The Ability And Molecular Mechanism Of Biodegradation Of Malachite Green By Cyanobacterium Synechococcus Elongatus PCC 7942 Engineered With A Triphenylmethane Reductase Gene

Malachite green(MG)is an alkaline triphenylmethane dye that was extensively used in paper printing,textile industry and also used as insecticides and fungicides in aquacultures around the world.However,MG is toxic and carcinogenic that accumulates in fish tissues and water,which potentially threatening human health and destroying the ecological environment.Bioremediation has the characteristics of low-cost and environmental friendliness,it is a sustainable treatment method for dye pollution.At present,a variety of microorganisms have been proven to have MG decolorizing activity,but they have not been applied due to their limited growth in polluted or aquatic environments.In contrast,cyanobacteria grow in freshwater that have the advantages of independent of organic carbon sources and energy sources,fast growth and reproduction under light conditions,which have received widespread attention in the field of environmental restoration.In addition,cyanobacteria is easy to genetic engineering,recombinantly expressing active enzymes in cyanobacteria is conducive to the internal degradation of MG on the basis of biosorption and enrichment,which could improve the repeated utilization of algae.Triphenylmethane reductase(TMR)is an oxidoreductase that specifically degrades triphenylmethane dyes into colorless low-toxic molecules.We previously characterized a novel TMR,KaTMR,from an endophytic bacterium Klebsiella aerogenes S27 that could efficiently degrade MG into small non-toxic products and restore the growth of plants in MG solution,it has the potential for bioremediation applications.However,the recombinant expression of katmr gene in commonly used engineering strain Escherichia coli BL21 is mainly in the form of inclusion bodies,which is not conducive to its industrial development and application.In this study,we took freshwater cyanobacteria Synechococcus PCC 7942 as the research object and first explored its decolorization activity and molecular mechanism to MG.Secondly,we constructed an engineered cyanobacteria PCC7942-KaTMR by heterologous expression of the katmr gene,which producing recombinant KaTMR as an active and soluble protein with a high production,then we studied the function and mechanism of engineered cyanobacteria in the degradation of MG.This study provided a theoretical basis for the application of cyanobacteria in the bioremediation of MG wastewater.The main research contents in this study were described as following:(1)The first report of the utilization of cyanobacterium in the adsorption of MG:our datas showed that the wild-type Synechococcus decolorized 99.5%of MG within 12 h at 30?and pH 6.0.(2)Synechococcus PCC 7942 absorbed and enriched MG,but can not degrade it:HPLC-MS analysis showed that MG can enter the vivo of Synechococcus cells,but no chemical reaction occurs.Meanwhile,the growth curve showed that MG significantly inhibited the growth of cells after permeation.(3)Synechococcus PCC7942-KaTMR has been successfully estabished:engineering cyanobacteria overexpressed soluble protein,the yield of recombinant KaTMR was approximately 720 mg/L.Furthermore,the recombinant KaTMR was purified by Ni-NTA agarose with a yield of 120 mg/L.Recombinant KaTMR has decoloring activity of MG and its optimal conditions of MG decolorization is pH 8.0 and 60?.HPLC-MS analysis showed that recombinant KaTMR degraded MG into(4-dimethylamino-phenyl)-Phenyl-methanone(m/z 226.98)and demethyl green(m/z 315.45)in vitro.(4)Synechococcus PCC7942-KaTMR possessed higher MG decoloring activity than wild-type Synechococcus.Firstly,comparing with the wild-type cyanobacteria,the engineered cyanobacteria possessed higher decoloring ability at the low cell concentration(4.5×10~6 cells/mL)and in stressful environments(NaCl 0.9-1.5 M,pH 4-6,temperature 42?),the decoloring rate increased 7.7%.Secondly,the engineered cyanobacteria showed a high sustainability in a fed-batch process,at 60 h,the remaining MG concentration of engineered cyanobacteria is 11.4 mg/L and the wild-type is 19.1mg/L respectively.(5)Synechococcus PCC7942-KaTMR can degrade MG,which verified the molecular mechanism of the advanced activities mentioned above:HPLC-MS analysis showed that the engineered cyanobacteria degraded MG into smaller molecules 4-methylaminobenzoic acid(m/z 226.98)and 4-hydroxyl-aniline(m/z 315.45)in vivo.(6)Synechococcus PCC 7942 is a promising tool in remediation of MG polluted water:the engineered cyanobacteria has significant detoxifying activity against MG,phytotoxicity experiments showed that the germination rate of the wheat seeds was 78.8%and the stem could not grow when co-incubated with 100 mg/L MG;comparingly,the germination rate increased to 97.7%and the stem length was 3.65 cm when the seeds co-incubated with the MG degradation products of engineered cyanobacteria cells.In summary,it was the first report to reveal the efficient decolorization of MG by Synechococcus PCC 7942.Furthermore,we established an engineered cyanobacteria by expression of the triphenylmethane reductase gene,katmr,which showed a high sustainability of MG decolorization and degradation that promoted the growth of the engineered cyanobacteria in MG polluted environment.These research results have enriched the application of cyanobacteria in bioremediation of toxic dyes and water,which is of great significance to the recycling of water resources,especially freshwater resources.