应用基因重组微藻生产生物活性物质

药物生物技术30Pharmaceutical Biotechnology 2001, 8(4): 230-233Production of Useful Substances fromRecombinant Microalgae by Using Genetic EngineeringQIN Song, ZoU Li-hong, JIANG Peng, DOU Chang-gui, Hiroyuki Kojima2. China Pharmaceutical Universithy, Nanjing, P R China; 3. Osaka National Research Institute, Osaka 563, Japan)Abstract Microalgae have unique features as photosynthetic microorganisms and produce a variety of substances froma2 with other inorganic nutrients. Genetic engineering has so far been applied in microalgae to make them usefulbioreactors to produce high-value substances. Recent developments of these attempts are reviewedKey words microalgae, genetic engineering, bioreactorCLC number: Q50 Document Code: A Article ID: 1005-8915(2001)04-0230-04Utilization of microalgae are essential for biotechnology against the mosquito larvae, the encoding gene was introducedbecause they grow as fast as microorganisms and perform oxyinto Synechocystis PCC7942 and the larvicidal activitygen-evolving photosynthesis like higher plants, which enables verified on Culer pipiens larvae. Endotoxin produced byries under simple Bacillus thuringiensis have long been studied for usage as insecand cheap conditions. Microalgae have a large diversity of over ticides, the encoding gene cry/VB was cloned and introducedten thousands species, which leads to further greater diversity into Synechococcus PCC700212). In another study severalof the primary and secondary metabolites. One can easily eval. copies of the same gene under the tobacco psba promoter werecharacteristics by productions of, for examples, un- introduced into Synechocystis PCC6803 31. In these attempts,the expression level was so low that the intact cyanobacterialnds and so on. However, the species currently culti- cells were not effective, the larvae died only when the sonicatvated for commercial productions are limited to only Chlorel. ed cells were fed. High level expressions were achieved inSynechococcus PCC7002 with crylVD4the transformedcellsToday we face serious demands for utilization of microal- were readily ingested by mosquito larvae and led the larvae togae from a view of the global problem of climate warming. To death in several days. Further higher mosquitocidal activitiesexpand microalgal biotechnology to new industrial fields we was obtained by a combination of the genes crylvA, crylVDneed to develop genetic engineering for each aspect of the ap. and p20 from B. thuringiensis war israelensis in Anabaenaplications: genetic recombination of a suitable species, mainte- PCC7120 51nance of the transformant, control of the expression, separaFor practical applications intion of the product, cultivation for long term and so on. Inthis paper we first review recent developments in applications mosquito breeding sites[6l. The commercialization seems stillof recombinant microalgae, and then useful methods develbeyond reach since the main difficulties are much lower level ofexpression in cyanobacteria than in B. thuringiensis and the1 Production of usefal substances from recombinant mi. insufficient effectiveness in natural fields1.2 Growth hormone(Gh)1.1 Mosquito larvicidesProduction of Gh in microalgae is interesting because re-illions of people in tropical regions died in malaria annu. combinant microalgae can be fed as dietary supplement forly. Mosquito Anopheles, in which the malaria-pathogen aquacultured fish and farmed animals. In general, Gh arePlasmodia parasitizes, mediates the infection. Cyanobacteria commercially produced in E. coli and yeast, only Kawata andgrow in aquatic habitats of mosquito larvae and are captured as his co-workers reported transformation of Synechococcusological controls are considered having advantage in PCC7002 with the salmon Gh genelong term effectiveness and limitation of target insectmmii evicts in denatured formThe toxin from Bacillus sphaericus shown highly active sion b中国煤化工 bCNMHGReceived date: 2000-08-08QIN Song et al: Production of Useful Substances from Recombinant Microalgae by Using Genctic Engineering 231microalgae and microalgal cell walls may be a good capsule for introduced the CGit gene from Bacillus macerans toGh prorection from digestion by gastric enzymesSynechococcus PCC7002 and converted starch to a-cyclodextrit1.3 Polyunsaturated fatty acids( PUFA)in cultivated medium. The conversion efficiency was still low,PUFA produced in microalgae are much interesting for but the conversion has special advantage to treat waste waterusages of pharmaceuticals