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.
Menaquinone (MK) was an attractive membrane-bound intracellular chemical. To enhance its production, we tried to find the relationship between its synthesis and the state of cell membrane in producing strain. Due to non-ionic surfactant-polyoxyethylene oleyl ether (POE) and plant oil-cedar wood oil (CWO) can typically increase extracellular secretion and intracellular synthesis of MK respectively, the effect of these two substances on cell morphology, physical properties of cell membrane was investigated. Finally, two engineering strains were constructed to verify whether the state of cell membrane can enhance MK synthesis. The result showed that the edge of cells was broken when POE added in the medium. Other physical properties such as total fatty acid content decreased by 40.7% and the ratio of saturated fatty acids to unsaturated fatty acids decreased from 1.58 ± 0.05 to 1.31 ± 0.04. Meanwhile, cell membrane leakage was enhanced from 7.14 to 64.31%. Different from POE group, cell membrane was intact in CWO group. Moreover, the ratio of saturated fatty acids to unsaturated fatty acids increased from 1.58 ± 0.05 to 1.78 ± 0.04 and the average lipid length decreased from 16.05 ± 0.08 to 15.99 ± 0.10. Two constructed strains, especially Escherichia coli DH5α FatB, exhibited strong MK secretion ability and the extracellular MK reached 10.71 ± 0.19 mg/L. An understanding of these functionary mechanisms could not only provide a new idea for the synthesis of MK, but also provide a reference to increase the yield of intracellular membrane-bound metabolites.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.