Metabolic Engineering Of Thermoanaerobacterium Aotearoense SCUT27 For Enhanced Hydrogen Production

The demand of energy keeps increasing for the rapid urbanization and industrialization of human being.Due to rapid consumption and non-renewable charactertistic of fossil fuels also with more and more serious pollution problems in our living environment,exploitation of alternative energy sources,which is renewable,abundant and clean,became a key research focus for researches in recent years.As one of the clean energy carriers,hydrogen has the potential to replace traditional fuels because of its high energy capacity and environmental friendliness,and the biological processes to produce hydrogen are more eco-friendly.The main products of T.aotearoense SCUT27 stored in our laboratory has been found to produce mainly ethanol,lactate acid,acetate acid and hydrogen using various sugars and showed industrial potential with utilization of even hemicellulose for the production of various compounds and the co-fermentation of pentose and hexose.But the concentration and yield of hydrogen are far from satisfactory for large-scale hydrogen production.In T.aotearoense SCUT27,NADH is the co-factor for the production of ethanol,lactate acid and hydrogen,and the NADH supply was first improved for the enhancement of hydrogen production.In T.aotearoense SCUT27,the Nfn AB catalyzes the transfer of electron from reduced ferredoxin and NADH to NADP⁺.Deletion of nfn AB would block NADPH production from NADH,which facilitates the accumulation of NADH.The nfn AB gene of T.aotearoense SCUT27 was deleted and obtained mutant SCUT27/△nfn AB.Hydrogen production by SCUT27/nfn AB with various sugars showed a 40%increase.The NADH/NAD⁺ratio in SCUT27/△nfn AB is always higher than in SCUT27 during the fermentation.No obvious inhibition effect was observed for either SCUT27 or SCUT27/?nfn AB when six types of lignocellulose hydrolysates pretreated with dilute acid were used for hydrogen production,and SCUT27/?nfn AB showed elevated hydrogen production than SCUT27.The concentration of hydrogen for strain SCUT27/?nfn AB from rice straw hydrolysate and corn cob hydrolysate reached 62.39±3.45 mmol/L and 69.76±2.78mmol/L,respectively.Subsequently,in 5-L bioreactor the hydrogen concentration reached 209.31 mmol/L when using rice straw hydrolysates as the substrate.The Adh E plays an important role in ethanol formation of T.aotearoense SCUT27.The adh E mutation may change the cofactor specificity of Adh E,which affects the production of ethanol.The adh E gene of T.aotearoense SCUT27 was site-directed mutated and obtained mutant SCUT27/adh E^G544D.The hydrogen production of SCUT27/adh E^G544Dreached 45.74 mmol/L,which was 33.94%higher than SCUT27.Then the nfn AB gene of SCUT27/adh E^G544Dwas deleted to further improve its hydrogen production.The hydrogen production of mutant SCUT27/adh E^G544D/nfn AB was 34.31%,20.66%and 74.11%higher than SCUT27/adh E^G544D,SCUT27/nfn AB and SCUT27,respectively.Finally,a Fe/C compounds was prepared and added to the fermentation medium.The addition of Fe/C compounds led to a 15.51%increase in hydrogen production for strain SCUT27/adh E^G544D/nfn AB.Homologous and heterologous genes related to hydrogen production were individually or co-expressed in T.aotearoense SCUT27.The genes selected were the hfs B hydrogenase gene and hyd G maturase gene from T.aotearoense SCUT27,the hyd G maturase gene（hyd Gc）,[Fe Fe]-hydrogenase gene（Fe Fec）and ech hydrogenase gene（echc）from Clostridium thermocellum.The individual expression of these genes could enhance the hydrogen production of SCUT27 at some extent,especially for the expression of genes hfs B,hyd Gc and Fe Fec,the hydrogen production of the mutants increase by 17.37%,25.82%and 17.82%,respectively.Then the genes hfs B and hyd Gc were co-expressed in T.aotearoense SCUT27,and the hydrogen production of the mutant SCUT27/hfs B/hyd Gc reached 49.84±2.96 mmol/L,which was 45.05%higher than SCUT27.The Cre/lox system was introduced to T.aotearoense SCUT27 and the operation procedure was simplified,which made it possible for repeatable gene manipulation in SCUT27.Then several hydrogen producing mutants were obtained based on this system.The hydrogen production of mutant SCUT27/adh E^G544D/nfn AB/ldh/hfs B/hyd Gc reached 148.67±9.65 mmol/L,which was 152%higher than its parent strain SCUT27/adh E^G544D/nfn AB.And it is 33.29%higher than that of SCUT27/adh E^G544D/nfn AB/ldh.Compared with the wild-type strain SCUT27,the hydrogen production of this mutant increases by 332.56%.And the hydrogen yield of this mutant reached 2.58 mol/mol glucose,which is 115%higher than SCUT27.In summary,the metabolic engineering strategy used in this study efficiently enhanced the hydrogen production of T.aotearoense SCUT27.The preliminary research on hydrogen production using several lignocelluloses provides a new idea for the development of hydrogen production by microbial fermentation.