8/3/2019 Plant Physiol. 2001 a 160 3

1/4

Plant Metabolic Engineering

Dean DellaPenna*

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan48824

Metabolic engineering is generally defined as theredirection of one or more enzymatic reactions toproduce new compounds in an organism, improvethe production of existing compounds, or mediatethe degradation of compounds. In highlightingprogress in plant metabolic engineering over the past25 years, it is first important to stress that it is in factquite a young science in plants. Our knowledge ofsubstrate-product relationships in plant pathways

was reasonably well advanced by 1975 as a result ofthe application of radiolabel tracer studies during theprevious decades. Attempts to use this knowledge toengineer metabolism in plants, however, first re-quired the development of basic molecular biologicaltechnologies such as cloning, promoter analysis, pro-tein targeting, plant transformation, biochemical ge-netics, and other areas of plant biology (describedelsewhere in this volume). Despite this delay signif-icant progress has been made since the mid-1980s inthe molecular dissection of many plant pathways andthe use of cloned genes to engineer plant metabolism.Although there are numerous success stories, there

has been an even greater number of studies that haveyielded completely unanticipated results. Such dataunderscore the fragmented state of our understand-ing of plant metabolism and highlight the growinggap between our ability to clone, study and manip-ulate individual genes and proteins and our under-standing of how they are integrated into and impactthe complex metabolic networks in plants. With anestimated 100,000 unique compounds produced inthe plant kingdom, elucidating these metabolic net-works is likely to be an exciting endeavor. The fewexamples cited in this article are meant to highlightcommon themes that have emerged in the field of

plant metabolic engineering, to exemplify the ad-vancements and limitations of current approachesand to provide a forward looking perspective of thisexciting area of plant biology over the coming years.

TECHNOLOGICAL ADVANCEMENTS IN GENEDISCOVERY HAVE HELPED TO DRIVEMETABOLIC ENGINEERING

The dependence of progress in plant metabolic en-gineering upon technological advancements is beau-tifully exemplified by research in lipid metabolism.

Though much was learned at the biochemical levelabout individual steps of plant lipid synthesis duringthe 1970s and early 1980s, progress in purifying andcloning many pathway enzymes, especially thosethat are membrane associated, was hindered due to

biochemical difficulties inherent in the target en-zymes. A major breakthrough in the field came fromgenetic dissection of the pathway in Arabidopsis (2).This pioneering work in plant biochemical geneticswas modeled after mutation-based approaches tostudy metabolism in bacterial systems. Earlier work

by Somerville and coworkers studying photorespira-tion in Arabidopsis proved the feasibility of using

biochemical genetics to dissect plant pathways (24).For lipid biosynthesis, over 10,000 mutated Arabi-dopsis plants were screened by gas chromatographyfor altered fatty acid profiles. This resulted in iden-tification of a suite of novel and informative muta-tions defining steps of the lipid biosynthetic path-way, which allowed genetic models of the plastidicand extraplastidic pathways to be developed andtested (2). The same mutants provided genetic targets

for subsequent cloning of several pathway genes bychromosome walking, T-DNA tagging, and varioushomology-based screening approaches (18). Our un-derstanding of the biosynthesis of other classes ofplant compounds such as amino acids, waxes, antho-cyanins, and ascorbic acid has been similarly ad-vanced by analogous molecular genetic approachesdissecting their respective pathways (5, 6, 13, 19, 20).

A second example of technologys impact on genediscovery for plant metabolic engineering comesfrom work on the carotenoid biosynthetic pathway.Though the pathway in plants had been known sincethe mid 1960s, the labile, membrane-associated en-

zymes remained recalcitrant to isolation and study.However, because carotenoids are also synthesized

by many photosynthetic and non-photosynthetic bac-teria, the development of molecular genetic tools inprokaryotes during the 1980s allowed plant research-ers to access carotenoid biosynthetic genes from pro-karyotes. Integrating prokaryotic systems into theirwork enabled researchers to finally clone the major-ity of carotenoid biosynthetic enzymes from plantduring the 1990s (for review, see 7).

