Optogenetic Regulation Of FNR Improves H₂ Production In Green Alga Chlamydomonas Reinhardtii

With the growth of population and the development of economic,the consumption of fossil energy has increased dramatically,which causing serious problems such as the depletion of fossil fuels and environmental pollution.Hydrogen gas is one of the highly versatile,efficient and sustainable clean energy carriers that may be used to replace the fossil fuels due to its high energy yield when compared to traditional hydrocarbon fuels.Microalgae are one of the groups of photosynthetic microorganisms suitable for the photo-biological production of hydrogen due to its high activity of hydrogenase,fast growth rate,and simple culture conditions.In this study,Chlamydomonas reinhardtii was used as an organism to establish its blue lightinducible system with which we achieved optogenetic regulation of target gene FNR expression.Here are the following results:1.An p Dp124-ami R-FNR expression vector was constructed through artifcial mi RNA approach,which activated the expression of ami RNA targeting FNR gene with a Psa D promoter.Then the recombinant plasmid was transformed into CC-849 strain by glass-bead method and transformants were screened with paromomycin and positive clones were purifed and verifed by sequencing.Quantitative PCR results showed the expression of ami R-FNR was detected a 32% decrease of target gene FNR.2.What is more,this transgenic alga accumulated about 3.8 times more H2 content than the wild-type strain CC-849 at the end of 7-days cultivation.The chlorophyll fluorescence parameters detected by Phyto-PAM showed that the relative parameters including Fv/Fm,Yield,ETR and q P of ami R-FNR,was 12.8%,16.4%,16.4% and 25.6% lower than that of the wild type individually,suggesting that downregulation of FNR affected the photosynthetic efficiency of C.reinhardtii.The gas chromatography results showed that the proportion of oxygen in ami R-FNR was much lower.The lowest level was only 9.1%,which was 41% lower than that of wild type.Quantitative PCR results showed that the relative expression of Hyd A1 and Hyd A2 gene transcript levels in transgenic algae ami R-FNR was a 84% and 75% increased than that in control individually.The expression of Fdx5 gene in ami R-FNR was increased by 16.76 times than that in control,suggesting ami R-FNR created an anaerobic environment for the algal cells,which induced a rapid and high expression of Fdx5.The expression level of Pet F in ami R-FNR was a 87% increased than that in control,suggesting that the down-regulation of FNR enhanced electrons transfer from Fd to hydrogenase,which promoted hydrogen synthesis.The total amount of starch in early growth phase of C.reinhardtii was detected,which showed that the content of starch in ami R-FNR was 42% higher than that in wild type,suggesting that the downregulation of FNR is favorable to the accumulation of starch in the early stage of C.reinhardtii and to promote the indirect biophotolysis increasing hydrogen production.3.A blue light-induced FNR expression system vector A(p Dh124-VP16-CRY2-USA-FNR)was constructed here,and vector B(p Db124-GAL4 BD-CIB1)has been constructed and stored by the lab.Vector A and vector B were transformed into ami RFNR through two steps and screened with different antibiotics.The final transformants ami R-FNR-B were purifed and verifed by sequencing.The transcript levels of FNR in CC-849 and transgenic algal strains were analyzed by quantitative PCR before and after white light irradiation.The results showed that after 2 hours of white light induction,the transcript levels of FNR in ami R-FNR-B was up-regulated by 47.2%.After returning to 3 hours of red light culture,the FNR in ami R-FNR-B was down-regulated by 63.7%.While it was almost unchanged in the wild type and ami R-FNR.The results demonstrated the successful application of the blue lightinducible expression system in ami R-FNR-B and the activation of FNR expression.4.The hydrogen production content was detected by alternately culturing CC-849 and transgenic algal strains in white and red light.The results showed that the initial hydrogen production yield of ami R-FNR-B was lower than that of ami R-FNR after 3 days of white light culture in exponential phase.While after alternating cultures through “8 h in red light-12 h in white light-8 h in red light”,the total hydrogen content of ami R-FNR-B was 19% higher than that of ami R-FNR and three times higher than that of wild-type.The difference was significant.It proved that the light regulation of FNR expression promoted the continuous and efficient production of hydrogen in C.reinhardtii.In summary,the light inducible system is reversible and non-toxic to green algae,and the operation is simple and convenient.The transgenic alga can be cultured in white light to recover well growth with the FNR expression,when turns to red light the FNR could be stop induced expressing,and down-regulated under the inner ami RNA suppression,causing hydrogen production.This optogenetic gene control system is a useful tool for gene regulation and also establishes a novel way to improve hydrogen production.