered organism demonstrated the complete conversion of PI113 to PII;\ with a negligible drop in overall production.
Rapid advances in the genetics of antibiotic biosythesis has led to the engineering of these pathways to produce novel products. In some CWZS. the genetic and catalytic structures are modular, allowing the rational design of pathways leading to new ‘unnatural’ natural products. This has been applied to nonribosomal peptide synAcsis (211 as well as polyketide biosynthesis [ZZ]. Several esamplcs have come from the study of 6-droxyerythronolide B synthase (DEBS), a multienzyme complex that produces h-droxyerythronolide B (6-dEB), the polykctide core of the broad spectrum antibiotic erythrom)-tin.
Leadlay and co-lvorkers [Z3] demonstrated that the modular nature of DEBS allous the rational .substitution of domains to produce novel products. DEBS normally produces 6-dEB, a polypropionate macrolactonc hy the step-wise condensation of meth);lmulon~I-Cor\ extender units. The sprciticitv for meth~lmalon)-l-CoA~~lon~l-~0,~~ resides in the six X)-I transferase domains (one for each extension step). LTsing a simplified model system, the researchers replaced a methylmalonl;l transferat: domain with a malonyl transferdse domain from another polvketide synthasr [Xl. The engineered construct incorporates malonyl-CoA at the expected position, producing the predicted ‘nor-methyl’ product. .\lore rcccntly similar acyl transferase substitutions have been performed in
New directions
the entire erythromycin pathway to generate biologically active analogs of this antibiotic [24,X]. Not only can functional domains be exchanged, but new functions may be added. Kao et N/. [26”] inserted dehydratase and enoylreductase domains system. %‘here the wild-type
into an engineered DEBS enzyme produces a hydroxyl
group, the engineered strain carries out two additional catalytic steps to generate a methylene group. Another recently reported method for the engineered biosynthesis of noye products involves the use of synthetic chemistry in combination with genetic engineering [27’]. hlutational inactivation of an early step in biosynthesis of 6-dEB generates an enzyme that requires exogenous addition of a synthetic intermediate. It was demonstrated that the mutant enzyme can incorporate non-natural analogs of the intermediate to produce corresponding analogs of 6-dEB. The ability to carry out these sorts of manipulation on natural products of proven medicinal relevance suggests the possibility of creating new small-molecule drug candidates via genetic engineering.
in metabolic
engineering
Jacobsen
and Khosla
135
might seem unlikely to limit the growth of higher plants, VHb expression in transgenic tobacco plants led to several beneficial metabolic changes [35’]. Germination times were reduced: this may be ascribed to increased available oxygen for respiration and growth. Levels of chlorophyll were increased 30-40% in the transgenic plants, possibly resulting from an increase in oxygen available for its biosynthesis. An increase in available oxygen also would explain the observed increase in the (oxygen-dependent) production of nicotine and concomitant decrease in production of anabasine, which are derived from the same precursors in an oxygen-independent reaction. Hetcrologous expression of other metabolic enzymes has been used advantageously to affect changes in growth and differentiation. Expression of a yeast invertase in potato tubers affects the size and number of tubers, although the effect is dependent on the localization of the invertase [36]. Cyosolic expression leads to increased numbers, whereas it has been shown that targeting the invertase to the apoplast leads to increased tuber size.
Control of growth and differentiation In fields as diverse as agriculture and biopharmaceutical production, metabolic engineering may be used to generate organisms with desirable growth characteristics. An early example of pathway engineering was the expression of I’heosrilh hemoglobin (VHb) in E. coli [28]. This oxygen-binding protein was shown to enhance growth under oxygen-limiting conditions, and a series of detailed experiments have begun to reveal the metabolic alterations induced by \‘Hb expression. Oxygen uptake in E. co/i grown under microaerobic conditions varies with \!Hb dosage, and VHb+ cells show decreased production of reduced metabolites, supporting an important role for \‘Hb in maintaining the cell in a more oxidized state [29]. Furthermore, metabolic flux analysis [30] showed that VHb expression perturbed central metabolism as well, directing increased flux through the pentose phosphate pathway [29], an effect that is consistent with increased intracellular oxygen levels. This appears to be mediated by increased expression and activity of cytochrome o (cyt o), a terminal oxidase in the electron transport chain with relatively lo\v oxygen affinit): A second terminal oxidase, cytochrome n (cyt (I?. displays higher oxygen affinity but lower proton-pumping efficiency and is normally active under lowoxygen conditions. \‘Hb increases both the expression and apparent os)-gen affinity of cyt 0, improving microherobic respiration and growth [31*]. YHb expression in a variety of hosts led to improved growth characteristics in several cases [32.33]. Of particular interest are examples in which improved growth is accompanied by increased flux through an oxygen-dependent pathlvay of interest. .4s a model for the oxidative detoxification of environmental pollutants, Stark and co-ivorkers [3-l] examined the effect of YtIb expression on the degradation of benzoic acid by X~~f/lorrrort~~ mzl~ophilicr, and noted improved conversion efficiency. Although oxygen
hletabolic engineering has been applied to the elimination of the requirement for animal serum in mammalian cell culture. Although serum is costly and can interfere with the recovery of biopharmaceuticals produced in cell culture, it is normally required in order to provide essential growth factors. To remove this constraint, Bailey and co-workers [37] first determined the cellular proteins that lvere produced in response to growth factors. Using two growth-stimulating factors for CHO cells, cellular protein levels were analyzed by two-dimensional gel electrophoresis and compared to cells grown in fetal calf serum. This powerful analytical technique identified three proteins that were upregulated by both growth factors. These were identified as cyclin Dl, cylin E, and EZF-1, proteins known to have roles in the cell cycle. The overexpression of either EZF-1 [38*] or cyclin E [39] in CHO cells eliminated the requirement for serum or growth factors in cell culture.
