Toward a Science of Metabolic Engineering
`
`JAMES E. BAILEY
`
`Application of recombinant DNA methods to restructure
`metabolic networks can improve production of metabo-
`lite and protein products by altering pathway distribu-
`tions and rates. Recruitment of heterologous proteins
`enables extension of existing pathways to obtain new
`chemical products, alter posttranslational protein pro-
`cessing, and degrade recalcitrant wastes. Although some
`of the experimental and mathematical tools required for
`rational metabolic engineering are available, complex cel-
`lular responses to genetic perturbations can complicate
`predictive design.
`
`T HE METABOLIC ACTIVITIES OF LIVING CELLS ARE ACCOM-
`plished by a regulated, highly coupled network of -1000
`enzyme-catalyzed reactions and selective membrane trans-
`port systems. However, metabolic networks that evolved in natural
`settings are not genetically optimized for the objectives important in
`practical applications. Hence, performance of bioprocesses can be
`enhanced by genetic modification of the cells.
`Metabolic engineering is the improvement of cellular activities by
`manipulation of enzymatic, transport, and regulatory functions of
`the cell with the use of recombinant DNA technology. The oppor-
`tunity to introduce heterologous genes and regulatory elements
`distinguishes metabolic engineering from traditional genetic ap-
`proaches to improve the strain. This capability enables construction
`of metabolic configurations with novel and often beneficial charac-
`teristics. Cell function can also be modified through precisely
`targeted alterations in normal cellular activities. Examples in the
`manipulation ofprotein processing pathways, as well as of pathways
`involving smaller metabolites, will be highlighted here.
`At present, metabolic engineering is more a collection ofexamples
`than a codified science. Results to date promise future technological
`benefits, as well as contributions to basic science, agriculture, and
`medicine. However, many studies have shown the feasibility of
`metabolic engineering methods without achieving the yields, rates,
`or titers (final concentrations) required for a practical process. Most
`experiments explore changes in a single gene, operon, or gene
`cluster. After a new strain has been created by such a manipulation,
`limitations arise that can in principle be addressed by subsequent
`genetic manipulation. An iterative cycle of a genetic change, an
`analysis of the consequences, and a design of a further change,
`analogous to that articulated for protein engineering (1), can be
`used to find an optimized strain. The few cases to date in which such
`a metabolic engineering cycle has been implemented have achieved
`success. An emerging base of strategies, tools, and experiences will
`aid in identifying, implementing, and refining which particular set of
`
`The author is the Chevron Professor ofChemical Engineering at the California Institute
`of Technology, Pasadena, CA 91125.
`
`1668
`
`genetic manipulations is most effective in accomplishing a desired
`change in cellular function.
`
`Recruiting Heterologous Activities for Strain
`Improvement
`Cloning and expression of heterologous genes can serve several
`useful purposes, including extending an existing pathway to obtain
`a new product, creating arrays of enzymatic activities that synthesize
`a novel structure, shifting metabolite flow toward a desired product,
`and accelerating a rate-determining step. Introduction of a function-
`al heterologous enzyme or transport system into an organism can
`result in the appearance of new compounds that may subsequently
`undergo further reactions. Difficulties in anticipating these further
`reactions are a central limitation of metabolic engineering.
`Expression of a heterologous protein does not guarantee appear-
`ance of the desired activity. The protein must avoid proteolysis, fold
`properly, accomplish any necessary assembly and prosthetic group
`acquisition, be suitably localized, have access to all required sub-
`strates, and not encounter an inhibitory environment. Despite these
`potential barriers to the successful recruitment of heterologous
`cellular activities, the number and scope of positive experiments
`encourage further application of this approach.
`Synthesis ofnew products is enabled by completion ofpartial pathways.
`The genetic and metabolic diversity that exists in nature provides a
`collection of organisms with a spectrum of substrate assimilation
`and product synthesis capabilities. However, many natural strains
`are imperfect from an applied perspective. Their performance can
`sometimes be enhanced by extension of their native pathways.