One general approach used mutant complementa-tion to identify and isolate carotenoid biosyntheticgenes based on their resistance to specific herbicides.

An early success was the cloning of phytoene desatu-* E-mail dellapen@msu.edu; fax 5173539334.

160 Plant Physiology, January 2001, Vol. 125, pp. 160163, www.plantphysiol.org 2001 American Society of Plant Physiologists

8/3/2019 Plant Physiol. 2001 a 160 3

2/4

rase (PDS) from the cyanobacterium Synechococcus-PCC7942 (4). Mutants resistant to an herbicide thatinhibits PDS activity were first selected, and a libraryof the mutant DNA were transformed into wild-typeSynechococcus. The mutant PDS gene was identified

by its ability to confer herbicide resistance in the

wild-type background. The evolutionary relationshipof cyanobacteria and plants quickly allowed isolationof PDS orthologs from a number of plant species. Asecond approach, termed color complementation, en-gineered carotenoid biosynthetic genes from bacte-rial, fungal, and plant sources onto a single plasmidfor expression in Escherichia coli, which normallylacks endogenous carotenoids and the associated en-zymes. Depending on which genes and portion of thepathway were engineered, accumulation of variouslycolored pathway intermediates resulted. Transforma-tion of plant cDNA expression libraries into such E.coli backgrounds allowed functional identification of

the rare cDNAs (often one in several hundred thou-sand) encoding the next enzyme of the pathway

based on the associated change in color of the carot-enoid product. Similar strategies utilizing heterolo-gous systems have also allowed function-based clon-ing of enzymes for the synthesis of plant sterols,amino acids, and vitamins (11, 20, 22, 23).

PREDICTING THE OUTCOME OF METABOLICENGINEERING IS A CHALLENGING JOB

Although progress in pathway gene discovery and

our ability to manipulate gene expression in trans-genic plants has been most impressive during thepast two decades, attempts to use these tools to en-gineer plant metabolism has met with more limitedsuccess. Though there are notable exceptions, mostattempts at metabolic engineering have focused onmodifying (positively or negatively) the expressionof single genes affecting pathways. In general, theability to predict experimental outcomes has beenmuch better when one is targeting conversion ormodification of an existing compound to anotherrather than attempting to increase flux through apathway. Modifications to metabolic storage prod-ucts or secondary metabolic pathways, which oftenhave relatively flexible roles in plant biology, havealso been generally more successful than manipula-tions of primary and intermediary metabolism (16,26). Some brief examples follow.

As was the case for gene discovery, the lipid bio-synthetic pathway was one of the earlier pathways to

be targeted for manipulation and represents one ofthe better examples of metabolic engineering inplants to date. Most enzymes for fatty acid synthesisin plants have been cloned and various academic andindustrial groups have modified their expression tomanipulate oilseed fatty acid composition. Space per-mits only a single example to be discussed here and

the reader is referred to the Somerville article for

additional discussions. The engineering of soybeanand canola to produce higher levels of mono-unsaturated fatty acids was undertaken because theiroils contain high levels of linolenic acid (18:2), whichis susceptible to oxidation and limits the shelf life andutility of these oils. Antisense inhibition of oleate(18:1) desaturase expression resulted in oil that con-tained 80% oleic acid (a mono-unsaturated fattyacid) and had a significant decrease in polyunsatu-rated fatty acids (12). The resulting mono-unsaturate-rich oils are more stable to oxidation, healthier in thehuman diet than the corresponding poly-unsaturatecontaining oils, and represent an excellent early ex-ample of the practical application of metabolic engi-neering in plants.