New approaches
to pathway engineering
hletabolic engineering has been defined as the use of genetic techniques to systematically manipulate the metabolic activities of living cells (401. Two recent reports challenge this definition by engineering metabolism in new nays. Bertozzi and co-workers [31’] made use of the intrinsic promiscuity of glycosyl transferdses to re-engineer cell-surface carbohydrates without making any genetic alterations. Using a chemical approach. synthetic carbohydrates bearing a reactive ketone group were incorporated metabolically into cell-surface oligosaccharides. Chemoselective reactions carried out on the engineered cell surface then allowed the introduction of new epitopes for the modulation of cellular recognition. To improve E. co/i resistance to the arsenate, Stemmer and co-workers [4P]
toxic effects of took a new, non-
136
Biocatalysis
and biotransformation
approach to genetic is a method for recombining
rational
engineering.
and selrcring
DNA shuffling advantageous
mutations. allo\ving the rapid evolution of function [43]. Starting u+rh 2 plasmid-borne arsenic resistance opcron. three cycles of fragmentation and recombination afford& a IO-fold incrcasc in resistance. Sequencing of the gcncs revealed a small number of mutations; ho\ve\,cr, the mechanism by lvhich these mucations confer improvementremains unknovm A major advanuge of this nonrarional approach to engineering is its ability to optimize the function of gcnch u+thout deuiled understanding of the metabolic pathw;l)-s.
tion of the genes for arablnose metabolism allowed the engmeered organism to metabolize this pentose. This work complements previous experiments in which the xylose metabolic pathway was introduced into this otgamsm, and represents a promising approach to the efficient utilization of llgnocellulosic biomass, a low-cost renewable resource. 7.
Lee SY: Bacterial 1996, 49:1-14.
8.
Hahn SK, Chang YK, Lee SY: Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eotrophus and recombinant Escherichia co/i. Appl Environ Microb/o/ 1995, 61:34-39.
9.
Pomer Y, Dennis DE, Klomparens K, Somerville C: Polyhydroxybutyrate. a biodegradable thermoplastic. in transgenic plants. Science 1992, 256:520~523.
10.
Conclusions LTntil recently, the field of metabolic engineering has largely been ;I collection of thematically related problems: holvevrr. thr swcesse~ and failures in this field art: gradually beginning to give rise to heuristics with predictivr power. Although the transformation of metabolic enginecring into ;I rigorous science must avxit the explanation of these (and other act-co-b~-disco~~r~~i) heurisCcs in terms of fundamental physicochcmical principles. t-hz gro\vth of this tield lvill unquestionably bc propelled by the mawration of some of rhe aboxx-mentioned problems into successful products and technologies rogcthcr lvith t-he emergcncc of ne\v and even mow inwllcctuall~ ‘I’he next few years arc likely to challenging problems. lvicnt-ss a significant cspansion of chc breadth and deprh of metabolic engineering.
References
and recommended
Papers of particular Interest, published have been hlghlighted as:
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Biofechnol
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John ME, Keller G: Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells. Proc NaU Acad SC; USA 1996, 93:12768-l 2773. The polyhydroxybutyrate synthesis pathway was expressed in cotton fiber cells and the resulting transgenic fibers were shown to contain low levels of PHB. Even at low levels, the PHB conferred measurably improved Insulating propertIes to the fiber. 12.
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Mermelstein LD, Papoutsakls ET: ln viva methylation in fscherichia co/i by the Bacillus subtilis phage 03T methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutykum ATCC 824. Appl Envrron Microb,o/ 1993. 59:1077-l 081.
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4.
Green EM. Boynton ZL. Hams LM, Rudolf FB, Papoutsakls ET, Bennett GN: Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutykum ATCC 824. Mcrobiology 1996, 142:2079-2086.
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Zhang M, Eddy C, Deanda K, Fmkelstein M. Plcataggio S: Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 1995, 267240. 243.
6. .
Deanda K. Zhang M. Eddy C, Plcatagglo S: Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl Environ Microbioi 1996, 62:44654470. Although Zymomonas mobIlis efficiently ferments the glucose component of ltgnocelluloslc feedstock to ethanol, It lacks the pathways for pentose metabolism necessary for efficient utllizatlon of these feedstocks. Introduc-
analysis Biotechnoi
19. .
Flares N. Xlao J, Berry A. Bollvar F, Valle F: Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Bfotechnology 1996, 14:620-623. Microbial production of aromatlc compounds may be Improved by dlrectlng the maYlmal amount of metabolic flux through DAHP and Into aromatlc ammo acid biosynthesis. This report describes a method for increasing the amount of PEP available for DAHP synthesis. The E. co/i PTS utlllzes one molecule of PEP for each molecule of glucose imported. By mutattonal InactIvatIon of the PTS and selection for growth on glucose. mutant strams were obtained that Internalize glucose by a non-PTS system while malntalnlng wild-type growth rates. Higher levels of DAHP production are observed, presumably due to a larger available supply of PEP. 20. .