and healthy food supplementsDesaturase is the key enzyme for synthesPUFA in2 Genetic engineering methods formicroalgae. Four acyl-lipid desaturases were cloned from2.1 VectorsSymechoc ysis PCC6803, and they were, respectively, specifiPlasmid Vectors Shuttle vectors based on endogenousto the△6,△9,△12anda3 positions of fatty acidsIntroductions of these genes into Synechococcus PCC7942 plasmids as well as broad host range bacterial plasmidsg incP and incQ can be used as plasmid vectorsresulted in changes of PUFA composition. An outstanding Typical broad-range bacterial plasmid such as RSF1010(incQchievement is production of eicosapentaenoic acid(EPA)incontaining, 8.68 kb)is not self-conjugal but mobilized with athe recombinant Synechococcus sp with the EPA gene coexisting conjugal plasmid(helper plasmid), including Rk2,clusteri9i. The yield marked 0. 64 mg/g dry wt when cells RP4. The replication system of inc Q seems independent fromgrown at 17Cthe host replication function: necessary genes are encoded onAnother practical approach is to use inhibitors of specifthe plasmid for initiation of replication. A replicon fromsteps in biosynthetic pathways or analogs of specific products. RSF1010 was maintained in Synechocystis strains PCC6803he resistant strains wouldPCC6714 and Symechoroecus strains PCC7942, PCC6301 14)mutation of specific enzymes so as to overprePlasmid vectors for nuclear transformation ofave been achived in Spirulina platensis // tion of proline Chlamydomonas reinhardtii were constructed using the yeastreplication origin and yeast arg 4 locus although they were notSince the phase transition temperature depended on the stable enough. The host was a mutant of argininosuccinatedegree of unsaturation of fatty acids of membrane lipids, it lyase-deficiency(arg7 locus in C. reinhardtii )complementeddesaturation of fatIntegrative vectors When no appropriate replicon isshowed increased tolerance of Symechocystis PCC7941, which available, it is necessary to integrate the exogenous gene intowas chilling sensitive, by introducing the A12 desaurase the genome. Although integrative vectors have not well beengene, des A, from chilling-tolerant Synechocystis PCC6803. developed as yet, they are more useful for practicalMoreover, they demonstrated that among various desaturation applications from the aspect of maintenance of transformantsreactions in cyanobacteria, the desaturation of u3 pasition of Chromosomal integrations give rather stable transformantsfatty acids was the most effective to the change in vectors for integration are devised based on transposons oremperaturehomologous recombinationsTransposon vectors have advantage in the wide host rangeCarotenoids are important microalgal products but so far and multiple duplication copies in the genome, butonly B-carotene from Dunaliella and astaxanthin from disadvantage in less stability of integrated genes. The mostHaematococcus are currently produced in mass cultivations. extensively developed transposon vectors in bacteria comesGenetic analyses of the biosynthetic pathways have extensively from Tn5. Transposition frequency of Tn5 is relatively highen made and most of responsive genes have been cloned from (103-10 2 per cell generation in E coli), but once Tn5 isanobacteria.A notable achievement is production of integrated, it is rather stable. A Tn5 based transposon vectorastaxanthin in Synechococcus PCC7942 transformed with the for searching an environmentally responsive promoter in theHaematococcus cto gene( 12). Although the conversion to Anabaena genome was constructed by Wolk and hiastaxanthin was low(2.7% of total carotenoids), this showed workers[16a possibility of mass production in future of a complexectors on the basis of homologous recombination arecarotenoid in recombinant cyanobacteriaconstructed simply using a homologous sequence to the host1.5 other substancesgenome and a selection marker. a gene of interest is firstRecombinant microalgae may be used for bio-conversion loaded on an E. coli plasmid vector and integrated into thein aquaculture. Suchappromade for production of chrom中国煤化工on, A problemcyclodextrin from starch catalyzed by cyclodextrin thisoccurs frequentlyglucanotransferase(CGTase ). Kojima and his co-workers" byYHCNMH Gous sequences232药物生物技术第8卷第4期workers/ found highly repetitive sequences of about 100 expression after reaching the stationary phase. The tac(orcopies per genome (named short tandem repeated repetitive se. Lac)promoter with lacI repressor gene is used widely inquences, STRR)in