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Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine

Paper ID Volume ID Publish Year Pages File Format Full-Text
31526 44807 2015 12 PDF Available
Title
Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine
Abstract

•Bacillus subtilis was engineered for production of vitamin B6 from 4-hydroxy-l-threonine (4-HO-Thr) and deoxyxylulose (DX).•B. subtilis was adapted at the genome level for improving conversion of 4-HO-Thr and DX to vitamin B6.•A better enzyme combination of the heterologous pathway led to enhanced Vitamin B6 production.

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5′-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5′-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28–34 mg/l PN from 4-HO-Thr while the wild-type parent produced only 12 mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65 mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.

Keywords
Vitamin B6; Pyridoxine; Bacillus subtilis; Antimetabolite; Promiscuous enzyme; 4-Hydroxy-l-threonine
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Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 29, May 2015, Pages 196–207
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us