Functional Characterization And Application Of The Transporter YdgF1 In Bacillus Licheniformis 9945a

D-alanine is an essential amino acid for the growth of gram-positive bacteria and participates in peptidoglycan biosynthesis.Gram-positive bacteria generally obtain D-alanine with D-alanine racemases converting L-alanine.In recent years,studies have found that some Gram-positive bacteria use transporters to assimilate extracellular D-alanine.Ala P from Bacillus subtilis and Ser P2 from Lactococcus lactis have been identified to have the function of assimilating D-alanine.At present,no transporter of Bacillus licheniformis has been identified to assimilate D-alanine.The existing research on alanine metabolism in Bacillus licheniformis is insufficient.In this study,the transporter Ydg F1that may assimilate D-alanine was found in Bacillus licheniformis 9945a genome by homologous alignment,then its structural analysis and functional characterization were conducted.The whole-cell catalyst was modified according to the function of Ydg F1,to analyze the effect of transport modification on the whole-cell catalytic synthesis ofα-methyl-L-serine by Bacillus licheniformis.The main results are as follows:（1）The strain 9945a/p HY300-Shu-ydg F1 with ydg F1 being overexpressed and the strain 9945aΔydg F1 with ydg F1 knocked out were constructed.The PDA medium containing only glucose,inorganic salt and D-alanine was used to compare growth differences among strains.The knockout of ydg F1 weakened cell proliferation of Bacillus licheniformis 9945a in PDA medium,and the overexpression of ydg F1 enhanced cell proliferation of Bacillus licheniformis 9945a in PDA medium.In cell assimilating experiments,the concentration of D-alanine assimilated by 9945aΔydg F1 was significantly lower than that assimilated by 9945a,while the concentration of D-alanine assimilated by 9945a/p HY300-Shu-ydg F1 was slightly higher than that assimilated by 9945a.It further proved that Ydg F1 can assimilate D-alanine in Bacillus licheniformis 9945a.The knockout or overexpression of ydg F1 had little effect on Bacillus licheniformis 9945a assimilating L-alanine,indicating the transporter Ydg F1 has chiral preference for D-alanine.In addition,Ydg F1 may also assimilate L-asparagine and amino acids with similar structures to L-asparagine.（2）RT-q PCR evaluated the relative expression of ydg F1 in 9945a and 9945aΔydg F1 at different growth phases in the LB medium.The relative expression of ydg F1 in 9945a increased gradually at the transition and stationary phases.β-acetylglucosaminidase Ybb D and peptidoglycan amidase Ybb E participating in peptidoglycan recycling had similar results as Ydg F1.CFU of 9945a and 9945aΔydg F1 at the anaphase in the LB medium were determined by viable plate counting.CFU of 9945aΔydg F1 in the anaphase was lower than that of 9945a,and the death rate of 9945aΔydg F1 was faster than that of 9945a.It suggested that the knockout of ydg F1 weakened the viability of Bacillus licheniformis 9945a in the anaphase.（3）α-Methylserine hydroxymethyltransferase of Paracoccus alcaliphilus was heterologously expressed in Bacillus licheniformis 9945a,which catalyzes D-alanine toα-methyl-L-serine.The synthesis ofα-methyl-L-serine whole-cell catalyzed by 9945a/p HY300-Shu-ydg F1-Shu-mshmt overexpressing both Ydg F1 andα-methylserine hydroxymethyltransferase in 1 h increased by 13.6%compared to that of 9945a/p HY300-Shu-mshmt overexpressing onlyα-methylserine hydroxymethyltransferase.When the initial concentration of D-alanine was 30 mmol·L^-1,9945a/p HY300-Shu-ydg F1-Shu-mshmt whole-cell catalyzed the synthesis of 20.39 mmol·L^-1α-methyl-L-serine in 3 h,and the conversion rate was 85.1%.This study preliminarily identified the transport function of the transporter Ydg F1 in Bacillus licheniformis 9945a,and explored the role of Ydg F1 in the anaphase survival of Bacillus licheniformis 9945a.Utilizing the function of the transporter to modify the cell membrane of whole-cell catalysts can improve their mass transfer efficiency,which provides a reference for the optimization of whole-cell catalysis in the future.