Effects And Mechanism Of Methionine Addition On Ethanol Production By Thermoanaerobacterium Aotearoense

The depletion of fossil energy and global warming are two major challenges for human society.It is extremely urgent to find a clean and renewable new energy source.Bioethanol is a new superior energy to replace non-renewable fossil fuels because of its low pollution and renewable characteristics.Saccharomyces cerevisiae and Z.mobilis,as important industrial strains for producing ethanol,have disadvantages such as narrow utilization of substrate.Instead Thermoanaerobacterium aotearoense can directly utilize lots of lignocellulose hydrolyzed monosaccharides,which is considered to be an ideal biocatalyst for the production of ethanol due to short fermentation cycles and wide substrate spectrum,whose growth and ethanol producing capacity are weak,and the fermentation cost is very high.In this study,first based on the effect of amino acids on the ethanol production and growth of T.aotearoense,the uptaking of exogenous methionine and endogenous methionine synthesis were enhanced by metabolic engineering,and then from the following three aspects to explore the mechanism of methionine affecting the growth of the strain:level of intracellular reducing power,the key enzyme activity of Pentose phosphate pathway and intracellular oxidative stress damage.Finally,the production cost was further reduced by optimizing the medium to realize the efficient production of ethanol.This study first compared the effects of different amino acids?histidine,phenylalanine,arginine,serine,glycine,threonine,cysteine,tyrosine,tryptophan,methionine?added to DMSZ solid medium on the growth of T.aotearoense.The results showed that methionine significantly promoted the growth of T.aotearoense SCUT27?ldh.Furthermore,fermentation of T.aotearoense with different titers of methionine in MTC in serum bottles were carried out.It was discovered that the ethanol production and the rate of xylose consumption reached a maximum of 4.57 g/L and 0.58 g/?L·h?,respectively,which was 69.26%and 56.76%higher than that of without methionine,when the concentration of methionine reached 20 mg/L with xylose as the substrate.In order to further improve ethanol production and sugar utilization,the methionine repressor gene by homologous recombination was knocked out in order to obtain resultant strain T.aotearoense SCUT27?ldh/?metJ,accompanying with strengthening intracellular methionine synthesis and extracellular methionine uptaking.When the fermentation of T.aotearoense SCUT27?ldh/?metJ with different carbon sources as substrates in MTC medium were carried out,the production,growth and sugar utilization were better than that of T.aotearoense SCUT27?ldh.Especially when xylose was the substrate,the ethanol production was increased by 30.74%compared with the control,and the xylose utilization rate was enhanced with 24.32%.Then the methionine addition amount was optimized.When the concentration of methionine was 30 mg/L with xylose as the substrate,T.aotearoense SCUT27?ldh/?metJ had the best fermentation performance.The growth ability and ethanol production and yield were significantly improved.OD₆₀₀=4.73,ethanol production was 7.45 g/L,and the yield reached0.38 g/g.In order to explore the mechanism of methionine promoting the growth and ethanol production in Thermoanaerobacterium aotearoense,this study first confirmed that the absorption capacity of methionine in engineering strains was enhanced,which was about 56.25%higher than that of the control?25.00 mg/L VS 16.36 mg/L?.The transcriptional levels for genes involved in methionine transport and uptake and key genes producing NADPH?metN,metQ,metI,G6PDH,6PGDH?,intracellular reducing power levels,oxidative stress levels,and key enzyme activity of pentose phosphate pathway were examined.The results showed that the genes expression were significantly up-regulated.The intracellular reductive force pool of T.aotearoense SCUT27?ldh/?metJ was increased.NADPH level was increased,resulting in improvement with ethanol production.High concentration of NADP⁺activated glucose-6-phosphate dehydrogenase,thereby enhancing pentose phosphate pathway.Decreasing intracellular oxidative stress is beneficial to enhance cell viability.In order to reduce the fermentation cost,so the medium composition was optimized.1 g/L yeast extract,4 g/L yeast extract and 2 g/L tryptone and different concentrations of corn steep liquor were added to the MTC medium for T.aotearoense.The results clarified that the fermentation performance of T.aotearoense SCUT27?ldh/?metJ in MTC with 4 g/L yeast extract and 2 g/L tryptone using different carbon sources as the substrate were best,whose fermentation performance was equivalent with the addition of 30 mg/L L-methionine.In order to further reduce the fermentation cost,DL-methionine was used instead of L-methionine.The ethanol production was 7.47 g/L when the addition of DL-methionine was 30 mg/L.Finally,the fed-batch free fermentation were carried out.The highest production of ethanol was reached27.85 g/L,and the maximum consumption rate of xylose was at the peak of 1.38 g/?L·h?.In summary,the addition of methionine can promote the growth and ethanol production of T.aotearoense.Knockout of methionine repressor protein can significantly enhance the uptake of exogenous methionine and synthesis of endogenous methionine,furether improving the fermentation performance of strain;methionine mainly affects the fermentation performance of the strain by strengthening the pentose phosphate pathway.