Sezonov G, Blanc V, Bamas-Jacques N, FrIedmann A. Pernodet J-L, Gu&neau M: Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes. Nat BYootechnol 1997, 15:349-353. Streptomyces pristmaespiralis normally produces a mixture of Pll~ and Its derlvatlve PII,. In this report the snaA and snag genes (which code for the
New directions
two components of the heterodimeric PllA synthase) were cloned behind the strong, constltuitlve ermf’ promoter and integrated into the chromosome. Chromosomal integration has the advantages of high stability and fixed single-copy status, which are desirable traits for Industrial application. The engineered organism demonstrated complete conversion of Plla to Pll~ with, negligible drop in overall production. This demonstrates an application of metabolic engineering to secondary metabolism in order to increase the production of a high-value product. 21.
Stachelhaus T, Schneider A, Marahiel MA: Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 1995, 269:69-72.
22.
Khosla C, Zawada RJX: Generation of polyketide libraries via combinatorial biosynthesis. fiends Biotechnol 1996, 14:335341.
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Oliynyk M, Brown MJB, Co&s modular polyketide synthase Chem Biol 1996, 3:633-639.
24.
Ruan X, Pereda A, Stassi DL, Zeidner D, Summers RG, Jackson M, Shivakumar A, Kakavas S, Staver MJ, Donadio S, Katz L: Acyltransferase domain substitutions in the erythromycin polyketide synthase yield novel erythromycin derivatives. I Bacterial 1997, 179:6416-6425.
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Kao CM, McPherson M, McDaniel RN, Fu H, Cane DE, Khosla C: Gain of function mutagenesis of the erythromycin polyketide synthase. 2. Engineered biosynthesis of an eight-membered ring tetraketide lactone. J Am Chem Sot 1997, 119:1133911340. The modular nature of deoxyerythronolide B synthase suggests that new functions may be engineered by rational domain substitutions. In this report, two domains from another polyketide synthase were inserted into DEBS, and new catalytic functions were added. Where the wild-type enzyme produces a hydroxyl group, the engineered strain carries out two additional catalytic steps to generate a methylene group. This has the addltional effect of allowing the product to form a novel eight-membered ring structure. This work is an excellent example of engineering new metabolic products by manipulation of a modular biosynthetic pathway. Jacobsen JR, HutchInson CR, Cane DE, Khosla C: Precursordirected biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 1997, 277:367-369. A genetic block was introduced into the erythromycin biosynthesis pathway. The engineered pathway requires an exogenous supply of synthetic intermediates and will utilize ‘unnatural’ precursors to generate corresponding analogs of erythromycin, some of which display antibiotic activity. This demonstrates the effective combination of metabolic engineering and organic synthesis In order to generate analogs of erythromycin and other structurally complex polyketide products. 26.
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Tsai PS, Hatzimanikatis V, Bailey JE: Effect of Vitreoscilla hemoglobin dosage on microaerobic fscherichia co/i carbon and energy metabolism. Biotechnol Bioeng 1996, 49:139-l 50.
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Liu S-C, Webster DA, Wei M-L, Stark BC: Genetic engineering to contain the Wfreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia. Biotechnol Boeng 1996, 49:101-l 05.
35. .
Holmberg N, Lilius G, Bailey JE, Bijlow L: Transgenic tobacco expressing Ktreoscilla hemoglobin exhibits enhanced growth and altered metabolic production. Nat Biotechnol 1997, 15:244247. Expression of VHb in tobacco plants was shown to have several beneficial effects. Chlorophyll production increased 30-404/o in the transgenic plants, possibly resulting from an increase in oxygen available for its biosynthesis. Nicotine production, which is oxygen dependent, was also increased at the expense of oxygen-independent production of anabasine. 36.
Sonnewald U, Hajlrezaei M-R, Kossmann 1, Heyer A, Trethewey RN, Wlllmitzer L: Increased potato tuber size resulting from apoplastic expression of a yeast invertase. Nat 5iotechnol 1997, 15:794-797.
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36. .
Lee KH, Sburlatl A, Renner WA, Bailey JE: Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovarv cells shifts Drotein oatterns and activates growth in prdtein-free medium. Bibrechnol Bioeng 1996, 50:273-279. In this report, a metabolic engineering approach was taken to eliminate the costly and inconvenient requirement for animal serum In mammalian cell culture. Recent advances in the understanding of the cell cycle suggested that deregulated expression of the transcription factor E2F-1 would bypass the requirement for growth factors normally obtained from serum. CHO cells were transfected with E2F-1 and the resulting overexpression of this protein allowed the cells to grow in serum-free media. 39.
Renner WA, Lee KH, Hatzimanikatis V, Bailey JE, Eppenberger HM: Recombinant cyclin E expression activates proliferation and obviates surface attachment in Chinese hamster ovary (CHO) cells in protein-free medium. Biotechnol Bioeng 1995, 47:476-482.
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41. .