`Native metabolites can be converted to preferred end products by
`the genetic installation of a few well-chosen heterologous activities
`(Table 1).
`For example, the final precursor in a current commercial process
`for ascorbic acid (vitamin C) synthesis is 2-keto-L-gulonic acid
`(2-KLG). One route to 2-KLG involves two successive fermenta-
`tions. The first converts glucose to 2,5-diketo-D-gluconic acid
`(2,5-DKG) in Erwinia herbicola; the second fermentation, carried
`out in a species of Corynebacterium, transforms 2,5-DKG to 2-KLG.
`Researchers devised a way to convert glucose to 2-KLG in a single
`fermentation step by cloning the Corynebacterium enzyme 2,5-DKG
`reductase, which catalyzes the 2,5-DKG to 2-KLG conversion, into
`E. herbicola (2). A similar goal was achieved for 7-aminocephalo-
`sporanic acid (7ACA), the precursor for several semisynthetic
`cephem antibiotics (3).
`Posttranslational modifications can influence the function of
`proteins. The types of modifications that occur can be affected by
`expression ofcloned protein processing enzymes. For example, expres-
`sion in Chinese hamster ovary (CHO) cells of P-galactoside a2,6-
`sialyltransferase (4) allows the formation of sialyl a2,6-galactosyl
`linkages on its surface glycoproteins. These terminal glycosylation
`linkages are normally absent from proteins produced in this industrial
`
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`cell line, including cloned erythropoietin. Thus, this strategy should
`enable erythropoietin made in recombinant CHO cells to more closely
`resemble human erythropoietin, which is rich in these linkages (5). In
`another study, mouse cells displayed the human H blood group
`antigen after transfection with human DNA (6).
`Transferring multistep pathways: Hybrid metabolic networks. The trans-
`fer of genes that encode entire biosynthetic pathways to a heterolo-
`gous host can provide more industrially robust strains, enhance
`productivity, or permit the use of less costly raw materials. Moreover,
`such experiments are useful for exploring the regulation and function
`of a multistep metabolic pathway in a particular species.
`Transferring entire antibiotic biosynthetic pathways to heterolo-
`gous hosts has been facilitated by the clustering of the genes
`involved (7). Genes for the biosynthesis of actinorhodin were
`transferred from Streptomyces coelicolor to Streptomyces tividans, en-
`abling the latter strain to produce actinorhodin (8). Subsequently,
`clustered erythromycin biosynthetic genes from Streptomyces eryth-
`reus were transferred to S. Iividans, which then synthesized an
`antibiotic indistinguishable from erythromycin A (9). Escherichia coli
`carrying this cloned gene cluster did not synthesize the antibiotic,
`possibly because of low transcriptional activity of Streptomyces
`promoters in E. coli. The fungi Neurospora crassa and Aspergillus
`niger, which normally do not produce P-lactam antibiotics, synthe-
`sized penicillin V after transformation with a cosmid containing
`Penicillium chrysogenum DNA that encoded enzymes in the penicillin
`biosynthetic pathway (10).
`Polyhydroxybutyrate (PHB), a storage product sequestered in
`large amounts by some bacteria under growth-limiting, carbon
`source-excess conditions, is a biodegradable polyester that already
`has small-scale applications. Alcaligenes eutrophus can produce not
`only PHB but, when supplied with different precursors, can synthe-
`size various polyhydroxyalkanoate copolymers as well (11). Meta-
`bolic engineering of the synthesis of these and related polymers
`should provide greater control over the nature and quantity of the
`polymer produced and should also offer alternative production
`organisms. The PHB synthesis operon from A. eutrophus, which
`encodes PHB polymerase, thiolase, and reductase activities, has been
`used to transform E. coli (12). As inA. eutrophus, this recombinant
`E. coli accumulates PHB when the nitrogen source is depleted; PHB
`concentrations in these cultures reach 50% of the dry cell weight.
