Studies On The Heterologous Expression Of Carbonic Anhydrase And The Mechanism Of CO₂ Fixation In Microalgae

In present study,a carbonic anhydrase?CAs?was cloned and characterised for carbon dioxide hydration,then an enzyme based CO₂ fixation system was conducted for algae cultivation.To further enhance the carbon fixation and utilization of carbon,the CAs sequences were constructed in expression vectors and integrated into Haematococcus pluvialis.Simultaneously,the transcriptome analysis was performed to elucidate the carbon metabolism,which could contribute to greenhouse gas emission solution and high value productions in microalgae.The major results are shown as follows:1.The target gene CAs was amplified and constructed into pCold-CAs vector for protein production with the optimization condition.The results showed that CAs could yield21.15mg/L with 1 mM IPTG under 16 ^o C for 24h induction.The fusion CAs was purified through Ni-chelating affinity chromatography and detected as 110kD by SDS-PAGE electrophoresis.The polypeptide sequence was identified as?-CA2 by mass spectrometry after the His-tag was removed by Factor Xa.2.The enzyme exhibited a specific activity of 726.8 WAU/mg proteins though the acid-base indicator?bromothymol blue?method.Then the CAs based CO₂ fixation way was obtained as follows:100?g protein in 1 M Tris?pH 9.5?incubated under 45 ^o C..The produces of carbon fixation calcium carbonate's mass was 21.00 mg.The results showed that CO₂ hydration efficiency reached 91.3%of sigma CAs.And the CAs still remained about 90.95%carbon fixation capacity on the seventh day,which is higher than commercial enzyme?77.61%?.3.Ble was select as the resistance marker gene,coupled with CAs sequences,pds promotor and 3'UTR fragments were constructed in expression vector pCAMBIA1301.Nine clones were observed in the agar plate with 30mg/L zeocin though agrobacterium tumefaciens mediated method.4.Transcriptome analysis was performed with Ilumina HiSeq?2000 sequencing platform to clarify the carbon sequestration and metabolic mechanism in H.pluvialis.The results showed that the carbon fixation pathway?calvin cycle?,glycolysis,fatty acids and astaxanthin biosynthesis was enhanced dramatically,explained the carbon flux from extracel ular CO₂ towards secondary metabolites,which could be contributed for microalgae based carbon fixation and high value added metabolites production.Figure 41;Table 20;Reference 67...