Microorganisms
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2025
·Vol. 13
·期 3
·536
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IF 4.2
Lina Yang,Can Tang,Yan Cui,Jianhua Zhang
Menaquinone-7 (MK-7) is recognized for its important biological activity, andBacillus subtilisis the preferred strain for its fermentative production. However, the limited phenotypic diversity among high-yielding strains complicates the development of rapid screening methods. To address this, we utilized the effect of MK-7 on transmembrane potential to develop a high-throughput screening (HTS) strategy for efficiently identifying strains with improved MK-7 production. Among various membrane potential fluorescent dyes tested, Rhodamine 123 was selected for quantifying intracellular MK-7 levels due to its effective staining and minimal impact on cell growth. By optimizing pretreatment protocols and staining conditions, we established an HTS protocol that combines fluorescence-activated cell sorting with HPLC to identify strains with increased MK-7 production. A linear correlation was observed between mean MK-7 content and average fluorescence intensity (R2= 0.9646). This approach was applied to mutant libraries generated through atmospheric room temperature plasma mutagenesis. After three cycles of mutagenesis and screening, the mutant AR03-27 was identified, showing an 85.65% increase in MK-7 yield compared to the original SJTU2 strain. Resequencing analysis revealed that the top three mutants contained mutations in genes related to membrane transport, suggesting their potential role in enhancing MK-7 yield.
研究领域:
微生物学
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生物化学
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合成生物学
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DOI
PubMed
高影响因子
Systems Microbiology and Biomanufacturing
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2023
·
IF 0
Li Chang-Long,Li Meng,Zhang Wei-Guo,Xu Jian-Zhong
Menaquinone-7 (MK-7) has an important role in preventing diseases such as cardiovascular disease and osteoporosis. In this study, a combination strategy of strain improvement and medium optimization is investigated to increase MK-7 production in Bacillus amyloliquefaciens . Conventional breeding method was first used to modify the biosynthetic pathway to construct a MK-7 high-producing strain by atmospheric and room temperature plasma mutagenesis and protoplast fusion. The resulted strain Ba-4 with resistance to sulfaguanidine, 1-hydroxy-2-naphthoic acid, menadione, 2-deoxy- d -glucose and rifampicin as well as sensitive to β-fluoropyruvate produced 73.57 ± 1.61 mg/L of MK-7, which was 1.36 times more than that of the parent strain H.β.D.R.-5 (i.e., 31.12 ± 1.40 mg/L). Subsequently, single-factor optimization and response surface methodology (RSM) were used to optimize the medium components for increasing MK-7 production by strain Ba-4. Strain Ba-4 produced 90.43 ± 1.32 mg/L of MK-7 under the single-factor optimized medium. Moreover, the results of response surface methodology indicated that glycerol, soy peptone and Tween-80 had significant effects on MK-7 production, and the highest MK-7 production (i.e., 95.03 ± 1.01 mg/L) was obtained under the optimized medium, which was 0.29 times higher than that of the initial medium. These results confirmed that the conventional breeding methods and fermenter control system are effective strategies in improving MK-7 production by B. amyloliquefaciens .
研究领域:
微生物学
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生物技术
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发酵工程
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DOI
BIOTECHNOLOGY LETTERS
·
2026
·Vol. 48
·期 5
·121
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IF 2.5
Chen Jiahui,Xu Han,Liu Tingting,Chen Ming,Yu Fang,Zhang Yue
Menaquinone-7 (MK-7), a highly preferred form of vitamin K2, has garnered extensive attention due to its long half-life in the human body and numerous nutritional and pharmacological benefits. To establish a cost-effective microbial platform for MK-7 production, we engineered the eukaryotic model organism Saccharomyces cerevisiae . The complete biosynthesis pathway was modularized into three parts: the endogenous mevalonate (MVA) pathway, the endogenous shikimate (SA) pathway, and a heterologous menaquinone (MK) module. Eleven key genes from the MK module were introduced into the yeast chassis using selection markers for His , Leu , and Met , creating the engineered strain YK-1. We then optimized the fermentation medium and conditions, and the MK-7 titer reached 0.413 mg/L. This represents a 31.1% increase over the non-optimized yield, demonstrating the successful de novo synthesis of MK-7 from simple carbon sources in a eukaryotic cell factory. This work not only expands the repertoire of chassis cells for MK-7 production but also provides a valuable framework for engineering biosynthesis pathways for other isoprenoids.