`Assembly of pathways for simultaneous degradation of chloro-
`and methylaromatics by combining and refining of cloned pathway
`segments and regulatory systems from several different organisms
`
`exemplifies the iterative design of an effective hybrid organism (13).
`The ultimate strain thus far constructed contains five pathway
`segments obtained from three organisms. The biochemical and
`metabolic complexities of the degradation of mixed substrates and
`the resulting rationale behind each portion of this construction offer
`useful general perspectives on metabolic engineering strategies (14).
`Creating new products and new reactants. Expression of biosynthetic
`genes for a secondary metabolite in a heterologous host that
`synthesizes its own different secondary metabolite can result in the
`construction of an array of enzymatic activities that yield novel
`products. Among the novel antibiotics that have been produced in
`recombinant strains of Streptomyces by such manipulations are
`mederrhodins A and B and dihydrogranatirhodin (15), 2-noreryth-
`romycins A, B, C, and D (16), and isovaleryl spiramycin (17).
`Compounds new to the cell that result from a heterologous
`activity often undergo further reactions. In some cases, such as in the
`biosynthesis of indigo by E. coli that express Pseudomonas putida
`naphthalene dioxygenase (18), these subsequent reactions are essen-
`tial components of the desired pathway. Another illustration of
`metabolic engineering to introduce a novel intermediate into a host
`involves recombinant E. coli that express the cloned tyrosinase gene
`from Streptomyces antibioticus (19). Synthesis by the recombinant E.
`coli strain of the pigment melanin, an ultraviolet light-absorbing
`compound with material and cosmetic applications, depends on a
`single critical catalytic step: the oxidation of tyrosine and L-dopa to
`dopaquinone by tyrosinase; the remaining reactions that yield melanin
`are apparently nonenzymatic. Melanin production is increased when
`another protein from S. antibiotcus is coexpressed with tyrosinase.
`Although definitive evidence is not yet available, this second protein
`may provide a copper-donor function that activates apotyrosinase.
`Thus, increasing the expression of a cofactor-requiring protein as part
`of a metabolic engineering scheme may require the engineering of an
`increased supply of the cofactor as well.
`Biodegradation of undesirable compounds can often be accom-
`plished by host enzymes after a heterologous activity provides the
`initial attack on the target compound or compounds. For example,
`the expression of Pseudomonas mendocina toluene monooxygenase in
`E. coli enabled the efficient degradation of trichlorethylene, a
`suspected carcinogen and widespread pollutant (20). In E. coli,
`degradation can be induced by isopropyl-l-thio-13-D-galactoside or
`by a temperature shift, rather than by toluene, as occurs in P.
`mendocina. In addition, the engineered E. coli has degradation
`kinetics (no competitive inhibition, as with toluene) and cosubstrate
`
`Table 1. Heterologous activities recruited to alter small metabolite and
`protein end products. The original metabolite serves as the substrate for the
`synthesis of the new product through a pathway involving the new interme-
`diate. It is difficult to prove that the inserted activity alone is responsible for
`
`an altered phenotype. In each case discussed here, the observed change in cell
`function is consistent with the expected consequence of the newly installed
`gene or genes. A. chrysogenum, Acremonium chrysogenum; GDP, guanosine
`diphosphate, F. solani, Fusarium solani; P. diminuta, Pseudomonas diminuta.
`
`Host orga nism
`
`Original metabolite
`
`E. herbicola (2, 21, 22)
`
`A. chrysogenum (3)
`
`2,5-DKG
`
`Cephalosporin C
`
`CHO cells (4)
`
`Mouse L cells (6)
`
`21 JUNE 1991
`
`Terminal P-galactosyl
`residues in
`N-acetyllactosamine
`sequences
`Unsubstituted type H,
`N-acetyllactosamine
`glycoconjugate end groups
`
`21 JUNE 1991
`
`Heterologous enzymes
`added (source organism)
`2,5-DKG reductase
`(Corynebacterium)
`D-Amino acid oxidase (F.
