| Bacterial cellulose(BC)is a kind of microbial extracellular polysaccharide,which is a polymer consisted of the ?-1,4-linked glucose unit catalyzed by BC synthase(BCS).Compare with plant cellulose,BC possesses high purity without pectin,lignin,hemicellulose and other heteropolysaccharides.In addition,BC has unique properties such as high crystallinity,high water-holding capacity,high mechanical strength and good biocompatibility.BC has been certified by the U.S.Food and Drug Administration(FDA)as a safe food additive since 1992,and is widely used as a food suspension,thickener and emulsifier.Furthermore,it is widely applied into medical,textile and papermaking industries,as it possesses unique character such as renewability,biodegradation and good biocompatibility.Komagataeibacter xylinus is a high-yield model species for the research of BC.Whereas the synthesis and regulation mechanism of BC in K.xylinus is little understood,and the industrial development of BC was limited by the slow progress of the genetic modification.In order to deepen the understanding of the regulation network of BC biosynthesis and provide a theoretical basis for the genetic modification of BCproducing strains,the high-yield strain P5 obtained by NTG mutagenesis was involved into this study as the research object.Firstly,the properties of BC produced by strain P5 and P1 were analyzed.Secondly,comparative genomic analysis and comparative transcriptome analysis were performed to explore the high-yield mechanism of BC production in strain P5.And a transposon mutation library of strain P5 was constructed for the screening and identification of the regulatory genes of BC synthesis.Finally,BC produced by strain P5 was applied in the preparation of meatball products with low carbohydrate and high dietary fiber.The main research contents and conclusions are displayed as follows:1.Characteristics of BC produced by the high-yield strain P5.In this study,BC produced by the wide-type(WT)strain P1 and the high-yield strain P5 were obtained by agitated and static culture,respectively.The analysis of scanning electron microscope(SEM)revealed that BC filaments produced by strain P5 were more thinner and uniform,showed that the average diameter of BC filaments were26.47 ± 5.94 nm(agitated culture)and 31.08 ± 7.15 nm(static culture),respectively.The functional groups type of BC produced by strain P5 remained unchanged,which appeared typical infrared absorption spectrum of BC.The results of X-ray diffraction(XRD),thermogravimetric analysis(TGA)and tensile test indicated that the crystallinity,thermal stability and the Young's modulus of BC produced by strain P5 decreased slightly,while the water-holding capacity improved significantly.2.Investigation of the high-yield mechanism of BC-producing strain P5 by comparative genome analysis.The genome of high-yield strain P5 was composed of a cyclic chromosome and a cyclic plasmid,with a total length of 3.59 Mbp and GC content of 62.66%,and 3306 coding genes was annotated.Genome-wide analysis showed that strain P5 could synthesize BC from a wide range of carbon sources.Besides,four types of BCS operons were found in the high-yield strain P5,with low nucleotide sequence similarity.Further comparison of genome revealed that the high-yield strain P5 possessed 136 single nucleotide variations(SNPs),1 multi-nucleotide variation(MNP)and 1 deletion(DEL).KEGG pathway analysis showed that the mutant genes were concentrated in the pathways related to carbohydrate metabolism and energy metabolism.Further analysis of mutated genes in related metabolic pathways suggested that the mutations of sdh B,fum A,ace F and ald B genes related to the tricarboxylic acid cycle(TCA cycle)may influence the synthesis of BC by affecting the energy metabolism.bcs C ? encodes the Bcs C subunit of the BCS.The mutation of this gene may affect the secretion of BC and directly lead to the change of the yield of BC.gdh and tkt A mutations may affect the synthesis of gluconate and fructose-6-phosphate,respectively,improving the synthesis of UDPG,which is the precursor of BC synthesis,and further affect the synthesis of BC.3.Exploration of the high-yield mechanism of BC in strain P5 by comparative transcriptome analysis.To further analyze the high-yield mechanism of BC in strain P5,differentially expressed genes(DEGs)between strain P5 and strain P1 were analyzed by comparative transcriptome analysis.And further classified and analyzed according to KEGG database to identify the DEGs that associated with the changes of BC yield.Compared