Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica

Liu, Yan; Ding, Xiu-min; Xue, Zhenglian; Hu, Liu-xiu; Cheng, Qian; Chen, Minghong; Su, Yun; Zhu, Bin; Xu, Ping

doi:10.1016/j.procbio.2017.05.002

Cited by 11 publications

(6 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…cremoris. Firstly, enhanced expression of menA result in around 2-fold higher MK production in E. coli, B. amyloliquefaciens, E. meningoseptica, and B.subtilis (Kong and Lee, 2011;Liu et al, 2017Liu et al, , 2018Xu et al, 2017;Ma et al, 2019;Yang et al, 2019) and we found that this also applies for L. lactis. More MenA activity will pull out products of both the menaquinone and MEV+PP pathways possibly leading to an increased flux through both pathways.…”

Section: Discussionmentioning

confidence: 62%

“…They include optimization of the fermentation process, increasing menaquinone secretion, improving bioreactor design, directed mutagenesis of strains or genetic engineering of strains (Ren et al, 2019). To enhance strain performance genomic changes has been made in order to maximize substrate pools, limit the production of by-products, overexpress key genes, express novel pathways, or combinations of the aforementioned strategies (Kong and Lee, 2011;Liu et al, 2017Liu et al, , 2018Xu et al, 2017;Ma et al, 2019;Yang et al, 2019). Most of these studies have been performed using B. subtilis, B. amyloliquefaciens, E. meningoseptica, or E. coli and seek to produce menaquinone on an industrial scale for use as a nutritional supplement or food additive.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Bøe

Holo

2020

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Cheese produced with Lactococcus lactis is the main source of vitamin K2 in the Western diet. Subclinical vitamin K2 deficiency is common, calling for foods with enhanced vitamin K2 content. In this study we describe analyses of vitamin K2 (menaquinone) production in the lactic acid bacterium L. lactis ssp. cremoris strain MG1363. By cloning and expression from strong promoters we have identified genes and bottlenecks in the biosynthetic pathways leading to the long-chained menaquinones, MK-8 and MK-9. Key genes of the biosynthetic menaquinone pathway were overexpressed, singly or combined, to examine how vitamin K2 production can be enhanced. We observed that the production of the long menaquinone polyprenyl side chain, rather than production of the napthoate ring (1,4-dihydroxy-2-naphtoic acid), limits total menaquinone synthesis. Overexpression of genes causing increased ring formation (menF and menA) led to overproduction of short chained MK-3, while overexpression of other key genes (mvk and llmg_0196) resulted in enhanced full-length MK-9 production. Of two putatively annotated prenyl diphosphate synthases we pinpoint llmg_0196 (preA) to be important for menaquinone production in L. lactis. The genes mvk, preA, menF, and menA were found to be important contributors to menaquinone levels as single overexpression of these genes double and more than triple the total menaquinone content in culture. Combined overexpression of mvk, preA, and menA increased menaquinone levels to a higher level than obtained individually. When the overproducing strains were applied for milk fermentations vitamin K2 content was effectively increased 3-fold compared to the wild type. The results provide a foundation for development of strains to ferment foods with increased functional value i.e., higher vitamin K2 content.

show abstract

Section: Discussionmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Bøe

Holo

2020

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…The method used for site-directed mutagenesis was similar to that used by [ 32 ]. The PCR reaction was conducted using the PrimeSTAR HS DNA polymerase (Takara, Japan) and the pHY-P43/SinR plasmid as the template DNA.…”

Section: Methodsmentioning

confidence: 99%

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis

Zhao

et al. 2021

Microb Cell Fact

Self Cite

View full text Add to dashboard Cite

Background Menaquinone (MK-7) is a highly valuable vitamin K2 produced by Bacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth. Results In this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants, sinR knock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquad maximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions of tapA, tasA and epsE were up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquad formed more wrinkly and smoother biofilms than BS168. The upregulated expressions of glpF, glpk, and glpD in this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especially sdhA, sdhB, sdhC and glpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway. Conclusions In this study, we report for the first time that SinRquad has significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.

show abstract

“…Therefore, acetyl-CoA may enter either the mevalonate pathway or polyketide pathway, which form the farnesyl tail precursor or ring precursor of AQ in A. camphorata S-29, respectively (Chou et al 2017 ). The CoQ2 and ubiA genes encode p -hydroxybenzoate polyprenyl transferase, which is required in the biosynthetic pathway of ubiquinone (Forsgren et al 2004 ; Liu et al 2017 ). P -hydroxybenzoate polyprenyl transferase, which is also referred to as the ‘ CoQ2 enzyme’, mediates the second step in the final reaction sequence of coenzyme Q biosynthesis, namely, the condensation of the polyisoprenoid side chain with 4- p -hydroxybenzoate (Forsgren et al 2004 ).…”

Section: Discussionmentioning

confidence: 99%

Comprehensive transcriptomic and proteomic analyses of antroquinonol biosynthetic genes and enzymes in Antrodia camphorata

et al. 2020

View full text Add to dashboard Cite

Antroquinonol (AQ) has several remarkable bioactivities in acute myeloid leukaemia and pancreatic cancer, but difficulties in the mass production of AQ hamper its applications. Currently, molecular biotechnology methods, such as gene overexpression, have been widely used to increase the production of metabolites. However, AQ biosynthetic genes and enzymes are poorly understood. In this study, an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify AQ synthesis-related genes and enzymes in Antrodia camphorata during coenzyme Q 0-induced fermentation (FM). The upregulated genes related to acetyl-CoA synthesis indicated that acetyl-CoA enters the mevalonate pathway to form the farnesyl tail precursor of AQ. The metE gene for an enzyme with methyl transfer activity provided sufficient methyl groups for AQ structure formation. The CoQ2 and ubiA genes encode p-hydroxybenzoate polyprenyl transferase, linking coenzyme Q 0 and the polyisoprene side chain to form coenzyme Q 3. NADH is transformed into NAD+ and releases two electrons, which may be beneficial for the conversion of coenzyme Q 3 to AQ. Understanding the biosynthetic genes and enzymes of AQ is important for improving its production by genetic means in the future.

show abstract

Site-directed mutagenesis of UbiA to promote menaquinone biosynthesis in Elizabethkingia meningoseptica

Cited by 11 publications

References 22 publications

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Engineering Lactococcus lactis for Increased Vitamin K2 Production

Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis

Comprehensive transcriptomic and proteomic analyses of antroquinonol biosynthetic genes and enzymes in Antrodia camphorata

Contact Info

Product

Resources

About