`solani), cephalosporin
`acylase (P. diminuta)
`
`P-Galactoside a2,6-
`sialyltransferase (rat)
`
`GDP-L-fucose:
`P-D-galactoside
`2-a-L-fucosyltransferase
`(A431 human cell line)
`
`New product (new
`intermediate)
`2-KLG
`
`7ACA
`[7-0-(5-carboxy-5-
`oxopentanamido)-
`cephalosporanic acid]
`Sialyl a2,6-galactosyl
`linkages
`
`H fucosyl al1- 2
`galactosy linkages
`
`1669~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`
`ARTICLES
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`1 669
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`requirements (glucose instead of toluene) that are superior to those
`of the native host of this toluene monooxygenase activity. This
`example illustrates that transfer of a crucial enzyme activity to a
`different regulatory environment can render that activity useful for
`biotechnology.
`New metabolites arising from the action of cloned heterologous
`enzymes may also undergo undesirable side reactions. The precursor
`of 7ACA in engineered Acremonium chrysogenum, produced as a
`consequence of the cloned D-amino acid oxidase, can also react with
`hydrogen peroxide to give a useless by-product, dramatically reduc-
`ing 7ACA yield (3). Cloned degradation enzymes have led to
`metabolic dead ends in the sense that the host cannot convert their
`products further; in some cases these recalcitrant intermediates
`inactivate key catabolic enzymes (14). Other unexpected complica-
`tions can arise when desired end products are similar to some native
`metabolite and are converted to another product by host enzymes.
`After observations of unexpectedly low yields of 2-KLG in a
`recombinant strain (2), it was found that 2-KLG was converted to
`L-idonic acid by endogenous 2-ketoaldonate reductase (2KR).
`Cloning, deletion mutagenesis, and homologous recombination of
`the mutated gene for 2KR into the chromosome were part ofseveral
`steps undertaken to develop an engineered organism able to accu-
`mulate large amounts of2-KLG (> 120 g/liter) (21, 22). The present
`engineered metabolic pathway involving these constructs (Fig. 1)
`shows complex interactions of enzymes and substrates that were
`identified, characterized, and engineered in an iterative process.
`Pefecting strains by altering nutrient uptake and metabolite flow.
`Increased growth rates, decreased nutrient demands for cell growth,
`and higher attainable cell densities have advantages in many different
`applications. The use of metabolic engineering to realize these
`objectives has been based on increasing the efficiency of nutrient
`assimilation, enhancing the efficiency of adenosine triphosphate
`(ATP) production, and reducing the production of inhibitory
`metabolic end products. In one of the earliest applications of
`recombinant DNA to the improvement of the metabolism of the
`
`Fig. 1. Summary of the enzymes, intermediates, and by-products encoun-
`tered in the synthesis of 2-keto-L-gulonic acid (2-KLG) from glucose in
`genetically engineered E. herbicola. The control of cloned (white printing on
`black) 2,5-DKG reductase activity, which requires the reduced form
`(NADPH) of nicotinamide adenine dinucleotide phosphate (NADP) sup-
`plied by the cell metabolism, directs metabolite flow to 2-KLG. Wide band
`with dots, cell membrane; straight bars, transport systems. Abbreviations are
`as follows: GDH, D-glucose dehydrogenase; GADH, D-gluconate dehydro-
`genase; 2KDGDH, 2-keto-D-gluconate dehydrogenase; DKGR, 2,5-diketo-
`D-gluconate reductase; IADH, L-idonate dehydrogase; 5KR(G), 5-keto-D-
`D-glucose;
`(D-gluconate-producing);
`GA,
`G,
`gluconate
`reductase
`D-gluconate; 2KDG, 2-keto-D-gluconate; 2,5KDG, 2,5-diketo-D-gluconate;
`IA, L-idonate; and 5KDG, 5-keto-D-gluconate. Reprinted by permission
`from (21).