Calothrix PCC7601 and other betrocys. coli and applicable for cyanobacteria(23). The repression isous strains. It is interesting to use such repetitive sequences completely released with isopropyl thiogalactoside(IPtG )orfor homologous recombination. Chromosomal integration a. methylthiogalactoside(TMG). These repressors suffice labo-curs even without any homologous sequence in a vector. ratory usethey are too expensive for cultures on largeDzelzkalns and bogorad(8 observed stable integrative trans. scaleformation for Synechocystis PCC6803 with UV irradiationGene dosage and codon usage were also the important facWe also experienced this with electroporation for Spirulina tors determining the expression level of the introduced geneplatensis although the transformation was not stable[i9.A When the endogenous plasmid is not cured from the host cell,similar mechanism as the Sos response induced such illegiti. it is preferable to reduce the plasmid copy number prior tomate recombination with the shock of uv or electric pulse2.2 Selection and maintenance of the transformantsbetter to use 5-fluoro-2-deoxyuridine or novobiocin prior toThe selection for transformants is attained usually using transformation for reducing the genome copy number. Codonantibiotics, typically ampicillin, chloramphenicol and usage was biased to some extent from that of most organismkanamycin for cyanobacteria, or spectynomycin and The effect of the bias is different with each gene. Aoyama( 24.kanamycin for eukaryotic microalgae. However we have ever showed that the codon usages of Ile(ATA), Val(GTA),Throbserved that the wild-type Synechococcus sp. PCC7002 lost (ACA)and Arg(AGA)were scarcely used in Synechococcusbut frequently in the clostridial hydrogenase genegrow with time Lag of a few days. This may be due to induc- 2.4 Extracellular release of gene productstion of the multiple-antibiotic-resistance designated MarIt is well known for Grarm-negative bacteria that somewhich was disoovered in E coli(20, 21). The cells showed de. enzymes are transported from the cytoplasm to the periplasmcreased susceptibility to many structurally unrelated anti-mi. via the signal peptide, and then are released to medium by os-crobial agents with time and the antibiotic efflux pump was ac- motic shock. Such process for release of recombinant genetivated.A similar mechanism is thought to exist in common to product to medium was verified using the gene of p-lactamase,all bacteria. One may distinguish the positive transformants a typical periplasm enzyme in E. coli introduced in Symeonly by the difference of initial growth rate with antibiotic re- chococcus PCC7002 25), as well as the transformation for cysistance, but may be unable to maintain them in culture for a clodextrin glucanotransferase gene 3)since cyanobacteria arelong period because the selection pressure with antibiotic bGram negative. These results show that the cyanobacteriumcomes ineffectivecan recognizes the heterologous signal and accumulates theIt is convenient to use a host of auxotrophic mutation: protein in the periplasm. In the case of Synechococcusthe target gene may be introduced into the mutant host with a PCC7002, osmotic shock is given by transferring the cellsgene for complement. However such mhardy avail. from the normal medium to the medium lacking NaCl.Mostable for most microalgae because of presence of multiple copies of the product is released to medium within 20 min and the re-of the genomic dna in a cell (over 10/cell for cyanobacteria; lease can be repeated after recovering incubation. It is possible100-10,000/cell for the chloroplast). Exceptional is glu- in principle to release any protein of interest extracellularly bytamine-auxotrophic mutants of Anabaena variabilis[z2Jmodifying the gene with a signal sequenceibitor-resistance, which encoding the mutated enzyme losingthe affinity to inhibitor, but keeping the enzymatic activity. [1] Tandeau de mn, de la Torre F, Saulmajester J. Expresion ofThe target gene was introduced into wild-type host with suchthe larvicidal gene of bacillus sphaericus 1593 M in thea mutated gene, followed inhibitor pressing to get the positivecyanobacterium Anacystis nidulans R2, Mod Gen Genet, 1987,clones. Advanage of this approach is that the selection is eff209:396[2] Angsuthanasombat C, Panym S Biosynthesis of 130-kilodaltontive even if the normai gene remains in other copies of the hostmosquito larvicide in the cyanobacterium Agmnedlum quadrgenome. Disadvantage is that the enzymatic activity may beplicatum PR-6. APp! Environment Microbiol, 1989decreased2.3 High conditional expression of exogenous ger[3]pression of target gene during growth phase and to depress theH中国煤化工 osquitoadal-protenCN GC6803 Cur Mi-QIN Song et al: Production of Useful Substances from Recombinant Microalgae by Using Genetic Engineering 23355:2:28for the cyanobacteria Synechocystis sp, strains FCC6803 andi3. ChungIatupornchai w. Expression of the mosquitocidal-proteinPCC714 or Synechococcus sp strains PCC7942 and P((:30genes of bacillus thuringiensis subsp. israelensis and theCarr Microbus. 