Other areas of plant metabolism with high poten-tial to benefit human health have also been success-fully engineered in recent years (8). In one example,the last enzyme in the synthesis of -tocopherol,-tocopherol methyltransferase (-TMT) was used toincrease the vitamin E activity of Arabidopsis seedoil (22). Arabidopsis seed, like most oilseed crops,contains a high proportion of -tocopherol, whichhas 10% of the vitamin E activity of -tocopherol.Expression of -TMT in Arabidopsis seed resulted inthe conversion of the large pool of -tocopherol to-tocopherol with a corresponding 10-fold increasein vitamin E activity. Engineering similar conver-sions in soybean, canola and maize would elevate thelevels of this important antioxidant/vitamin in thediet and potentially have significant health conse-quences for the general population (10). In an exam-

ple of metabolic engineering of plant vitamin contenttargeted at the developing world, -carotene (provi-tamin A) was recently engineered into rice en-dosperm (29). Vitamin A deficiency is a serioushealth issue in many parts of the developing world.Rice is a major staple in developing countries, but isa poor source of many essential vitamins and miner-als, including -carotene (provitamin A). Efforts toengineer -carotene production in rice benefited di-rectly from the identification of carotenoid biosyn-thetic genes in model systems described earlier.Three carotenoid biosynthetic enzymes (two fromplants and one from bacteria) were engineered for

simultaneous expression in rice endosperm. The re-sulting first generation transgenic rice produced yel-low endosperm (so-called golden rice) containing-carotene at levels that would provide 10% of therecommended daily allowance with an average dailyrice intake. Subsequent manipulation may allow thevitamin A recommended daily allowance to be ap-proached with a daily rice intake and potentiallyprovide relief from vitamin A deficiency for millionsworldwide.

When faced with the novel experimental possibili-ties that molecular, genomic, and transgenic ap-proaches have presented over the past two decades,

researchers can be tempted to become fixated on

Plant Metabolic Engineering

Plant Physiol. Vol. 125, 2001 161

8/3/2019 Plant Physiol. 2001 a 160 3

3/4

producing transgenic plants and lose appreciationfor the important roles enzyme kinetics play at indi-vidual reaction steps and within entire pathways.The results of careful consideration of enzyme kinet-ics in metabolic engineering were elegantly demon-strated in research directed at modifying starch syn-thesis by manipulating ADP-Glc pyrophosphorylase(ADPGPP). Plant ADPGPP is sensitive to allostericeffectors and has been proposed to be a key regulatorlimiting starch synthesis. Escherichia coli ADPGPP isinvolved in glycogen synthesis and is also sensitiveto allosteric effectors. Mutations affecting allostericregulation cause an increase in glycogen levels in E.coli. Stark et al. (25) engineered wild-type and mutantE. coli ADPGPP for expression in plants and assayedthe effect on starch accumulation. Tubers from potatoplants transformed with the wild-type E. coli enzymehad starch levels similar to wild-type plants, whereasthose transformed with the allosterically insensitive

E. coli ADPGPP enzyme had starch levels up to 60%higher than wild type. The effect was only observedwhen the mutant protein was targeted to the chloro-plast and driven by a tuber specific promoter; con-stitutive expression was lethal. Such results demon-strate the importance of considering the targettissues, subcellular localization, and kinetics of en-zymes when engineering plant metabolism.

Attempts to manipulate the Lys content of seeds(Lys is a limiting amino acid in most seeds used forfood or feed) illustrate that one needs to considercatabolic, as well as anabolic, variables when tryingto engineer a particular metabolic phenotype in

plants. A key step in Lys synthesis is carried out bydihydrodipicolinate synthase, which is feedback in-hibited by the pathway end product, Lys, and thusplays a key role in regulating flux through the path-way. Engineering plants to overexpress a feedbackinsensitive bacterial dihydrodipicolinate synthase,similar to the approach with ADPGPP described ear-lier, greatly increased flux through the Lys biosyn-thetic pathway. However, in most cases this did notresult in increased steady-state Lys levels as theplants also responded by increasing flux through theLys catabolic pathway (1, 9). Substantial increases inLys only occurred in plants where flux increased to

such a level that the first enzyme of the catabolicpathway became saturated.