`
`1670
`
`commercial strain, the goal was improvement of the efficiency of
`carbon conversion into cell mass by Methylophilus methylotrophus, a
`strain developed as an animal feed material. The native route of
`nitrogen assimilation used by this bacterium is the glutamate
`synthase pathway, which consumes one ATP per nitrogen incorpo-
`rated into glutamate. Nitrogen assimilation by means of glutamate
`dehydrogenase, a process absent from this organism, does not
`require ATP. In an effort to improve cell yield, glutamate dehydro-
`genase from E. coli was expressed in a glutamate synthase mutant of
`M. methylotrophus (23). The efficiency of carbon conversion was
`increased 4 to 7%.
`End products of carbon catabolism (acetate, ethanol, and lactate)
`that inhibit cell growth are produced by bacteria, yeasts, and
`mammalian cells under conditions of oxygen limitation or carbon
`source excess. The final optical density of E. coli grown under
`shake-flask aerobic conditions was increased threefold after intro-
`duction of a plasmid that expressed pyruvate decarboxylase and
`alcohol dehydrogenase from Zymomonas mobilis (24). The former
`activity, absent in unmodified E. coli, redirects catabolite fluxes from
`pyruvate and results in a shift from acetate production, which
`strongly inhibits cell growth, to production of ethanol, which is less
`inhibitory.
`Microbial catabolic products such as ethanol, acetone, and buta-
`nol are important industrial chemicals. Large increases in ethanol
`yields from pentose and hexose sugar substrates from E. coli (25, 26)
`and Erwinia chrysanthemi (27) have been achieved by transformation
`with plasmids that encode pyruvate decarboxylase from Z. mobilis,
`in some cases coexpressed with Z. mobilis alcohol dehydrogenase.
`The E. coli so engineered have the potential practical advantages of
`rapid and efficient conversion of several sugars found in biomass
`(26).
`oa-Acetohydroxy acids, synthesized during fermentation by brew-
`ers' yeast, leak into the medium where spontaneous hydroxylation
`produces diacetyl, which has an undesirable flavor. On the basis of
`suggestions that the time required for beer lagering is determined by
`the time required for the enzymatic reduction of diacetyl by the
`yeast, genes for the enzyme a-acetolactate decarboxylase (a-ALDC)
`were cloned from Kiebsiella terrigena or Enterobacter aerogenes, fused
`to yeast promoters, and inserted into Saccharomyces cerevisiae on
`multicopy plasmids. This enzyme converts ca-acetolactate to acetoin,
`rather than diacetyl; acetoin influences flavor only at relatively high
`concentrations. Pilot brewing studies with these engineered strains
`that express a-ALDC yielded beer of quality equal to that produced
`by controls, but in a process time of 2 weeks, as compared to 5
`weeks for the conventional process. The lagering step could be
`omitted when the recombinant brewers' yeasts were used because of
`low diacetyl production by these organisms (28).
`Enabling a cell to utilize alternative materials as nourishment is
`another capability of metabolic engineering. In order to produce
`microbial surfactants from industrial waste raw materials, E. coli
`p-galactosidase and lactose permease were stably integrated into the
`chromosome of two Pseudomonas aeroginosa strains. These recombi-
`nant strains synthesize biosurfactants when grown in lactose and
`whey-based minimal media (29).
`Yeast ornithine decarboxylase was cloned and expressed in cul-
`tured roots of Nicotiana rustica in order to direct a greater metabolite
`flux from ornithine to putrescine, a precursor of nicotine (30). Some
`clones showed approximately two times as much nicotine accumu-
`lation as the controls. Rearrangement of the native fluxes in the
`hyoscyamine-rich Atropa belladonna was motivated by greater com-
`mercial demand for scopolamine, the 6,7-epoxide of hyoscyamine.
`Expression of Hyoscyamus niger hyoscyamine 63-hydroxylase in an
`A. belladonna hairy-root clone produced three to ten times as much
`scopolamine as did wild-type clones (31).
