Introduction ofNewCarotenoids into theBacterial Photosynthetic Apparatus byCombining theCarotenoid Biosynthetic Pathways of Erwinia herbicola andRhodobacter sphaeroides

Carotenoids havetwomajorfunctions inbacterial photosynthesis, photoprotection andaccessory light harvesting. Thegenes encoding manycarotenoid biosynthetic pathways havenowbeenmappedandcloned in several diferent species, andtheavailability ofcloned genes whichencode thebiosynthesis ofcarotenoids not found inthephotosynthetic genusRhodobacter opensupthepossibility ofintroducing awider rangeofforeign carotenoids intothebacterial photosynthetic apparatus thanwouldnormally beavailable byproducing mutants ofthenative biosynthetic pathway. Forexample, thecrtgenes fromErwinia herbicola, agram-negative nonphotosynthetic bacterium whichproduces carotenoids inthesequence ofphytoene, lycopene, ,8-carotene, I-cryptoxanthin, zeaxanthin, andzeaxanthin glucosides, areclustered within a 12.8-kb region andhavebeen mappedandpartially sequenced. Inthis paper, partoftheE.herbicola crtcluster hasbeenexcised and expressed invarious crtstrains ofRhodobacter sphaeroides. Thishasproduced light-harvesting complexes with anovel carotenoid composition, inwhichtheforeign carotenoids suchas,8-carotene function successfully in light harvesting. Theoutcome ofthecombination ofthecrtgenes inR.sphaeroides withthose fromE.herbicola has,insomecases, resulted inaninteresting rerouting oftheexpected biosynthetic sequence, whichhasalso provided insights into howthevarious enzymes ofthecarotenoid biosynthetic pathway mightinteract. Clearly this approach hasconsiderable potential forstudies onthecontrol andorganization ofcarotenoid biosynthe- sis, aswellasproviding novel pigment-protein complexes forfunctional studies. Carotenoids havetwomajor functions inbacterial photosyn- thesis, photoprotection andaccessory light harvesting (6,25). Spectroscopic studies designed tounravel themolecular details ofthephotophysical reactions involved inthese processes have beengreatly aided bytheavailability ofmutants withthesame pigment-protein complexes butwithaltered carotenoid com- position (for examples, seereferences 4,7,8,and24). Initially, these mutants wereproduced byrandom chemical mutagenesis orUV treatment (5,26); however, withthedevelopment of molecular genetic methods, these mutations cannowbeintro- duced inamorecontrolled andsystematic way,forexample, by interposon ortransposon mutagenesis (9, 11,28). Inphotosyn- thetic bacteria suchasRhodobacter capsulatus, these forms of insertional mutagenesis haveproduced mutations incrtI, -C, and-D,forexample, yielding strains which accumulate phy- toene, neurosporene, andneurosporene derivatives, respec- tively, rather thanthenormal endproducts ofthebiosynthetic pathway, spheroidene andspheroidenone (11). Thegenes encoding manycarotenoid biosynthetic pathways havenowbeenmappedandcloned inseveral different species (9,11,13,18,21,23,28), andinthecaseofR capsulatus, for example, thecomplete DNA sequence ofthecrtgenecluster is available (2). Theavailability ofcloned genes which encode the biosynthesis ofcarotenoids notfoundinRhodobacter spp. opensupthepossibility ofintroducing awider range offoreign carotenoids intothebacterial photosynthetic apparatus than wouldnormally bemadeavailable byproducing mutants ofthe