Effects Of BofA And BglG Genes On Sugar's Utilization In Thermoanaerobacterium Aotearoense SCUT27

Biomass is one of the major new energy sources in the 21st century.After being pretreatment,largr numbers of fermentable sugars such as glucose and xylose can be obtained from the abundant biomass and the mass ratio?glucose and xylose?is about 2:1.For converting biomass resources to high value-added clean bioenergy,a biocatalyst,which can efficiently co-utilize glucose and xylose,is urgently needed.Thermoanaerobacterium aotearoense SCUT27 with co-utilizing xylose and glucose is a promising candidate for lactic acid,ethanol and hydrogen production from lignocellulosic feedstocks.So exploration on the molecular mechanism of xylose and glucose co-utilization,further improving the ability of xylose and glucose utilization efficiency,has an important theoretical research significance and application value.Based on the transcriptome data of SCUT27 under glucose,xylose and mixed sugar as carbon sources,bofA gene encoding pro-sigmak processing inhibitor BofA,which is special expressed in mixed sugar and bglG gene encoding transcription antiterminator Bg1G,which is highly expressed with xylose in the culture medium were selected for function verification by gene knock-out and overexpression.Based on the sequence of 1c1|NC₀17992.1_cdsid_YP₀06391243.1?bofA?and Tsac₂568?bglG?,primers of both genes' homologous arms were designed and pBluescript ?SK?+?was the basic knockout plasmid,in which,kanamycin gene acted as resistance marker and homologous arms were ligated at both sides of marker.After achieving the targeting vector,gene knock out was carried out and both PCR results and Southern blot verification proved that SCUT27/?bofA and SCUT27/?bglG were successfully obtained.Using pIKMI as basic expression vector in which kanamycin gene as resistance marker,we successfully obtained overexpression engineering strain SCUT27/bglG.When xylose is used as sole carbon source,the xylose metabolic rate of SCUT27/?bofA and the productivity of lactic acid increase by 51.43%and 23.53%,respectively,compared with the parent strain.When glucose and xylose?glucose:xylose=1:1?are used as carbon source,xylose metabolic rate of SCUT27/?bofA and the productivity of lactic acid and ethanol are 44.44%,43.75%and 20.00%higher than those of the parent strain,respectively.Therefore,pro-sigmak processing inhibitor BofA is a negative regulator for xylose utilization and its functions need further exploration.When xylose is used as the sole carbon source,compared with SCUT27,the xylose metabolism rate of SCUT27/?bglG decreases by 68.57%and the xylose metabolism rate of SCUT27/bglG increases by 31.74%;When mixed sugar is used as the carbon source,the xylose metabolism rate of SCUT27/?bglG decreases by 33.33%and xylose metabolism rate of SCUT27/bglG increases by 33.33%.It is concluded that bglG gene is a positive regulator for xylose metabolism.When xylose is used as the sole carbon source,in the late logarithmic phase,the xylose isomerase activity of SCUT27/?bglG enhances 61.54%compared with SCUT27;When mixed sugar is used as the carbon source,in the late logarithmic phase,the xylose isomerase activity of SCUT27/?bglG enhances 17.03%compared with SCUT27.These results are in accordance with the improved transcriptional level of gene xi.When xylose or mixed sugar is used as carbon source,RNA transcriptional level of gene xylf which is related to xylose transporter proteins in SCUT27/?bglG is down-regulated significantly.We speculate that BglG may be related to the genes about xylose transporter proteins.In summary,pro-sigmak processing inhibitor BofA is a negative regulator for xylose utilization.After releasing the negative control of bofA genes,the transcriptional level of genes related to xylose metabolism such as xi,xk and xylf are upgraded significantly.Therefore,BglG is a positive regulator for xylose metabolism to which the positive effect may be related to xylose transport.