`
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`frequently complicated by the thick broths that result from growth
`of filamentous fungi and Streptomyces. Success of the strategy for
`enhancing aerobic metabolism in other bacteria prompted cloning
`and intracellular expression of VHb in two different Streptomyces
`species (41). Streptomyces lividans with a multicopy hemoglobin
`expression plasmid achieved final cell densities up to 54% greater
`than the untransformed host in shake-flask cultivations. The pres-
`ence of cloned intracellular VHb in S. coelicolor markedly increased
`secondary metabolite accumulation, without affecting cell growth
`relative to a control strain containing a mutated VHb gene (Fig. 2).
`These examples suggest a general genetic strategy for addressing
`stresses and corresponding productivity limitations encountered in
`bioprocessing: after identifying a response in nature to a similar
`stress (most likely involving a different organism), genes that specify
`that response can be transferred to the organism of choice.
`
`Redirecting Metabolite Flow
`Typically the route of reactions to a desired product passes several
`forks where intermediates can enter alternative pathways. At such
`bifurcations of metabolite flow, a common resource-for example,
`substrate, enzyme, transport system, or ribosome-contributes to
`two or more parallel processes. Maxmizing product formation
`requires that the desired route at each fork be made a priority and
`that traffic in alternative pathways be miimizd to the extent
`possible without decreasing cell viability.
`Directing traffic toward the desired branch. Amplification of the
`activity initiating a desired process at a fork in a metabolic flow is a
`common strategy of metabolic engineering. Whereas isolation of
`mutant enzymes that are desensitized to feedback repression was
`achieved with classical methods, such mutants may now be obtained
`more rapidly with the use ofcloned genes. This approach also avoids
`the complication of uncharacterized additional mutations that are
`often obtained with classical, whole-cell mutagenesis.
`The past decade has seen a new generation ofstrain improvements
`in amino acid-producing coryneform bacteria with metabolic engi-
`neering (also called molecular breeding) (42, 43). Central to the
`success achieved was the development of new vectors and transfor-
`mation procedures.
`Genetic engineering of improved threonine production by Brevi-
`bacterium lactofermentum illustrates some of the strategies useful in
`redirecting metabolite flow to the desired product. Figure 3 presents
`an abbreviated diagram of the reactions involved in the synthesis of
`the aspartate family of amino acids and a few key reactions that feed
`into the synthesis pathway for this family. Homoserine dehydroge-
`nase (HD) was amplified by cloning and transformation into a
`threonine- and lysine-producing mutant (designated M-15). This
`mutant organism was selected for its lack of feedback inhibition of
`aspartokinase by threonine and lysine and of HD by threonine (44).
`The respective final concentrations of threonine, homoserine, and
`lysine from benchtop fermentations were 25.0, 2.8, and 1.1 g/liter
`for the recombinant strain compared to 17.5, 0.5, and 12.1 for
`M-15. Subsequent fiuther engineering to coexpress cloned homo-
`serine kinase (HK) with HD further increased the final threonine
`concentration to 33 g/liter and reduced homoserine and lysine levels,
`relative to the strain with cloned HD alone (45). In another study,
`the coryneform gene for HD was mutagenized to eliminate feedback
`inhibition by threonine. Introduction ofthis mutated HD gene into
`a lysine producer shifted the final lysine concentration from 65
`g/liter to 4 g/liter and the final threonine concentration from 0 g/liter
`to 52 g/liter (43). Threonine production by M-15 was increased
`12% by the expression of cloned phosphoenolpyruvate (PEP)
`carboxylase (PEPCase) (46). This manipulation was motivated by
`ARTICLES
`1671
`
`Systematic genetic manipulation of protein processing pathways
`has proven effective in increasing the quantity of active protein
`recovered. Overexpression of the E. coli chaperone proteins GroES
`and GroEL provided a five- to tenfold increase in assembled
`cyanobacterial Rubisco (D-ribulose-1,5-bisphosphate carboxylase/
`oxygenase) enzyme coexpressed in E. coli. In vitro studies of
`interactions among Rubisco and the GroE proteins implicate Mg2+-
`ATP as a requirement for assembly (32). The failure to achieve
`assembly of Rubisco from higher plants in altered E. coli signals
`future challenges in the transfer of heterologous protein processing
`pathways (33). Other challenges include genetic manipulations of
`processing pathways of bacteria that alter the solubility of recombi-
`nant proteins (34). In addition, opportunities exist for extending
`such strategies to eukaryotic hosts (35).