Mahal LK, Yarema KJ, Bertozzi CR: Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997, 276:l 125-l 126. The intrinsic promiscuity of glycosyl transferases was exploited to re-engineer cell surface carbohydrates. Using a chemical approach, synthetic carbohydrates bearing a reactive ketone group were incorporated metabolically into cell-surface oligosacchandes. Chemoselective reactions carried out on the engmeered cell surface allowed the introduction of new epltopes for the modulation of cellular recognition. This was demonstrated by an example of selective drug delivery. This approach demonstrates metabolic engineering through chemistry rather than genetic alteratlon.
Tsai PS, NIgeli M, Bailey JE: Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic fscherichia co/i metabolism through elevated concentration and specific activitiy of cytochrome o. Biotechnol Bioeng 1996, 49:151-l 60. Expression of VHb in f. co/i and other organisms appears to increase the amount of oxygen available for metabolic processes. This paper shows that this observed effect is medlated by increased expression and activity of cyt o, a terminal oxtdase in the electron transport chain with relatively low oxygen affinity. A second terminal oxidase, cyt d, displays higher oxygen affinity but lower proton-pumping efficiency and is normally active under low oxygen conditions. VHb increases both the expression and apparent oxygen affinity of cyi o, improving microaerobic respiration and growth.
Crameri A, Dawes G, Rodriguez E Jr, Silver S, Stemmer WPC: Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat Biotechnol 1997, 15:436-436. This study demonstrates a nonrational approach to the metabolic engineering of E. co/i to improve resistance to the toxic effects of arsenate. DNA shuffling is a method for recombining and selecting advantageous mutations, allowing rapid evolution of function. Starting with a plasmid-borne arsenic resistance operon, three cycles of fragmentation and recombination afforded a 40.fold increase in resistance. Sequencing of the genes revealed a small number of mutations; however, the mechanism by which these mutations confer improvement remains unknown. A major advantage of this nonrational approach to engineering is its ability to optimize the function of genes without detailed understanding of the metabolic pathways.
32.
43.
31. .
Magnolo SK, Leenutaphong DL, DeModena JA. Curtis JE, Bailey JE, Galauo JL, Hughes DE: Actinorhodin production by Sfreptomyces coelicolor and growth of Streptomyces lividans
42. ..
Stemmer WPC: DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Nat/ Acad SC; USA 1994, 91 :10747-l 0751.
New directions in metabolic engineering John R Jacobsen*
Metabolic term
engineering
typically
biochemistry ones.
refers
is a rapidly to the
to introduce
Recent
microbiology
progress and
new
field.
modification
properties
The
advances
in this field. results
are constantly
being
developed.
Addresses *Department
of Chemical
Engineering,
CA 94305.5025, USA ‘Departments of Chemical
Engineering,
Stanford
Unlverslty,
Chemistry
Stanford University, Stanford, CA 94305.5025, e-mail: [email protected] Current
Opinion
in Chemical
Biology
1998,
Stanford,
and Biochemlstty,
USA
2:133-l
37
http://biomednet.com/elecref/1367593100200133 0 Current
Biology
Ltd ISSN
1367-5931
Abbreviations cytd cyto DAHP E4P 6-dEB DEBS HV PllA PIIB PEP PHA PHB PTS VHb
cytochrome d cytochrome o 3-deoxy-o-arabino-heptulosonate-7-phosphate erythrose-4-phosphate 6-deoxyerythronolide 6-deoxyerythronolide hydroxyvalerate
lvill emphasize new approaches Lvhich have helped to broaden
to metabolic engineering the scope of this field.
existing
biology,
to yield exciting
and technologies
Khoslat
of cellular
or modify
molecular
are driving
areas continue
and new problems
evolving
genetic
in genetics,
chemistry
Many well-studied
and Chaitan
B B synthase
pristinamycin IlA pristinamycin 11~ phosphoenolpyruvate polyhydroxyalkanoate polyhydroxybutyrate phosphotransferase system Vitreoscilla hemoglobin
Introduction The redirection of cellular metabolism to create or enhance desirable attributes has been accomplished using a variety of techniques and applied co an even greater variety of goals. The challenges associated with metabolic engineering vary with the goal. For the production of relatively inexpensive chemicals such as solvents, economic competition demands emphasis on product yields, utilization of inexpensive feedstocks, and separation costs. Xlost products of interest in this category are derived from primary metabolism. In contrast, for most structurally complex (bio)pharmaceuticals, the identification of the end product of a biosynthetic pathway is of the greatest Optimizing the pathway itself, though not importance. irrelevant, is typically not the primary determinant of success. Products in this category are typically secondary metabolites (or heterologous proteins, which may be considered as a special category of secondary metabolites). We will focus on advances of the past two years, and
Primary metabolites I\ major challenge for metabolic
engineering is to provide nc\j’ routes to produce commodity chemicals that arc economically competitive with established methods. hlthough the industrial production of butanol and acetone by Clos~t.inj~m cl~efobgylinlm is currently unable to compete with the economics of synthesis from petroleum, environmental concerns are generating renewed interest in fermentation-based production. :\dvances in metabolic engineering of solvent production have followed from the development of important tools for the genetic manipulation of L’. crretohrrr)~/imm [1,2]. Cloning of the genes responsible for acetone formation in this organism allowed overexpression of the rate-limiting enzymes and a corresponding rise in solvent production [3]. hlore recently. integrational plasmid technology has been used to disrupt the pathways leading to (undesirable) carboxylic acid production redirecting butyryl-CoA toward butanol production
[q].
