Effects Of Xylose And Other Precursors On The Yield Of Gentamicin C1a By A Synthetic Medium

Gentamicin C1a(GM C1a)is an aminoglycoside antibiotic which has broad-spectrum antibacterial activity.It is widely used in animal husbandry and medicine treatment.GM C1a,the main component of gentamicin,is mostly produced by Micvomonospora purpurea.At present,studies on gentamicin C1a mainly focus on the strain selection and fermentation optimization using composite medium.And there are few in-depth investigations of the metabolic characteristics of M.purpurea.This results in poor stability of industrial production,which is not conducive to the promotion of competitiveness of enterprises.In this study,the effect of adding different substances such as xylose to the yield of gentamicin C1a.Then,a synthetical medium for GM C1a by M.purpurea was developed.Finally,a steady-state 13C labeling experiment was conducted to analyze the mechanism of xylose addition on the metabolic characteristics.Firstly,the amino acids,methylation precursors and synthetic pathway precursors were added to the complex medium in order to improve the yield of GM C1a,The addition time and the concentrations are also optimized.The results showed that the addition of 0.1 g/L xylose at 24 h can increase the yield of gentamicin C1a to 660.3 ?g/mL,which is 31.6%higher than the control(502 ?g/mL),the yield increased 35.5%by adding eight precursors.Secondly,A high-throughput screening method was used to perform a single-factor verification experiment on 20 amino acids,10 purine pyrimidines,7 vitamins,5 metal ions,and 2 synthetic precursors,in which,20 substances had significant effect on the yield of gentamicin C1a.Using the PB experiment,the concentrations of these substances were optimized,and a synthetic medium suitable for the gentamicin C1a production by M.purpurea was obtained.The experimental results showed that the addition of xylose and betaine were beneficial to the increase of gentamicin C1a production.The addition of asparagine and 2-deoxy-D-glucose would inhibit the growth of bacteria cell,while the addition of phenylalanine,glutamine,methionine,and choline had no significant effect on fermentation.Finally,in the steady-state 13C labeling experiment,the intracellular metabolic flux was analyzed according to the measured intracellular metabolites abundance information and extracellular macroscopic data.The results showed that,the flux of ED pathway and the glyoxylic acid circulation pathway is very low.Compared with the control,the flux of the gentamicin C1a synthesis pathway increased 42.8%by adding the xylose.And the flux of the NADPH and NADH exchange reaction doubled.71%of the xylose entered central carbon metabolism through the HMP pathway and 29%of the xylose directly participated in product synthesis.The flow of HMP significantly increased.In this research,the understanding of the intracellular metabolic characteristics of M.purpurea,deepened.Furthermore,it provides theoretical bases for the genetic engineering modification and the fermentation process optimization.