US 2026/0146269 A1Published application

Genetically engineered bacteria for producing d-pantothenic acid, construction method and application

AssigneeZhejiang University of Technology

InventorsBo Zhang, Zhoulin He, Zhiqiang Liu, Mengna Tang, Yunying Xiao, Pan Shen, Yuguo Zheng

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Abstract

The present disclosure relates to genetically engineered bacteria for producing D-pantothenic acid, a construction method thereof, and application of the genetically engineered bacteria in preparation of the D-pantothenic acid through microbial fermentation. Mainly, (1) carbon flux is further directed toward the synthesis of the D-pantothenic acid by increasing the copy number of key genes in the synthesis pathway of pantothenic acid in an Escherichia coli (E. Coli) organism; and (2) in order to further enhance the carbon flux to direct toward the synthesis of the D-pantothenic acid, glycolysis front-end genes are activated by weakening a nitrogen limitation-negative regulatory transcription factor to drive the carbon flow, phosphoenolpyruvic acid is saved, the central carbon flow entering TCA is reduced, and a plasma-free and antibiotic-free genetically engineered bacterium strain for producing D-pantothenic acid is obtained. Finally, D-pantothenic acid shake flask titer is increased by 87.2% compared with an original strain, which reaches 5.43 g/L; and fermentation is performed with a 5 L fermentation tank for 84 hours, and the yield of the D-pantothenic acid can reach 94.2 g/L.

Genetically engineered bacteria for producing D-pantothenic acid, constructed through the following method comprising:

(1) adopting genetically engineered bacteria ZJUTDPAL5 as chassis bacteria, increasing the copy number of an EcilvD gene regulated by a promoter pTrc on a genome of the genetically engineered bacteria, and obtaining an engineered bacteria DPAL6 derivative, yjiV::Ptrc-EcilvD and denoting the same as engineered bacteria DPAP7;
(2) increasing the copy number of a Bacillus subtilis BspanBA gene regulated by the promoter pTrc on a genome of the engineered bacteria DPAP7 in a gene knockin manner, and obtaining an engineered bacteria DPAP7 derivative, flik::Ptrc-BspanBA and denoting the same as engineered bacteria DPAP8;
(3) increasing the copy number of a Corynebacterium glutamicum CgpanC gene regulated by the promoter pTrc on a genome of the engineered bacteria DPAP8 in a gene knockin manner, and obtaining an engineered bacteria DPAP8 derivative, ompT::Ptrc-CgpanC and denoting the same as engineered bacteria DPAP9;
(4) increasing the copy number of a Bacillus subtilis alsS gene regulated by the promoter pTrc on a genome of the engineered bacteria DPAP9 in a gene knockin manner, and obtaining an engineered bacteria DPAP9 derivative, yjiP::Ptrc-alsS and denoting the same as engineered bacteria DPAP10;
(5) further increasing the copy number of a Bacillus subtilis BspanBB gene regulated by the promoter pTrc on a genome of the engineered bacteria DPAP10 in a gene knockin manner, and obtaining an engineered bacteria DPAP10 derivative, ydeU::Ptrc-BspanBB and denoting the same as engineered bacteria DPAP11;
(6) replacing an initiator codon ATG of a nac gene in a genome of the engineered bacteria DPAP11 with GTG, and obtaining an engineered bacteria DPAP11 derivative, nacGTG and denoting the same as engineered bacteria DPAP12;
(7) knocking out in-situ promoters P3, P4 and P5 located upstream of gene clusters ptsH, ptsI and crr in a genome of the engineered bacteria DPAP12, and obtaining an engineered bacteria DPAP12 derivative, ΔP345ptsH and denoting the same as engineered bacteria DPAP13;
(8) replacing an initiator codon ATG of a gltA gene in a genome of the engineered bacteria DPAP13 with GTG, and obtaining an engineered bacteria DPAP13 derivative, gltAGTG and denoting the same as engineered bacteria DPAP14;
(9) replacing an initiator codon GTG of a gltA gene in a genome of the engineered bacteria DPAP14 with TTG, and obtaining an engineered bacteria DPAP14 derivative, gltATTG and denoting the same as engineered bacteria DPAP15;
and
(10) replacing an in-situ promoter of a pfkB gene in a genome of the engineered bacteria DPAP15 with Ptrc, and obtaining an engineered bacteria DPAP15 derivative, PpfkB::Ptrc and denoting the same as engineered bacteria DPAP16, namely the genetically engineered bacteria for producing the D-pantothenic acid.

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Abstract

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