Another option for process improvement via metabolic engineering is to produce a desired chemical from less expensive feedstocks. The conversion of low-cost lignocellulosic feedstocks to ethanol has been targeted by pathway engineering in Zynomonns mobil’ir. Although Z. mobilis efficiently ferments the glucose component of these feedstocks to ethanol, it lacks the pathways for pentose metabolism necessary for the efficient utilization of the feedstock. In separate experiments, Picataggio and co-workers introduced genes for the metabolism of xylose [j] and arabinose (6’1 permitting efficient utilization of these
pentoses.
Polyhydroxyalkanoates (PH‘4s) have received considerable attention as biodegradable polymers with useful material properties (reviewed in [7]). PHAs are produced as energy reserves in microorganisms such as Alm/&er~es eutr-oph. As with solvent production, cheaper, more efficient
economic methods
considerations demand of PHA production. In
addition, the material properties of PHAs need to be controlled and optimized for industrial applications. ?ittempts to increase PHA yields have focused on the expression of the three-enzyme pathway in the natural host as well as E&e?%& roli [8] and plants [9,10]. Recently, the pathway has been polyhydroxybutyrate (PHB) synthesis expressed in cotton fiber cells and the resulting transgenic fibers were shown to contain low levels (0.34% by weight) of polyhydroxybutyrate [ll’]. Even at this level, the PHB conferred measurably improved insulating properties to the fiber.
134
Biocatalysis
The
material
and biotransformation
properties
of
polymers
are
strongly
in-
fluenccd by their composition as well as by the ;1verage molecular weight and molecular weight distribution. For example, co-polymers of hydroxybut)-rate and hydroxyvalerdte (H\‘) exhibit superior material properties to PHB. It \vas recently demonstrated that poly-(3hydroxybutyrate-ro-3-hydroxyvalerate is produced by a recombinant E. co/i strain harboring a plasmid-encoded PHB synthesis pathway without further genetic modifications [12]. The induction of appropriate enzymes in the host organism lvith acetate, propionate or oleate allowed the production of HV from propionate, resulting in co-polymers with an Hi7 content as high as 33%. Polymer size and polydispersity are affected by the expression level of polyhydroxyalkanoate synthase, the enzyme responsible for the polymerization of the monomers. Overexpression of the synthase under the control of an inducible promoter resulted in a threefold to fivefold lower average molecular weight and greater polydispersity, although the overall level of PHA produced was not significantly affected [ 13’1. ‘I-his suggests a model in which each enzyme unit is responsible for the synthesis of a single polymer chain.
An area that has received considerable attention due to its utility as well as its complexity is the biosythcsis of aromaric metabolites. ‘l’he large number of branch points that must be controlled in order to direct the maximal amount of metabolic flux to the product or products of interest make this an extraordinarily challenging problem. A central focus of this field is increasing the flux through 3deox~-I)-arabino-hepculosonate-7-phosI~h~te (DMIP). the first committed intermediate in the synthesis of aromatic amino acids. DAI~IP is derived from cr~throse-4-phosphate (E4P) and phosphoenolpyrurate (PEP) in a reaction catalyzed by Dr\HP synthase. Overexpression of DAHP synthase shifted the focus to increasing available UP and PEP as well as control of the downstream steps [lq,lS]. Overexpression of transketolase [16] or PEP synthase [ 171 also increases DAHP production. presumably b) increasing the available supply of E4P and PEP. hlore recent studies have focused on the role of transaldolase. which also products E3P. Transaldolase overexpression increases obtained
DAHP production: however. no advantage is in cells that overexpress translketolase. suggest-
ing that transketolase overexpression saturating levels of EIP [ 181.
To further
increase
the available
is adequate
supply
to reach
of PEI’, \‘alle
and
co-workers [ 19’1 examined the glucose uptake pathnay. Internalization is driven by the glucose phosphotransferase system (P’I’S). lvhich converts glucose to glucose-6-phosphate using PEP 3s a phosphate donor. By inacti\.ation of the PTS and selection for growth on ,glucose. mutant by a strains \vere obtained that internalize glucose non-PTS system while maintaining wild-type growth rata [ 19’1. Higher levels of D.4llP production are observed, presumably due to 21larger available supply of PEP.
Secondary metabolites The challenges associated lvith engineering the biosynthesis of secondary metabolites are qualitatively different from those associated with bulk chemicals. Products of interest are often complex molecules that are necessarily derived from biological sources. In this arena. pathlvay engineering may increase the efficiency of existing production methods but may also lead to the development of new products. The relatively high vdur of these products shifts the emphasis from economics and efficiency to innovation. The antibiotic pristinamyin. produced by J’tqptorqres p/-istimNe.spir-,l/is, consists of a mixture of compounds includin,. pristinamycins 11,~ and 1113(PII,., and PIIll), Ivhich arc normally present in the ratio SO:20 (Pll,~:Pll~~). PII. is derived from the oxidation of PI113 in a reaction catalyzed by Pll,~ synthase. A new semisynthetic derivative of pristinamycin with improved properties utilizes PII, as a starting material. and it nas therefore desirable to increase the yield of PII. by engineering the complete conversion of Pll~+ The .wnA and snnB genes (which code for the two components of the heterodimcric PII. synthase) were cloned behind the strong. constituitive em/E* promoter and intcgratcd into the chromosome using the pSAhl2 integrative vector [20’]. Chromosomal integration has the advantages of high stability and fixed single-copy status, which arc desirable traits for industrial application. The engineered organism demonstrated the complete conversion of PI113 to PII;\ with a negligible drop in overall production.
