Heterologous Expression Of A Thermophilic Ferulic Acid Esterase Gene From Clostridium Thermocellum In Plants

Lignocellulose is an important form of biomass energy,which is abundant,sustainable and environment-friendly.The solid anti-degradation barrier of plant cell walls is a key limiting factor to the efficient conversion and utilization of lignocellulose.Ferulic acid is a bridge between covalently cross-linked hemicelluloses molecules or between covalently cross-linked hemicellulose and lignin molecules,which plays a key role in the formation of complex polymer network structures in plant cell walls,thereby restricting the degradation of lignocellulose by degrading enzymes.The heterologous expression of the ferulic acid esterase gene from microorganisms in plant cells can effectively improve the degradation and transformation efficiency of lignocellulose,but the thermesophilic enzyme will express its hydrolysis activity during plant growth and development and as a result has an interference or toxic effect on the host cell.In this paper,the genetic transformation of a thermophilic ferulic acid esterase gene(FAE)derived from Clostridium thermocellum was applied to achieve the dual goals of safe plant growth and cell wall degradation.The main results are as follows:1)In order to improve the expression efficiency of FAE enzyme in plant cells,the codons of this gene are optimized,including replacing the rare codons and increasing the GC%content.Plant expression vectors with recombinant FAE gene,pBINPLUS-ImpactVectors-FAEx,were contructed to express this FAE enzyme in different subcellular compartment,such as cytoplasm,apoplast,endoplasmic reticulum,chloroplast or mitochondria.2)Arabidopsis thaliana was genetically transformed by Agrobacterium-mediated method.The transgenic Arabidopsis thaliana plants were screened by PCR and the positive individual T0 plants for FAE1(cytoplasmic expression),FAE2(apoplast expression),FAE4(chloroplast expression),and FAE5(mitochondrial expression)were 5,6,4 and 7,respectively.3)An efficient in vitro regeneration system of Miscanthus sinensis was established.Based on this,the thermophilic FAE gene was introduced into embryonic calli of M.sinens is by Agrobacterium-mediated method,and five transgenic lines for different subcellularly expressed FAE enzyme were obtained and identified by PCR.4)The agronomic traits comparing the flowering time,plant height,100-seed weight,fresh biomass weight and biomass water content for different transgenic lines of Arabidopsis thaliana was determined and compared.The results showed that the flowering time of FAE1 or FAE2 line was not significantly different from that of the wild type,while there was a significant difference between the FAE4 or FAE5 line and the wild type because they had a flowering time of 6 days and 10 days earlier than the wild type,respectively.The plant height,fresh biomass weight and 100-seed weight in each transgenic line were not significantly different from the wild type,but the water content in biomass of FAE5 line was reduced.5)The recombinant thermophilic FAE enzyme activity and the degradation efficiency of lignocellulosic biomass were determined by HPLC,and it indicated that the thermophilic FAE enzyme was successfully expressed in different transgenic lines and their enzyme activities at 70 °C were much higher than those at 25 °C,with the highest expression level of 1152.23 U/mg in the cytoplasmically expressed transgenic plants.Under different pretreatment strengths and different hydrolysis temperatures,the yield of reducing monosaccharide in transgenic plants expressing thermophilic FAE enzymes was significantly higher than that in wild-type controls,and the reduction monosaccharide yield after pretreatment at 70 °C were much higher than those at 25 °C.In particular,when the thermophilic FAE enzyme was expressed in the cytoplasm,the highest yield of reducing monosaccharide was obtained at different pretreatment strengths,ranging from 49.55% to79.53%,which was 2.55 to 2.75 times that of the wild type control.