The manipulation of well-characterized rate-limiting enzymes of primary carbon metabolism tostudy their role in regulating pathway flux has pro-vided some of the more surprising results from met-abolic engineering in plants (for review, see 26).These experiments drive home the point that a thor-ough understanding of the individual kinetic prop-erties of enzymes may not be informative as to theirrole in complex metabolic pathways. Potential regu-latory enzymes are generally identified based ontheir catalyzing irreversible reactions and being reg-

ulated by appropriate effector molecules for a path-

way; traditional biochemical hallmarks of rate con-trolling enzymes. When the highly regulated Calvincycle enzymes Fru-1, 6-bisphosphatase and phosh-phoribulokinase were reduced 3- and 10-fold in ac-tivity, respectively, surprisingly minor effects wereobserved on the photosynthetic rate. In contrast, aminor degree of inhibition of plastid aldolase, whichcatalyzes a reversible reaction and is not subject toallosteric regulation, led to significant decreases inphotosynthetic rate and carbon partitioning. Thusaldolase, an enzyme seemingly irrelevant in regulat-ing pathway flux, was shown to have a major controlover the pathway. Analogous surprises were alsofound when manipulating presumed rate limitingenzymes of glycolysis. Such data has called intoquestion many of the longstanding ideas about fluxregulation in plants and is forcing a reassessment ofthe role of individual enzymes in the process. Thesestudies also make clear the caution that must be

exercised when extrapolating individual enzyme ki-netics to the control of pathway flux.

TRANSCRIPTIONAL REGULATORS MAY ALLOW

MANIPULATION OF ENTIRE PATHWAYS

Thus far we have only discussed manipulatingstructural genes for pathway enzymes to affectchanges in metabolism. An intriguing approach formetabolic engineering and increasing our under-standing of the coordinate changes in gene expres-sion needed to regulate entire pathways is to identify

and study transcriptional factors controlling path-ways or branches of metabolism (14, 15, 17, 28). Manyof the transcriptional regulators affecting plant bio-chemistry and development were originally identi-fied by chemical- or transposon-based mutant screensin maize, snapdragon or Arabidopsis. The cloning ofsuch loci has provided the opportunity to use thesegenes to manipulate plant biochemistry in the hostorganism or in other plants. One of the early in-stances of using this approach to manipulate plant

biochemistry was the engineering of Arabidopsis toexpress the maize transcription factors C1 and R,which regulate production of anthocyanins in maize

aleurone layers (17). Expression of C1 and R togetherfrom a strong promoter caused massive accumula-tion of anthocyanins in Arabidopsis, presumably byactivating the entire pathway. More recently, themaize transcriptional regulators C1, R, and P wereexpressed in cell cultures and the effect on anthocy-anin biochemistry and global gene expression ana-lyzed (3). Novel insights into the anthocyanin path-way, its regulation, and additional differentiallyexpressed targets of these regulatory genes were ob-tained. Such expression experiments hold greatpromise and may eventually allow the determinationof transcriptional regulatory networks for biochemi-

cal pathways.

DellaPenna

162 Plant Physiol. Vol. 125, 2001

8/3/2019 Plant Physiol. 2001 a 160 3

4/4

ELEPHANTS, BLIND MEN, AND A BRIEF DIP INTHE METABOLIC POOL

This article is too brief to effectively cover manyaspects of metabolism and metabolic engineering inplants during the past 25 years. Suffice it to say, it has

been a tremendously exciting quarter century thathas set the stage for a revolution in the way we thinkabout and approach metabolism and metabolic engi-neering in plants. The pace of gene discovery in plantmetabolism has increased dramatically during thepast decade and will only quicken in coming years asthe public deposition of expressed sequence tags andcomplete genomes allows plant researchers to movetheir pathways and experiments with increasing ease

between organisms and through evolutionary time.During the last 15 years we have also refined ourability to engineer changes in gene expression intransgenic plants to a point where manipulations canoften be targeted to the appropriate tissue and devel-opmental stage. However, during this same time pe-riod our ability to analyze the global effects of suchmodifications on metabolism has lagged behind. Wehave been very much like the blind men and theelephant; often only able to touch one small portionat a time of the beast we call metabolism!

