Recombinant Bacillus subtilis for Menaquinone 7 Yield
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Solution Overview
Problem
Current biological fermentation methods for producing menaquinone 7 (MK-7) are limited by complex processes, long fermentation periods, low product purity, and insufficient metabolic flux in Bacillus subtilis, making it challenging to increase MK-7 yield effectively.
Innovation Solution
Recombinant Bacillus subtilis strains are engineered by replacing natural promoters with P43 and Phbs promoters, introducing exogenous genes, and knocking out specific genes to enhance the metabolic flux and biosynthetic pathway for MK-7 production, resulting in strains BS1-BS14 that significantly increase MK-7 yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If natural promoters are used in Bacillus subtilis for MK-7 synthesis, then the metabolic flux is insufficient, but replacing with strong promoters (P43, Phbs) increases the complexity of genetic engineering
Solution Approach 1:
The patent applies parameter changes by replacing natural promoters with strong constitutive promoters (P43, Phbs) to dramatically increase the expression levels of key enzymes in the MK-7 biosynthetic pathway. This promoter substitution strategy transforms the metabolic flux parameters, enabling high-yield MK-7 production (up to 3.53 times higher than wild-type) while managing genetic engineering complexity through systematic promoter replacement.
2Productivity
If exogenous genes (entC, ppsA) are introduced to enhance metabolic flux, then MK-7 synthesis increases, but the complexity of the biosynthetic pathway regulation increases
Solution Approach 1:
The patent segments the MK-7 biosynthetic pathway into distinct regulatory modules, each controlled by specific promoters. Exogenous genes (entC, ppsA) are introduced as separate functional units with independent promoter control (P43, Phbs), allowing modular optimization of metabolic flux without overwhelming system-wide complexity. This segmentation enables precise control over pathway regulation.
Solution Approach 2:
The strong constitutive promoters (P43, Phbs) used in this patent serve multiple functions: they drive high-level expression of different genes (menF, menB, menE, tkt, entC, ppsA) throughout the pathway, and they provide consistent, high-level expression across various growth conditions. This universality simplifies the overall regulatory complexity by using a standardized promoter system for multiple pathway components.
3Productivity
If ptsG gene is knocked out to redirect metabolic flux, then MK-7 production increases, but the strain stability and adaptability may be affected
Solution Approach 1:
The patent converts the potential harm of ptsG knockout (reduced adaptability) into a benefit by redirecting all glucose metabolic flux toward MK-7 production. The knockout eliminates competing pathways, forcing carbon flow through the desired biosynthetic route. This is compensated by the robustness of the strong promoters and the essential nature of the introduced genes, maintaining strain stability while maximizing productivity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered recombinant strains BS6-BS14 achieve MK-7 yields 1.59 to 3.53 times higher than the wild-type strain, providing a method to enhance MK-7 production and offering a theoretical basis for constructing high-yielding strains.
Implementation Method 1
the natural promoters of a menaquinone-specific isochorismate synthase gene menF and a dihydroxynaphthoic acid synthetase gene menB on a chromosome are replaced with P43 promoters; the natural promoters of an O-succinylbenzoic acid-CoA ligase gene menE and a transketolase gene tkt on the chromosome are replaced with Phbs promoters
Implementation Method 2
performing fermentation production using the recombinant strain
Data Source
AI summary
The present disclosure provides a recombinant Bacillus subtilis for increasing the yield of menaquinone 7 (MK-7) and application thereof, and belongs to the field of genetic engineering. In the present disclosure, 14 recombinant strains BS1-BS14 are constructed through the modification of genes related to the biosynthetic pathway of MK-7 on a chromosome of Bacillus subtilis, wherein BS6-BS14 significantly increase the yield of the MK-7, reaching up to 33.5 mg/L, which is 3.53 times the yield of the original strain of wild-type Bacillus subtilis 168. The present disclosure further provides a method for modifying the MK-7 biosynthetic pathway in microorganisms to increase the yield of the MK-7, providing a theoretical basis for constructing a high-yielding strain of the MK-7.