Mass Production And Harvest Of Engineered Synechococcus Harbouring VP28 Gene

White spot syndrome virus(WSSV) was detected in Taiwan in 1992 and rapidly spread to the world. Most shrimp aquaculture industry has been hampered by WSSV and sustained seven billion losses in all. None of the anti-WSSV protections has been commercialized. Specific envelope protein VP28 plays a crucial role in WSSV infection. It has been reported that recombinant VP28 was a promising candidate against WSSV via shrimp oral administration or injection. After feeding with engineered cyanobacteria harbouring vp28, we found that the shrimp gained the ability against WSSV in 30 days. As a promising product, engineered Synechococcus was prepared for mass production. However, the commercial development was limited by three factors: 1) low biomass from mass production and undeveloped manufacturing technique; 2) no available method for optimizing expression of recombinant protein; 3) no available efficient harvesting method.1. Light intensity control for biomass of engineered Synechococcus To improve biomass of engineered Synechococcus, light intensity(I) was regulated via two factors: 1) the interaction with temperature(T) and p H; 2) the attenuation effect in photobioreactor(PBR). First, via single factor experiment, as net photosynthesis rate(NPR) reached extreme value, the range of I, T and p H was 200-400 μmol/m2 s, 28-48 °C, 6.5-8.5 respectively; via response surface test, as optimum I, temperature and p H was 300±10 μmol/m2 s, 37 °C, 7.5 respectively, the maximum NPR was 342μmol O2/mg Chlah. High density cultivation was performed in two PBR models(5L/108 W outer illuminated, 100L/120 W inner illuminated)with 37 °C and p H 7.5. In 5L-PBR, I range was 250-300 μmol/m2 s in logarithmic phase and final biomass was 1.5 g/L after three days; in 100L-PBR, I range was 180-250 μmol/m2 s in logarithmic phase and final biomass was 0.4 g/L after seven days. 100L-PBR was reshaped: light power was added to 300W; light distribution number was from three to six. Through cultivation experiment in reshaped 100L-PBR, I range in 50% PBR area reached 300±10 μmol/m2 s in logarithmic phase. The total logarithmic phase was increased by 36 h and final biomass was 0.8g/L after 7 days. Thus, I was a crucial factor for production efficiency in PBR, optimum I power and distribution improved final biomass. 2. Light quality control for recombinant protein VP28 Yield of biomass and recombinant protein VP28 was improved by light quality regulation. High cell density cultures were performed with variant ratios of white(W), red(R) and blue light(B) in the 5L-PBR. Yields of biomass, expressions of vp28 and transcription levels of psb A were detected. 1) As light quality consisted of 50% W, 33.3% B and 16.7% R, final expression of vp28 was 2.4% and 3-folds as that of 100% W; relative accumulation of VP28 was 2-folds as that of W; biomass reached 1.22 g/L; transcription of psb AII and psb AIII were improved. 2)As 50% W, 33.3% R and 16.7% B, final expression of vp28 was 0.5%; relative accumulation of VP28 was only 5% as that of high ratio B in 10 μg protein; transcription of psb AII and psb AIII were inhibited as psb AI was improved; biomass reached 1.68 g/L. Results indicated that high ratio B facilitated accumulation of recombinant protein as inhibited biomass, while high ratio R has the opposite effect. The regulation mechanism of light quality interrelated with host gene psb A.3. Study of the efficient harvesting method for engineered Synechococcus To develop a safety and efficient harvest method, study the feasibility of undissolved precipitates induced flocculation, including optimal inducers and dose, the removal of algal organic matter(AOM) and control of protein loss after flocculation. 1) As magnesium concentration in suspension was increased by 0.25 m M, flocculation efficiency(FE) was over 90% at p H 11. 2) AOM in suspension was removed by 4000 rpm centrifugation and cells were resuspended with fresh culture solution(0.3 m M magnesium), then FE was over 95% at p H 10.5. 3) As light quality consisted of 50% W, 33.3% B and 16.7% R, final AOM in suspension was below 2 mg/L. 4) As with 100% W, AOM was around 4.5 mg/L. 5) As with 50% W, 33.3% R and 16.7% B, AOM was over 5 mg/L. 6) With 5 °C、20 °C and 35 °C water bath, total protein in supernatant fluid was detected as 35 μg/ml, 24 μg/ml and 10 μg/ml respectively. Soluble protein was separated by electrophoresis and molecular weight was below 20 KDa. VP28 protein was not detected by antibody in soluble protein. 7) After flocculation, supernatant protein concentration was decreased to 12μg/ml in 30 min with modulating p H from 10.5 to 7.5. These results indicated that magnesium induced FE was decreased by AOM significantly. High ratio B inhibited the AOM formation and high ratio R had the opposite effect. After flocculation, algal protein loss was affected by temperature and solution p H. With low temperature and regular p H, algal protein loss during flocculation was reduced by 50%.In sum, as mass production of engineered Synechococcus was performed in PBR, optimum light intensity and quality improved yield of biomass and recombinant protein; reformative magnesium induced flocculation was an available harvesting method with safeness and no recombinant protein loss.