The Study Of Two Key Technologies For Preparation Of Microalgae Biodiesel By Genetic Engineering

In recent years, with the continuous improvement of fossilfuel consumption caused more and more serious energy crisis andenvironmental problems, microalgae biodiesel as a new energy has beenpaid more and more attentions. The purpose of thisresearch is to study two key technologies of the development andutilization of microalgae biodiesel, and provide technical support for thewidespread use of microalgae biodiesel. In the preparation of biodiesel bymicroalgae, we can effectively improve the lipid content of microalgal bygenetic engineering. And the selections of species and genes are the keyfactors of genetic engineering. In this study, we selected the marineChlorella sp. which has higher growth rate and can grow in seawater asthe host. And we selected the marine Chlorella sp. pepc2geng as thetarget gene. At frist, marine Chlorella sp. pepc2gene was cloned andanalyzed the structural and functional characteristics of marine Chlorellasp. PEPCase2by bioinformatics method. And then, we expressed thepepc2gene fragment from marine Chlorella sp. in Escherichia coli andanalysed its function. In addition, this study also exploredmicroalgae mass cultivation by photoreactor. We used the100Lphotoreactor to culture microalgae and analyzed its growth law andregulation.Phosphoenolpyruvate carboxylase (PEPCase: EC4.1.1.31) is animportant enzyme of photosynthetic carbon metabolism, and catalyzesthe β-carboxylation of phosphoenolpyruvate (PEP), generatingoxaloacetate (OAA) and inorganic phosphate (Pi). In the recent20 years, the study found in non C4and CAM plants, such as algaeis alsofound this enzyme, and prove the existence of two types ofPEPC enzymes in eukaryotic algae, but its function is not clear. In orderto further understand the characteristics of algae PEPC enzyme, the pepc2gene into theseawater Chlorella To further understand the characteristicsof algae PEPCase, in this paper, marine Chlorella sp. pepc2gene wascloned and analyzed the structural and functional characteristics ofmarine Chlorella sp. PEPCase2by bioinformatics method. The resultsshow that:1. Phylogenetic analysis showed that the genetic relationshipof marine Chlorella sp. pepc2and Chlamydomonas reinhardtii pepc2ismore close; analysis of amino acid sequence showed thatmarine Chlorella sp. PEPCase2polypeptide consists of five conservedsequences, and its N-terminal lack of a phosphorylation sites, and itsC-terminal is a RNTG structure. So we can conclude that marineChlorella sp. PEPCase2is a bacterial-type PEPC enzyme;2. Thesecondary structure of marine Chlorella sp. PEPCase2mainly includesα-helix, β-turn, random coli and extended strand, and Its tertiarystructure external to the α-helix and random coil, is conducive tomaintaining the stability of PEPCase structure, a parallel β-barrelstructure in the centre is the reaction center of PEPC enzyme;3. MarineChlorella sp. PEPCase2has many important physiological functions,mainly related to a variety of important material synthesis.In this work, the pepc2gene fragment from marine Chlorella sp. wasexpressed in the E. coli with antisence expression vector, to inhibitPEPCase activity and increase lipid content. The pepc2gene fragment（611bp） was cloned with PCR amplification from the marine Chlorellasp. and sequenced. The sequence similarity is100%with marineChlorella sp. pepc2gene. The cloned pepc2gene fragment was inserted into the vector pET-28a to form prokaryotic sense expression vector(pET-28a-forw-pepc2) and antisense expression vector(pET-28a-reve-pepc2). Both sense and antisense expression vectors weretransformed into E. coli to produce the forward mutant withpET-28a-forw-pepc2and the reverse mutant with pET-28a-reve-pepc2,respectively. Contrasted with the wild type, when inducing at6thhours,the relative expression of pepc gene, the soluble proteins and thePEPCase activity increased to215.74%,5.59U/g protein and216.96mg/gbiomass, respectively, and the lipid content decreased to3.67%in theforward mutant (compared with the wild type), while decreased to41.23%,2.83U/g protein and139.19mg/g biomass, respectively, and thelipid content increased24%in the reverse mutant. It indicated that pepcgene plays an important regulatory role in protein and lipid metabolism inE. coli.This study also used an airlift vertical column type photoreactor inmicroalgae culture. The Anabaena variabili7120was cultured in theBG-11medium containing nitrogen, at35℃, with continuousillumination. The results show that the periodic supply of CO2ensuresthe supply of inorganic carbon source, and on the other hand, it madethe pH remained in the7.1~7.9and effectively removed the dissolvedoxygen accumulation, maintain the suitable conditions for microalgaeculture. To determine the optical density and biomassof microalgae, under the same culture conditions, the growth rate oftwo transgenic microalgae was higher than that of wild typemicroalgae, but the reason remains to be further study.