The good news is that technology continues to holdgreat promise for the future of plant metabolic engi-neering. We are now able to analyze the conse-quences of transgenic or genetic alterations on theexpression of thousands or tens of thousands ofgenes simultaneously. With advances in proteomicswe should also be able to simultaneously quantify

the levels of many individual proteins or follow post-translational alterations that occur. What are nowneeded are analogous analytical methods for catalog-ing the global effects of metabolic engineering onmetabolites, enzyme activities and fluxes. Nuclearmagnetic resonance and metabolite profiling arelikely candidates to fill part of this void (21, 27).Integrating global analyses from transcription to pro-teins to metabolites may finally allow us to see theelephant in all its glory! A lofty goal to be sure butone has only to look back at the progress in DNAsequencing the past 25 years to realize that anythingis possible.

LITERATURE CITED

1. Brinch-Pedersen H, Galili G, Knudsen S, Holm PB(1996) Plant Mol Biol 32: 611620

2. Browse J, Sommerville C (1991) Annu Rev PhysiolPlant Mol Biol 42: 467506

3. Bruce W, Folkerts O, Garnaat C, Crasta O, Roth B,Bowen B (2000) Plant Cell 12: 6580

4. Chamovitz D, Pecker I, Hirschberg J (1991) Plant MolBiol 16: 967974

5. Conklin PL, Norris SR, Wheeler GL, Williams EH,Smirnoff N, Last RL (1999) Proc Natl Acad Sci USA 96:41984203

6. Conklin PL, Saracco SA, Norris SR, Last RL (2000)Genetics 154: 847856

7. Cunningham FX, Gantt E (1998) Annu Rev PhysiolPlant Mol Biol 49: 557583

8. DellaPenna D (1999) Science 285: 3753799. Falco SC, Guida T, Locke M, Mauvais J, Sanders C,

Ward RT, Webber P (1995) Biotechnology 13: 57758210. Grusak M, DellaPenna D (1999) Annu Rev Physiol

Plant Mol Biol 50: 13316111. Hartmann M (1998) Trends Plant Sci 3: 170175

12. Hitz WD, Yadav NS, Reiter RS, Mauvais CJ, KinneyAJ (1995) In JC Kader, P Mazliak, eds, Plant LipidMetabolism. Kluwer Academic Publishers, London, pp506508

13. Holton TA, Cornish EC (1995) Plant Cell 7: 1071108314. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schaben-

berger O, Thomashow MF (1998) Science 280: 10410615. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K,

Shinozaki K (1999) Nat Biotechnol 17: 28729116. Kinney AJ (1998) Curr Opin Plant Biol 1: 17317817. Lloyd AM, Walbot V, Davis RW (1992) Science 258:

1773177518. Ohlrogge J, Browse J (1995) Plant Cell 7: 957970

19. Post-Beittenmiller D (1996) Annu Rev Physiol PlantMol Biol 47: 40543020. Radwanski ER, Last RL (1995) Plant Cell 7: 92193421. Roberts JK (2000) Trends Plant Sci 5: 303422. Shintani D, DellaPenna D (1998) Science 282:

2098210023. Smirnoff N (2000) Curr Opin Plant Biol 3: 22923524. Somerville C (1986) Annu Rev Physiol Plant Mol Biol

37: 46750725. Stark DM, Timmerman KP, Barry GF, Preiss J,

Kishore GM (1992) Science 258: 28729226. Stitt M, Sonnewald U (1995) Annu Rev Physiol Plant

Mol Biol 46: 34136827. Trethewey RN, Krotzky AJ, Willmitzer L

(1999) CurrOpin Plant Biol 2: 838528. van der Fits L, Memelink J (2000) Science 289: 29529729. Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P,

Potrykos I (2000) Science 287: 303305

Plant Metabolic Engineering

Plant Physiol. Vol. 125, 2001 163