`Transfer of promising natural motifs: Vitreoscilla hemoglobin. Be-
`cause of the constant drive toward maximum cell densities to
`maximize volumetric productivity, growth and product synthesis in
`many industrial processes are limited by oxygen supply. The Gram-
`negative aerobic bacterium Vitreoscilla, which lives in poorly aerated
`environments, synthesizes increased quantities of a hemoglobin
`molecule in oxygen-limited cultures (36). Although the function of
`this protein in its natural host has not been established, this pattern
`of regulation of expression, combined with the oxygen-binding and
`release characteristics of the protein, suggest a possible beneficial
`physiological activity in poorly oxygenated environments.
`Motivated by this hypothesis and the premise that this beneficial
`function might be genetically transferred to industrial microorga-
`nisms, the gene for Vitreoscilla hemoglobin (VHb) was cloned and
`expressed in E. coli (37). Escherichia coli that carried a single copy of
`this gene integrated in the chromosome synthesized total cell
`protein more rapidly than an isogenic wild-type strain in oxygen-
`limited cultivations (Fig. 2), a response attributed to an increased
`efficiency of net ATP synthesis in the hemoglobin-expressing strain
`(38). Facilitation of oxygen transfer to the respiratory center (39)
`and modification of some aspect of cellular redox chemistry (38)
`have been suggested as contributing mechanisms for these phenom-
`ena. Coexpression of VHb increases the expression of cloned
`13-galactosidase, chloramphenicol acetyltransferase (CAT) (38), and
`a-amylase (40) by 1.5- to 3.3-fold relative to controls in oxygen-
`limited E. coli cultures, probably as a result of enhanced net ATP
`synthesis.
`Aeration of bioreactors used in the synthesis of antibiotics is
`
`Fig. 2. (A) Time trajec-
`tories of total E. coli cell
`protein
`(dashed
`lines)
`and cloned CAT activity
`(solid lines) in the wild
`type (open symbols) and
`an engineered host that
`expresses VHb from a
`single gene copy inte-
`grated in the chromo-
`some (closed symbols)
`in oxygen-limited fed-
`batch fermentations [re-
`printed from (38)]. (B)
`(dashed
`Cell
`densities
`lines) and concentration
`of actinorhodin in the
`medium (solid lines) in
`batch fermentations of
`S. coelicolor. Closed sym-
`bols are from a transfor-
`mant expressing active
`VHb; open symbols are from a control transformant not expressing VHb
`[reprinted from (41)].
`
`O.
`~.73
`50
`&b.
`
`-0.
`
`0.
`
`4
`4BB
`
`10
`
`16
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`22
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`
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`122
`
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`
`0
`
`0
`
`80
`40
`Time (hours)
`
`120
`
`21 JUNE 1991
`
`27'
`
`3._500
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`.9
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`E.
`
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`
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`Fig. 3. Pathways of the biosynthesis of the aspar-
`tate family of amino acids. Metabolite abbrevia-
`tions are as follows: acetyl CoA, acetyl coenzyme
`A; TCA, tricarboxylic acid cycle; Asp, aspartate;
`ASA, aspartate semialdehyde; Hse, homoserine;
`Lys, lysine; Met, methionine; Hse-P, 0-phospho-
`homoserine; and Thr, threonine.
