Fructosylated chondroitin serves as a crucial precursor for the enzymatic synthesis of chondroitin sulfate (CS).However, its current biosynthesis faces significant challenges, such as low synthesis efficiency, complex extraction processes, and reliance on antibiotics during production, which hinder its large-scale production and application.To overcome these limitations, Escherichia coli K4, a strain capable of naturally synthesizing and transporting fructosylated chondroitin, was employed as the chassis cell.A plasmid expression vector, pMUT2 ΔHyp3, was developed, demonstrating genetic stability for up to 9 days without antibiotic pressure.By recombinantly overexpressing the UDP-glucose dehydrogenase gene (ugdA2) from Corynebacterium glutamicum and the UDP-N-acetylglucosamine pyrophosphorylase gene (glmU) from Bacillus subtilis, the precursor synthesis pathway was enhanced, increasing fructosylated chondroitin production in shake-flask cultures to 0.95 g/L.Further optimization of the fermentation medium components elevated the yield to 1.35 g/L.Finally, antibiotic-free fermentation in a 3 L bioreactor achieved a production level of 7.5 g/L within 48 hours, with a yield of 4.4%.This study not only achieved the biosynthesis of fructosylated chondroitin and eliminated antibiotic dependency in recombinant E.coli K4 fermentation systems but also laid the groundwork for scalable, environmentally friendly enzymatic production of chondroitin sulfate.
SUN Jieyu
,
ZHU Yuhao
,
SHI Zhicheng
,
XIAO Sen
,
HUANG Ziyang
,
XU Zhibing
,
DU Guocheng
,
CHEN Jian
,
KANG Zhen
. Construction of an antibiotic-free expression system in Escherichia coli K4 and optimization of fructosylated chondroitin biosynthesis pathway[J]. Food and Fermentation Industries, 2025
, 51(22)
: 1
-9
.
DOI: 10.13995/j.cnki.11-1802/ts.042436
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