Rapid advances in the genetics of antibiotic biosythesis has led to the engineering of these pathways to produce novel products. In some CWZS. the genetic and catalytic structures are modular, allowing the rational design of pathways leading to new ‘unnatural’ natural products. This has been applied to nonribosomal peptide synAcsis (211 as well as polyketide biosynthesis [ZZ]. Several esamplcs have come from the study of 6-droxyerythronolide B synthase (DEBS), a multienzyme complex that produces h-droxyerythronolide B (6-dEB), the polykctide core of the broad spectrum antibiotic erythrom)-tin.
Leadlay and co-lvorkers [Z3] demonstrated that the modular nature of DEBS allous the rational .substitution of domains to produce novel products. DEBS normally produces 6-dEB, a polypropionate macrolactonc hy the step-wise condensation of meth);lmulon~I-Cor\ extender units. The sprciticitv for meth~lmalon)-l-CoA~~lon~l-~0,~~ resides in the six X)-I transferase domains (one for each extension step). LTsing a simplified model system, the researchers replaced a methylmalonl;l transferat: domain with a malonyl transferdse domain from another polvketide synthasr [Xl. The engineered construct incorporates malonyl-CoA at the expected position, producing the predicted ‘nor-methyl’ product. .\lore rcccntly similar acyl transferase substitutions have been performed in
New directions
the entire erythromycin pathway to generate biologically active analogs of this antibiotic [24,X]. Not only can functional domains be exchanged, but new functions may be added. Kao et N/. [26”] inserted dehydratase and enoylreductase domains system. %‘here the wild-type
into an engineered DEBS enzyme produces a hydroxyl
group, the engineered strain carries out two additional catalytic steps to generate a methylene group. Another recently reported method for the engineered biosynthesis of noye products involves the use of synthetic chemistry in combination with genetic engineering [27’]. hlutational inactivation of an early step in biosynthesis of 6-dEB generates an enzyme that requires exogenous addition of a synthetic intermediate. It was demonstrated that the mutant enzyme can incorporate non-natural analogs of the intermediate to produce corresponding analogs of 6-dEB. The ability to carry out these sorts of manipulation on natural products of proven medicinal relevance suggests the possibility of creating new small-molecule drug candidates via genetic engineering.
in metabolic
engineering
Jacobsen
and Khosla
135
might seem unlikely to limit the growth of higher plants, VHb expression in transgenic tobacco plants led to several beneficial metabolic changes [35’]. Germination times were reduced: this may be ascribed to increased available oxygen for respiration and growth. Levels of chlorophyll were increased 30-40% in the transgenic plants, possibly resulting from an increase in oxygen available for its biosynthesis. An increase in available oxygen also would explain the observed increase in the (oxygen-dependent) production of nicotine and concomitant decrease in production of anabasine, which are derived from the same precursors in an oxygen-independent reaction. Hetcrologous expression of other metabolic enzymes has been used advantageously to affect changes in growth and differentiation. Expression of a yeast invertase in potato tubers affects the size and number of tubers, although the effect is dependent on the localization of the invertase [36]. Cyosolic expression leads to increased numbers, whereas it has been shown that targeting the invertase to the apoplast leads to increased tuber size.
Control of growth and differentiation In fields as diverse as agriculture and biopharmaceutical production, metabolic engineering may be used to generate organisms with desirable growth characteristics. An early example of pathway engineering was the expression of I’heosrilh hemoglobin (VHb) in E. coli [28]. This oxygen-binding protein was shown to enhance growth under oxygen-limiting conditions, and a series of detailed experiments have begun to reveal the metabolic alterations induced by \‘Hb expression. Oxygen uptake in E. co/i grown under microaerobic conditions varies with \!Hb dosage, and VHb+ cells show decreased production of reduced metabolites, supporting an important role for \‘Hb in maintaining the cell in a more oxidized state [29]. Furthermore, metabolic flux analysis [30] showed that VHb expression perturbed central metabolism as well, directing increased flux through the pentose phosphate pathway [29], an effect that is consistent with increased intracellular oxygen levels. This appears to be mediated by increased expression and activity of cytochrome o (cyt o), a terminal oxidase in the electron transport chain with relatively lo\v oxygen affinit): A second terminal oxidase, cytochrome n (cyt (I?. displays higher oxygen affinity but lower proton-pumping efficiency and is normally active under lowoxygen conditions. \‘Hb increases both the expression and apparent os)-gen affinity of cyt 0, improving microherobic respiration and growth [31*]. YHb expression in a variety of hosts led to improved growth characteristics in several cases [32.33]. Of particular interest are examples in which improved growth is accompanied by increased flux through an oxygen-dependent pathlvay of interest. .4s a model for the oxidative detoxification of environmental pollutants, Stark and co-ivorkers [3-l] examined the effect of YtIb expression on the degradation of benzoic acid by X~~f/lorrrort~~ mzl~ophilicr, and noted improved conversion efficiency. Although oxygen
hletabolic engineering has been applied to the elimination of the requirement for animal serum in mammalian cell culture. Although serum is costly and can interfere with the recovery of biopharmaceuticals produced in cell culture, it is normally required in order to provide essential growth factors. To remove this constraint, Bailey and co-workers [37] first determined the cellular proteins that lvere produced in response to growth factors. Using two growth-stimulating factors for CHO cells, cellular protein levels were analyzed by two-dimensional gel electrophoresis and compared to cells grown in fetal calf serum. This powerful analytical technique identified three proteins that were upregulated by both growth factors. These were identified as cyclin Dl, cylin E, and EZF-1, proteins known to have roles in the cell cycle. The overexpression of either EZF-1 [38*] or cyclin E [39] in CHO cells eliminated the requirement for serum or growth factors in cell culture.
