Identification Of Salecan Biosynthesis Genes And Preparation Technology Of Salecan Oligosaccharide

Salecan,a soluble ?-1,3-D-glucan produced by a salt-tolerant strain Agrobacterium sp.ZX09,has multiple bioactivities and unusual rheological properties in solution,leading to its broad potential application in food,industry,feeding,agriculture and pharmaceuticals.However,little is known about the molecular mechanism of salecan biosynthesis.In order to deeply understand salecan biosynthesis pathway,it is necessary to clone and identify the salecan biosynthesis gene cluster.First of all,we cloned the salecan biosynthesis gene cluster(sle)by transposon mutagenesis and SEFA-PCR.The sle gene cluster contained 19646 bp encoding 16 ORFs.The functional structure of the sle gene cluster had the regulation gene(sleX),chain length determination protein gene(sleH),glycosyltransferase gene(sleCDEF,sleU,and sleW)modifying enzyme gene(sleA and sleV),and export protein gene(sleT).The putative gene products of the sle gene cluster showed high sequence identity with those of the succinoglycan biosynthesis exo cluster genes in various Rhizobium and Agrobacterium species.The mutations in sleB,sleC,sleD and sleH genes suppressed salecan biosynthesis.RT-PCR analysis revealed that the transcription of sleABCDEFGH was consecutive and at the same cistron.The analysis of promoter activity showed that the promoter upstream of sleX was strongest;the second strong promoter was upstream of sleA.In addition,weak promoters were identified upstream of sleW?sleT and sleC,the promoter upstream of sleG was weakest.The putative pyruvyl transferase gene(sleV)and succinyltransferase gene(sleA)were found in the salecan biosynthesis gene cluster from Agrobacterium sp.ZX09.Furthermore,we found the signals of noncarbohydrate substituents in the NMR spectra of enzymatic salecan fragments using a ?-glucanase.Both succinyl and pyruvyl substituent groups on salecan were identified by HPLC and MS analysis.Disruption of the putative succinyltransferase sleA gene resulted in the absence of succinyl substituent groups on salecan.This defect could be complemented by expressing the sleA cloned in a plasmid.We next investigated the influence of succinyl substituents on the rheological properties of salecan solution.The viscosities of succinate-free salecan solutions were significantly lower than that of native salecan at the same concentration.Loss of succinyl substituents on salecan lowered the sensitivity to temperature and salt concentration.SleH protein encoded by chain length determination protein gene sleH in the sle gene cluster contained a Wzz chain length determination conserved region,a G-rich domain on putative tyrosine kinase and a P-loop NTPase domain.SleH,belonging to Polysaccharide Co-Polymerase family(PCP),was an inner membrane-embedded protein with a transmembrane domain located around amino acid positions 36-60.The N-terminal hydrophilicity region was located in cytoplasm;the C-terminal region was located in periplasm.The sleH mutant Agrobacterium sp.MUH could secrete some oligosaccharide with the relatively uniform molecular weight.On the basis of spectroscopic analysis,including PMP-HPLC,ESI-MS,ID-and 2D-NMR techniques,the backbone structure of the oligosaccharide was ?-D-Glcp-(1?3)-[?-D-Glcp-(1?3)-?-D-Glcp-(1?3)]3-D-Glccp with a pyruvyl substituent group attached to the second glucose residue from the reducing end and the succinyl substituents occasionally attached to the third and fourth glucose residue.The structure was in conformity with that of salecan.Therefore,the oligosaccharide was proved to be the repeating unit of salecan and SleH was involved in salecan biosynthesis and polymerization.On the basis of Htm medium,the medium optimization for salecan oligosaccharide(SO)production was performed by single factor experiment and response surface methodology.The optimized medium was:NaH2PO4 1 g,KNO3 3 g,MgCl2 0.2 g,CaCl2 0.07 g,FeCl20.0125 g,MnSO4 0.003 g,ZnCl2 0.0075 g,Sucrose 50 g,Tryptone 0.1 g,Water 1000 mL,pH 9.0.The SO yields reached up to 12.3 g/L when inoculated with 4%(v/v)of the seed culture and cultivated at 30? for 144 h.We first cloned and identified the salecan biosynthesis gene cluster.Using the methods of genetic engineering,we obtained Agrobacterium sp.MUH as the production strain of salecan oligosaccharide with the relatively uniform molecular weight,indicating that Agrobacterium sp.MUH could be used in the industrial fermentation of salecan oligosaccharide.