`
`PEPCase
`
`OAA
`
`X
`
`Asp
`
`Aspartokinase
`
`PEP
`
`-OAcetyl CoA
`
`O CItrate
`
`o to TCA cycle
`
`HK
`
`o Hse-P
`
`o Thr
`
`HD
`- OASA - O Hse
`
`I
`
`Lys
`
`I
`
`Met
`
`the researchers' desire to increase oxaloacetate (OAA) production
`and thereby to increase carbon flow into amino acid production.
`Further improvements in rates of amino acid synthesis and yields
`will depend on a better understanding of mechanisms of regulation
`of gene expression and metabolite flow in these bacteria (47, 48).
`Because of metabolic engineering, E. coli has become an indus-
`trially important producer of amino acids. Transformation by
`multicopy plasmids that contain tryptophan (49) and threonine (50)
`biosynthetic genes have increased production of these amino acids.
`A project to engineer phenylalanine production in E. coli showed
`that overexpression of some genes in the phenylalanine biosynthetic
`pathway could cause a decrease in phenylalanine production and
`that inducible excision vector technology can be used to manipulate
`the biosynthesis of tyrosine, an inhibitor of the desired pathway
`(51). An intermediate in a metabolic pathway can be overproduced
`by combining a mutation that blocks that intermediate's use by the
`cell and by genetic augmentation of precursor flow into that
`pathway; although this concept has been used extensively in classical
`genetic production of organisms that are amino acid overproducers,
`it can be implemented in other contexts by metabolic engineering
`(52).
`The gene eryF in Saccharopolyspora erythrae encodes the first
`enzyme in the pathway from 6-deoxyerythronolide B to the antibi-
`otic erythromycin. After the targeted disruption of this gene using
`an integrative plasmid, 6-deoxyerythronolide B was converted to an
`erythromycin derivative that is more stable at the low pH of the
`stomach (53).
`Because enzyme activities involved in secondary metabolite pro-
`duction are regulated at both the gene and protein levels, identifying
`genetic changes that accelerate synthesis of these metabolites is
`challenging. One successful strategy is based on measuring the
`biosynthetic pathway intermediate concentrations in the growth
`medium. Relatively high extracellular concentrations of the inter-
`mediate penicillin N suggested that the activity that converts this
`intermediate to cephalosporin C (encoded in cefEF) may limit the
`rate of the overall pathway (54). Thus, expression of cefEF was
`elevated through increased gene dosage in a production strain of
`Cephalosporium acremonium. This recombinant fungus exhibited a
`15-fold reduction in penicillin N production and an increase of
`-15% in cephalosporin C production.
`Routing through protein processing pathways has also been
`altered by manipulation of host genes. The overproduction lethality
`3-galactosidase fusion proteins
`commonly observed with exported
`in E. coli is suppressed by the overproduction of E. coli prlF (55),
`and the expression of E. coli DnaK enables export of lacZ-hybrid
`proteins that are otherwise confined to the cytoplasm (56). An
`NH2-terminal methionine often differentiates cloned polypeptides
`synthesized in E. coli from their native human counterparts. Coex-
`pression of cloned E. coli methionine aminopeptidase with human
`interleukin-2 in E. coli has substantially reduced the fraction of
`product with methionine at its NH2-terminus (57). Observation of
`large quantities of a variant of human tissue plasminogen activator
`1672
`
`(tPA) associated with GRP78 in the rough endoplasmic reticulum
`of CHO cells suggested that GRP78 binding was a rate-limiting
`step in tPA secretion. (GRP78 is the 78-kD glucose-regulated
`protein, one of the stress-response proteins.) Coexpression of
`antisense GRP78 message resulted in smaller quantities of GRP78
`and faster tPA secretion (58).
`A quantitative study was conducted of S. cerevisiae isolates that
`contain different numbers of the phosphoglycerate kinase (PGK)
`gene (PGK1). In some cases a 10 to 15% increase in PGK activity
`gave rise to a higher (30%) overall cell mass yield when the yeast
`were grown on glucose. However, in an