New approaches
to pathway engineering
hletabolic engineering has been defined as the use of genetic techniques to systematically manipulate the metabolic activities of living cells (401. Two recent reports challenge this definition by engineering metabolism in new nays. Bertozzi and co-workers [31’] made use of the intrinsic promiscuity of glycosyl transferdses to re-engineer cell-surface carbohydrates without making any genetic alterations. Using a chemical approach. synthetic carbohydrates bearing a reactive ketone group were incorporated metabolically into cell-surface oligosaccharides. Chemoselective reactions carried out on the engineered cell surface then allowed the introduction of new epitopes for the modulation of cellular recognition. To improve E. co/i resistance to the arsenate, Stemmer and co-workers [4P]
toxic effects of took a new, non-
136
Biocatalysis
and biotransformation
approach to genetic is a method for recombining
rational
engineering.
and selrcring
DNA shuffling advantageous
mutations. allo\ving the rapid evolution of function [43]. Starting u+rh 2 plasmid-borne arsenic resistance opcron. three cycles of fragmentation and recombination afford& a IO-fold incrcasc in resistance. Sequencing of the gcncs revealed a small number of mutations; ho\ve\,cr, the mechanism by lvhich these mucations confer improvementremains unknovm A major advanuge of this nonrarional approach to engineering is its ability to optimize the function of gcnch u+thout deuiled understanding of the metabolic pathw;l)-s.
tion of the genes for arablnose metabolism allowed the engmeered organism to metabolize this pentose. This work complements previous experiments in which the xylose metabolic pathway was introduced into this otgamsm, and represents a promising approach to the efficient utilization of llgnocellulosic biomass, a low-cost renewable resource. 7.
Lee SY: Bacterial 1996, 49:1-14.
8.
Hahn SK, Chang YK, Lee SY: Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eotrophus and recombinant Escherichia co/i. Appl Environ Microb/o/ 1995, 61:34-39.
9.
Pomer Y, Dennis DE, Klomparens K, Somerville C: Polyhydroxybutyrate. a biodegradable thermoplastic. in transgenic plants. Science 1992, 256:520~523.
10.
Conclusions LTntil recently, the field of metabolic engineering has largely been ;I collection of thematically related problems: holvevrr. thr swcesse~ and failures in this field art: gradually beginning to give rise to heuristics with predictivr power. Although the transformation of metabolic enginecring into ;I rigorous science must avxit the explanation of these (and other act-co-b~-disco~~r~~i) heurisCcs in terms of fundamental physicochcmical principles. t-hz gro\vth of this tield lvill unquestionably bc propelled by the mawration of some of rhe aboxx-mentioned problems into successful products and technologies rogcthcr lvith t-he emergcncc of ne\v and even mow inwllcctuall~ ‘I’he next few years arc likely to challenging problems. lvicnt-ss a significant cspansion of chc breadth and deprh of metabolic engineering.
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Biofechnol
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John ME, Keller G: Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells. Proc NaU Acad SC; USA 1996, 93:12768-l 2773. The polyhydroxybutyrate synthesis pathway was expressed in cotton fiber cells and the resulting transgenic fibers were shown to contain low levels of PHB. Even at low levels, the PHB conferred measurably improved Insulating propertIes to the fiber. 12.
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6. .
Deanda K. Zhang M. Eddy C, Plcatagglo S: Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl Environ Microbioi 1996, 62:44654470. Although Zymomonas mobIlis efficiently ferments the glucose component of ltgnocelluloslc feedstock to ethanol, It lacks the pathways for pentose metabolism necessary for efficient utllizatlon of these feedstocks. Introduc-
analysis Biotechnoi
19. .
Flares N. Xlao J, Berry A. Bollvar F, Valle F: Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Bfotechnology 1996, 14:620-623. Microbial production of aromatlc compounds may be Improved by dlrectlng the maYlmal amount of metabolic flux through DAHP and Into aromatlc ammo acid biosynthesis. This report describes a method for increasing the amount of PEP available for DAHP synthesis. The E. co/i PTS utlllzes one molecule of PEP for each molecule of glucose imported. By mutattonal InactIvatIon of the PTS and selection for growth on glucose. mutant strams were obtained that Internalize glucose by a non-PTS system while malntalnlng wild-type growth rates. Higher levels of DAHP production are observed, presumably due to a larger available supply of PEP. 20. .