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高影响因子
Bioresources and Bioprocessing
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2025
·Vol. 12
·期 1
·1-18
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IF 5.1
Zhang Rui,Wang Han,Wang Li,Zheng Zhiming
Menaquinone-7, a form of vitamin K 2 , plays a critical role in the treatment of hemorrhagic diseases caused by vitamin K deficiency and in the prevention of bone fractures. Microbial fermentation has emerged as a promising method for MK-7 production due to its high product optical purity and significant physiological activity. However, the current production efficiency of MK-7 remains insufficient to meet industrial demands. In this study, we employed a combination of single-factor experiments, Plackett-Burman design, steepest ascent experiments, and Box-Behnken design to optimize the fermentation medium for MK-7 production by Bacillus subtilis BS - Δ ackA . Through regression analysis and consideration of practical production constraints, the optimal fermentation medium parameters were determined as follows: 20 g/L sucrose, 20.7 g/L glycerol, 47.3 g/L soy peptone, 4 g/L yeast extract, and 1.9 g/L KH 2 PO 4 , 0.1 g/L MgSO 4 ·7 H 2 O. Under these optimized conditions, the MK-7 yield reached 154.6 ± 1.32 mg/L. The experimental results demonstrated excellent stability and reliability, aligning closely with the model predictions. This study significantly enhances MK-7 production at the shake-flask fermentation level, providing valuable insights for large-scale industrial applications.
研究领域:
微生物发酵
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生物技术
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生物工程
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应用微生物学
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营养科学
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DOI
PubMed
高影响因子
BRITISH JOURNAL OF NUTRITION
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2023
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IF 3.6
Fan Du,Min Yan,Lili Duan,Guolong Xie,Xiuhua Yao,Wenjing Hu,Yu Liu,Min Meng,Jiepeng Chen,Di Shao
Menaquinone-7 (MK-7), a multipotent vitamin K2, possesses a wide range of biological activities, a precise curative effect and excellent safety. A simple and rapid LC-APCI-MS/MS method for the determination of MK-7 in human plasma with single liquid–liquid extraction (LLE) extraction and 4·5-min analysis time has been developed and validated. Four per cent bovine serum albumin (BSA) was used as surrogate matrix for standard curves and endogenous baseline subtraction. This method was reproducible and reliable and was used to analyse of MK-7 in human plasma. The endogenous circadian rhythm and bioavailability of MK-7 were investigated in two randomised single-dose, open, one-way clinical trials (Study I and Study II). A total of five healthy male subjects were enrolled in Study I and 12 healthy male subjects in Study II. Single-dose (1 mg) of MK-7 was given to each subject under fasting condition, and all eligible subjects were given a restricting VK2 diet for 4 d prior to drug administration and during the trial. The experiment results of Study I demonstrated that endogenous MK-7 has no circadian rhythm in individuals. Both studies showed MK-7 are absorbed with peak plasma concentrations at about 6 h after intake and has a very long half-life time.
研究领域:
药理学
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营养学
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临床研究
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分析化学
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DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
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2024
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IF 3.7
Xilin Huang,Xuli Gao,Junbao Huang,Yani Luo,Wei Tao,Mingyu Guo,Yongyuan Liu,Jing Wu,Yu Chen,Yan Liu
A recombinant strain (BS168-Δ bdhA ) that increased co-production of MK-7 and NK was constructed by knocking out the 2,3-butanediol dehydrogenase gene ( bdhA ) of Bacillus subtilis 168 to reduce the carbon spillover. The results showed that the 2,3- butanediol content in the recombinant strain was reduced by 64% to 2.76 g/L. The recombinant strain had an MK-7 production of 30.6 mg/L and an NK activity of 6.5 FU/mL. RNA-Seq analysis showed that: bdhA knockdown blocked the carbon flux of 2,3-butanediol and promoted glycerol uptake, and the carbon flux flowed more to the MK-7 and NK synthesis pathways. The negative regulator of NK, codY , was down-regulated by 2.19-fold. secA , which is responsible for the energy supply of transmembrane proteins, was down-regulated by 0.37-fold, the Tat secretion pathway was up-regulated by 2.8-fold and 0.5-fold for tatAD and tatC , respectively. This study revealed the mechanism of co-production of MK-7 and NK in recombinant strains for the first time and revealed potential molecular targets to promote the co-production of MK-7 and NK, which will provide a new strategy for metabolic engineering to increase the production of MK-7 and NK.
研究领域:
微生物学
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分子生物学
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代谢工程
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DOI
高影响因子
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