Enhancing the capability of Klebsiella pneumoniae to produce 1, 3-propanediol by overexpression and regulation through CRISPR-dCas9.

Publisher: Wiley-Blackwell Country of Publication: United States NLM ID: 101316335 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1751-7915 (Electronic) Linking ISSN: 17517915 NLM ISO Abbreviation: Microb Biotechnol Subsets: MEDLINE

Publication: Hoboken, NJ : Wiley-Blackwell
Original Publication: Oxford : Blackwell, c2008-

Clustered Regularly Interspaced Short Palindromic Repeats* ; Klebsiella pneumoniae*/genetics; Fermentation ; Glycerol/metabolism ; Propylene Glycol/metabolism ; Propylene Glycols/metabolism

Klebsiella pneumoniae is a common strain of bacterial fermentation to produce 1, 3-propanediol (1, 3-PDO). In general, the production of 1, 3-PDO by wild-type K. pneumoniae is relatively low. Therefore, a new gene manipulation of K. pneumoniae was developed to improve the production of 1, 3-PDO by overexpressing in the reduction pathway and attenuating the by-products in the oxidation pathway. Firstly, dhaB and/or dhaT were overexpressed in the reduction pathway. Considering the cost of IPTG, the constitutive promoter P32 was selected to express the key gene. By comparing K.P. pET28a-P32-dhaT with the original strain, the production of 1, 3-PDO was increased by 19.7%, from 12.97 to 15.53 g l ^-1 (in a 250 ml shaker flask). Secondly, three lldD and budC regulatory sites were selected in the by-product pathway, respectively, using the CRISPR-dCas9 system, and the optimal regulatory sites were selected following the 1, 3-PDO production. As a result, the 1, 3-PDO production by K.P. L1-pRH2521 and K.P. B3-pRH2521 reached up to 19.16 and 18.74 g l ^-1 , which was increased by 47.7% and 44.5% respectively. Overexpressing dhaT and inhibiting expression of lldD and budC were combined to further enhance the ability of K. pneumoniae to produce 1, 3-PDO. The 1, 3-PDO production by K.P. L1-B3-PRH2521-P32-dhaT reached 57.85 g l ^-1 in a 7.5 l fermentation tank (with Na ⁺ neutralizer), which is higher than that of the original strain. This is the first time that the 1, 3-PDO production was improved in K. pneumoniae by overexpressing the key gene and attenuating by-product synthesis in the CRISPR-dCas9 system. This study reports an efficient approach to regulate the expression of genes in K. pneumoniae to increase the 1, 3-PDO production, and such a strategy may be useful to modify other strains to produce valuable chemicals.
(© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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0 (Propylene Glycols)
6DC9Q167V3 (Propylene Glycol)
PDC6A3C0OX (Glycerol)

Date Created: 20220317 Date Completed: 20220706 Latest Revision: 20220718

20240829

PMC9249332

10.1111/1751-7915.14033

35298861