1994, 28: 14erbicide- resis ance gene bar in Symechaxystis PCC6803. Cury [15] Debuchy R. Purton S. Rochaix J.D. The argininosuccinateMicrobiol. 1990. 21: 283lyase gene of Chlamydomonas reinhardti: an importan: tooi for4. Murphy R C. Stevens Jr S E Cloning and expression of thenuclear transformation and for correlating the genetic andrrv/vD gene of Bellus thuringiensis subsp. israelensis in themolecular maps o! the ARG7 locus. EMBO J. 1989, 8: 2803cyanobactenun Agmenellum quadraplicatum PR-6 and its [16] Wulk C P. Vonshak A, Kehoe P, et al. Construction of shuttleresulting larvicidal activity, Appi Enuronment Microhnovectors capable of conjugative transder from Eschericha coli tonitrogen-fixing filamentous cyanobacteria. Prox Nail Aud Set[51 Xiaoqiang W, Venison S], Huirong L, Mosquito larvicidalUSA.1984,81:1561activity of transgenic Anabaena strain PCC7120 expressing [17] Mazel D, Houmard J. Castets A M, et al. Highly repetitivecombinations of genes from arillus thuringiensis subspDNA sequences in cyanobacterium genomes. J Bactenol, 1990udensis. Appl Environ Microbiol, 1997, 63: 4971172:2755:6 Thiery I, Nicolas [, Rippka R, ef al. Selection of cyanobacteria 18: DzelzkaIns V, Bogorad L. Stable transformation of thecyanobacterium Synechocystis sp. PCC6803 induced by UVmosquito larvae. Appl Environ, Microbiol 1991, 57:rAdiation,j Bacteriol, 1986, 165: 9641354[191 Kawata Y, Yano S, Kojima H. 1993. Transformation of[7. Kawata Y, Yamano N, Kojima H, et al. Expression of salmonpirulina platensis by chromosomal integration. Proceedings ofgrowth hormone in the cyanobacterium Agmenellunm6th International Conference on Applied algology, pp, 72uadruplicatum. Biotechnol Letf, 1991, 13: 851Ceske rudejovice, Czech Republic[8 Reddy A S, Nuccio ML. Gross L. M, et al. Isolation of a 46. [20] George A M, Levy S B. Amplifiable resistance to tetracycline.om the cyanobacterium Synechchloramphenicol and other antibiotics in Escherichia coli:rain PCC6803 by gain function expression in Anabaena spinvolvement of a non-plasmid-determined efflux of tetracyclinetrain PCC7120. Plant Mol Biol, 1993, 27: 293j Bacterio! 1983, 155: 531[9] Takeyama H, Takeda D, Yazawa K, et al. Expression of the [21:Cohenlachler H, Levy S B. Genetic and functionaleicosapentaenoic acid synthesis cluster from Shewanella sp. in aanalysis of the multiple antibiotic resistance(mar)Locus in Ecati. J Bacteriol. 1993. 175: 1484Microbiol,1997,143(8):2725-2731[22] Spiller H, Latorre C, Hassan M E, et al. Isolation and[10 GIovanna R, Sora S, Ciferri. Production of amino acids bycharacterization of nitrogenase derepressed mutant strains ofanalog reststantts of the cyanobacterium spirulinacyanobacterium Anabaena variabilis, J Bacterin, 1986, 16platensis. J Bacteriol, 1981, 147: 1002.[11] Wada H. Gombos Z, Murata N. Enhancement of chilling [23] Stark M J R. Multicopy expression vectors carrying the LacLolerance of a cyanobacterium by genetic manipulation of fattyrepressor gene for regulated high level expression of genes in Ecoli.ent,1987,51:255.[123 Harker M. Hirschberg. Biosynthesis of ketocarotenoids in [24] Aoyama K, Miyake M, Yamada J, et al. Application of vectornsgenic cyanobacteria expressing the algal gene for B-4E4.9 carrying a strong promoter to the expression of foreignoxygenase, crO. FEBS Lett, 1997, 404: 129proteins in Synechococcus PCC7942. J Mar Biotechnol, 199613 Kojima H, Kawata Y, Yano S. 1996. Bioreactors ofrecombinant blue-green algae. Proc. of ITIT Symposium. [25 1 Yano S, Kawata Y, Kojima H. Development of host-vectorMicroaigal Biotechnology, ()aka National Res. Inst. Japsystems for microalgae IL. Expression and induction of Lacz inSynechococcus. Bull Osaka Natl Res Inst, 1992, 43[14] Mermet-Buavier P, Chauvet F. A conditional expression vector应用基因重组微藻生产生物活性物质秦松,邹立红,姜鹏,窦昌贵2, Hiroyuki Kojima(1中国科学院海洋研究所,青岛26071;2中国药科大学,南京210038;3大阪国立研充所,大阪563,日本)摘要微藻是指进行光合自养的微型藻类其种类繁多代谢途径与日夕栏,日想的基因工程生物反应器。文章综述了微藻应用基因工程技术,开发生产高价值产品的中国煤化工关键词微藻;基因工程;生物反应器CNMHG