Lactic acid bacteria gene engineering strain for producing malic acid as well as construction method and application of lactic acid bacteria gene engineering strain

By blocking the expression of specific enzymes and proteins in lactic acid bacteria through genetic engineering, a genetically engineered strain of lactic acid bacteria was constructed, which solved the problem of low malic acid content during lactic acid bacteria fermentation and realized the efficient application of lactic acid bacteria in the production of food-grade organic acids.

CN121653031APending Publication Date: 2026-03-13INNER MONGOLIA AGRICULTURAL UNIVERSITY
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Patent Information

Application Number
CN202511906376.3
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-12-17
Publication Date
2026-03-13

AI Technical Summary

Technical Problem

In existing technologies, lactic acid bacteria have low malic acid content during fermentation, which is difficult to meet the requirements of food-grade organic acid biomanufacturing. Furthermore, lactic acid bacteria have insufficient food safety and acid resistance, which limits their application in food systems.

Method used

By using genetic engineering techniques, the expression or activity of lactate dehydrogenases LCAZH_0554, LCAZH_2512 and cluster proteins LCAZH_2364, LCAZH_2365, LCAZH_2366, and LCAZH_2367 in *Lacticaseibacillus casei* Zhang were blocked to construct a genetically engineered strain of lactic acid bacteria. Gene knockout of this strain was then performed using CRISPR/Cas9 D10A protein and sgRNA.

Benefits of technology

It significantly increased the yield of malic acid produced by lactic acid bacteria fermentation, provided new ideas for the engineering modification of lactic acid bacteria and the production of malic acid, and enhanced the biomanufacturing capacity of food-grade organic acids.

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Abstract

The invention provides a lactic acid bacteria genetic engineering strain for producing malic acid as well as a construction method and application thereof, and belongs to the technical field of genetic engineering and microbial fermentation. The lactic acid bacterium gene engineering strain is a lactic acid bacterium gene engineering strain with two lactic dehydrogenase genes and one cluster protein gene being knocked out; the two lactic dehydrogenases are respectively a lactic dehydrogenase LCAZH0554 and a lactic dehydrogenase LCAZH2512, and the clusterin is a clusterin containing genes of LCAZH2364, LCAZH2365, LCAZH2366 and LCAZH2367. The invention further discloses a preparation method of the clusterin. Three target knockout plasmids are sequentially transferred on the basis of an original lactic acid bacterium strain for gene knockout, the lactic acid bacterium gene engineering strain is finally obtained, and the malic acid yield is obviously increased compared with that of the original strain when the lactic acid bacterium gene engineering strain is adopted for fermentation.
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