Sezonov G, Blanc V, Bamas-Jacques N, FrIedmann A. Pernodet J-L, Gu&neau M: Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes. Nat BYootechnol 1997, 15:349-353. Streptomyces pristmaespiralis normally produces a mixture of Pll~ and Its derlvatlve PII,. In this report the snaA and snag genes (which code for the
New directions
two components of the heterodimeric PllA synthase) were cloned behind the strong, constltuitlve ermf’ promoter and integrated into the chromosome. Chromosomal integration has the advantages of high stability and fixed single-copy status, which are desirable traits for Industrial application. The engineered organism demonstrated complete conversion of Plla to Pll~ with, negligible drop in overall production. This demonstrates an application of metabolic engineering to secondary metabolism in order to increase the production of a high-value product. 21.
Stachelhaus T, Schneider A, Marahiel MA: Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 1995, 269:69-72.
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Tsai PS, Hatzimanikatis V, Bailey JE: Effect of Vitreoscilla hemoglobin dosage on microaerobic fscherichia co/i carbon and energy metabolism. Biotechnol Bioeng 1996, 49:139-l 50.
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Liu S-C, Webster DA, Wei M-L, Stark BC: Genetic engineering to contain the Wfreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia. Biotechnol Boeng 1996, 49:101-l 05.
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Holmberg N, Lilius G, Bailey JE, Bijlow L: Transgenic tobacco expressing Ktreoscilla hemoglobin exhibits enhanced growth and altered metabolic production. Nat Biotechnol 1997, 15:244247. Expression of VHb in tobacco plants was shown to have several beneficial effects. Chlorophyll production increased 30-404/o in the transgenic plants, possibly resulting from an increase in oxygen available for its biosynthesis. Nicotine production, which is oxygen dependent, was also increased at the expense of oxygen-independent production of anabasine. 36.
Sonnewald U, Hajlrezaei M-R, Kossmann 1, Heyer A, Trethewey RN, Wlllmitzer L: Increased potato tuber size resulting from apoplastic expression of a yeast invertase. Nat 5iotechnol 1997, 15:794-797.
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Lee KH, Sburlatl A, Renner WA, Bailey JE: Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovarv cells shifts Drotein oatterns and activates growth in prdtein-free medium. Bibrechnol Bioeng 1996, 50:273-279. In this report, a metabolic engineering approach was taken to eliminate the costly and inconvenient requirement for animal serum In mammalian cell culture. Recent advances in the understanding of the cell cycle suggested that deregulated expression of the transcription factor E2F-1 would bypass the requirement for growth factors normally obtained from serum. CHO cells were transfected with E2F-1 and the resulting overexpression of this protein allowed the cells to grow in serum-free media. 39.
Renner WA, Lee KH, Hatzimanikatis V, Bailey JE, Eppenberger HM: Recombinant cyclin E expression activates proliferation and obviates surface attachment in Chinese hamster ovary (CHO) cells in protein-free medium. Biotechnol Bioeng 1995, 47:476-482.
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41. .
Mahal LK, Yarema KJ, Bertozzi CR: Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997, 276:l 125-l 126. The intrinsic promiscuity of glycosyl transferases was exploited to re-engineer cell surface carbohydrates. Using a chemical approach, synthetic carbohydrates bearing a reactive ketone group were incorporated metabolically into cell-surface oligosacchandes. Chemoselective reactions carried out on the engmeered cell surface allowed the introduction of new epltopes for the modulation of cellular recognition. This was demonstrated by an example of selective drug delivery. This approach demonstrates metabolic engineering through chemistry rather than genetic alteratlon.
Tsai PS, NIgeli M, Bailey JE: Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic fscherichia co/i metabolism through elevated concentration and specific activitiy of cytochrome o. Biotechnol Bioeng 1996, 49:151-l 60. Expression of VHb in f. co/i and other organisms appears to increase the amount of oxygen available for metabolic processes. This paper shows that this observed effect is medlated by increased expression and activity of cyt o, a terminal oxtdase in the electron transport chain with relatively low oxygen affinity. A second terminal oxidase, cyt d, displays higher oxygen affinity but lower proton-pumping efficiency and is normally active under low oxygen conditions. VHb increases both the expression and apparent oxygen affinity of cyi o, improving microaerobic respiration and growth.
Crameri A, Dawes G, Rodriguez E Jr, Silver S, Stemmer WPC: Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat Biotechnol 1997, 15:436-436. This study demonstrates a nonrational approach to the metabolic engineering of E. co/i to improve resistance to the toxic effects of arsenate. DNA shuffling is a method for recombining and selecting advantageous mutations, allowing rapid evolution of function. Starting with a plasmid-borne arsenic resistance operon, three cycles of fragmentation and recombination afforded a 40.fold increase in resistance. Sequencing of the genes revealed a small number of mutations; however, the mechanism by which these mutations confer improvement remains unknown. A major advantage of this nonrational approach to engineering is its ability to optimize the function of genes without detailed understanding of the metabolic pathways.
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Magnolo SK, Leenutaphong DL, DeModena JA. Curtis JE, Bailey JE, Galauo JL, Hughes DE: Actinorhodin production by Sfreptomyces coelicolor and growth of Streptomyces lividans
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