with the gene transcription level of the WT strain P1,177 genes were up regulated and363 genes were down regulated in the high-yield strain P5.Among them,the up regulation of bcs B ? and bcs C ? encoding BCS indicated that the synthesis level of BC was up regulated in strain P5,which directly promoted the synthesis of BC.The down regulation of gluconate-related genes gcd,gdh and gnt K revealed that the synthesis level of by-product gluconate in strain P5 was down regulated,and more glucose was used for UDPG synthesis.The down regulation of gene3141 encoding cellulase indicated that the degradation level of BC in strain P5 decreased.And the down regulated expression of ace gene cluster and its regulator chv I suggested that the synthesis level of other soluble polysaccharides in strain P5 decreased,UDPG was more directly supplied to the synthesis of BC,leading to the increase of BC production.In addition,the expression level of genes associated with energy metabolism in strain P5 was down regulated and the energy metabolism level decreased,suggesting that further improvement of energy metabolism level may further enhance the production of BC.4.Screening and identification of genes related to the synthesis of BC in strain P5.On the basis of the exploration of the high-yield mechanism of BC synthesis in strain P5,the BC synthesis regulatory genes were screened through transposon mutant library,which providing theoretical support for the targeted genetic modification of strain P5 to further improve BC production.Firstly,a transposon mutation library of strain P5 containing 8000 mutant strains was successfully constructed by conjugation transfer.And strains P5 was used as receptor strain,Escherichia coli SMl0?pir/p SCl89 was used as donor strain.Subsequently,a high-throughput screening method for BC production was developed to screen BC synthesis related gene mutant strains.33 mutant strains with significant BC yield changes were obtained through screening 3415 mutant strains and rescreening 88 mutant strains in shaker flask.The flanking sequence was amplified by thermal asymmetric interlaced PCR(TAIL-PCR),and inserted mutant genes were identified by sequencing.The inserted mutant gene in high-yield strain strain 7-74 was mnt H,which encoding divalent metal cation transporter.The mutant gene of low-yield mutant strains 1-1,1-9 and 4-52 was ett A,which encoding Ett A protein to regulate intracellular protein synthesis.And the low-yield mutant strains 10-95 and 12-93 were inserted with opr B,encoding carbohydrate porin.In addition,lowyield mutant strain 11-133 was inserted into the plasmid gene p A?gene0061,which encodes Tau E/Saf E family proteins responsible for sulfite output.5.Application of BC in low carbohydrate and high dietary fiber meat products.Finally,BC obtained by the high-yield strain P5 under different culture conditions were added to chicken,fish and beef to make meatballs with low carbohydrate and high dietary fiber respectively,and the texture of those meatballs were determined.The results of texture analysis showed that BC obtained by static culture improved the hardness,springiness,chewiness,resilience,gumminess and cohesiveness of chicken meatballs effectively,which possess a good improvement effect.And the BC obtained by agitated culture effectively improve the hardness,springiness,chewiness,resilience,gumminess and cohesiveness of fish meatballs and beef meatballs.Suggesting that as a dietary fiber,BC replaced 6% starch successfully,which increased the dietary fiber content and reduced the calorie of meatball,and improved the texture characteristics of meatball products.In conclusion,the characteristics of BC produced by the high-yield strain P5 were analyzed in this study.The mutation sites related to BC synthesis in strain P5 were preliminarily identified through comparative genome analysis.Comparative transcriptome analysis revealed energy metabolism in strain P5 was reduced and DEGs associated with increased BC production were identified.Subsequently,a transposon mutation library of strain P5 was constructed and 9 genes related to the BC synthesis were further identified.Finally,BC produced by strain P5 was applied to the production of low carbohydrate and high dietary fiber meatball products.This study laid a theoretical foundation for the genetic modification of high-yield strains to further improve the yield of BC,and provided a reference for the production of meatball products with low carbohydrate and high dietary fiber. |
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