As a natural biological macromolecule,
γ-polyglutamic acid
(γ-PGA) plays a significant role in medicine, food, and cosmetic
industries owing to its unique properties of biocompatibility, biodegradability,
water solubility, and viscosity. Although many strategies have been
adopted to increase the yield of γ-PGA in Bacillus
subtilis, the effectiveness of these common approaches
is not high because the strong viscosity affects cell growth. However,
dynamic regulation based on quorum sensing (QS) has been extensively
applied as a fundamental tool for fine-tuning gene expression in reaction
to changes in cell density without adding expensive inducers. A modular
PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study,
first, we analyzed the DegU-based gene expression regulation system
in B. subtilis 168. We constructed
a promoter library of different abilities, selected suitable promoters
from the library, and performed mutation screening on the selected
promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU
QS system to dynamically control the synthesis of γ-PGA in BS168.
Cell growth and efficient synthesis of the target product can be dynamically
balanced by the QS system. Our dynamic adjustment approach increased
the yield of γ-PGA to 6.53-fold of that by static regulation
in a 3 L bioreactor, which verified the effectiveness of this strategy.
In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated
with biocatalytic functions to achieve dynamic metabolic pathway control
in BS168, which can be stretched to a large number of microorganisms
to fine-tune gene expression and enhance the production of metabolites.
Vitamin K2, as a menadione compound, has been a hot topic in international research in recent years. Previous work mainly focused on the manipulation of several genes in metabolic pathway, but did not pay attention to the overall regulation, which always failed to make the fermentation yield of the strain meet people's requirements. On this basis, we shifted the research focus from the knockout and overexpression of several genes in a single metabolic pathway to the overall regulation of bacteria. The effect of transcription regulation system on MK-7 production in Bacillus subtilis was studied by mining transcription regulation factors. We constructed a set of PhrG-RapG-DegU regulatory system by using quorum sensing signal molecules PhrG pentapeptide, RapG protein and regulatory factor DegU. By fine-tuning the expression of related genes, the content of MK-7 in the finally constructed BS06 in a 250 mL conical flask reached 102.47 mg/L, and the cell density was 28.677, which was basically the same as that of the original bacteria. Our experiment realized the efficient synthesis of MK-7 in Bacillus subtilis and ensured the stable growth of bacteria, which proved the effectiveness of this control strategy.
Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. It plays an important role in the treatment of coagulation, osteoporosis, promotion of liver function recovery and prevention of cardiovascular diseases. In this study, in order to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of metabolic synthesis of MK-7 by mutant strain Bacillus subitilis 168 KO-SinR (BS168 KO-SinR) was analyzed by adding surfactants. The results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm, as indicated by scanning electron microscopy and flow cytometry. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. qRT-PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the results of electron microscope showed that the addition of surfactant changed the permeability of cell membrane. The research results of this paper provide a certain reference value for the industrial development of MK-7 prepared by fermentation.
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