How to Produce Q10 Enzyme?

Coenzyme Q10, an important hydrogen transporter in the respiratory chain of living cells, is widely found in animals, plants and microorganisms. Coenzyme Q10 was first isolated from the mitochondria of bovine hearts by Frederick crane of Wisconsin, USA, in 1957; in the same year, Morton of the UK obtained the same compound from the livers of vitamin A-deficient rats and named it coenzyme Q10; and in 1958 karl Folkers of Merck Inc. determined its structure and chemically synthesized it for the first time.

In 1958, Karl Folkers of Merck Inc. determined its structure and chemically synthesized Coenzyme Q10 for the first time. In 1977, the production of Coenzyme Q10 by microbial fermentation was industrialized in Japan. In 1978, Peter Mitchell was awarded the Nobel Prize for his use of chemical osmosis theory to explain the important proton transfer role of Coenzyme Q10 in energy conversion systems [1].

Currently, the main production methods of coenzyme Q10 include plant and animal tissue extraction, plant and animal tissue culture, microbial fermentation and chemical synthesis. Compared with other methods, microbial fermentation is characterized by high product activity, low raw material cost, easy control and large-scale production [2].

With the extensive research on the medical value and clinical application of coenzyme Q10, it has been widely used in the treatment of various diseases such as heart disease, diabetes, cancer, acute and chronic hepatitis, and Parkinson's disease. In addition, it has the ability to block the peroxidation of proteins. As a scavenger of oxygen radicals, Coenzyme Q10 is also widely used in health and beauty care [3-4].

1 Coenzyme Q10 Biosynthetic Pathway

1 . 1 Structure and Precursors

Coenzyme Q10 is a quinone compound. Its structure is shown in Figure 1. From its structure, it can be seen that it is based on 2,3-dimethoxy 5-methylbenzoquinone as the core, with a poly(2-methylbutene(2)-based) side chain attached [5].

The precursor of the quinone nucleus, p-hydroxybenzoic acid, is synthesized in bacteria and higher eukaryotes using branching acid and tyrosine as precursors, respectively, but in yeast, both substances can be used as precursors for the synthesis of p-hydroxybenzoic acid. In yeast, both substances can be used as precursors for the synthesis of p-hydroxybenzoic acid. The synthesis of poly(2-methylbutene)-butylene (2)-based side chains is based on two isomeric precursors, dimethylpropyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) [6].

Fig 1 The structure of coenzyme Q10

1 . 2 Aromatic Ring Synthesis Pathway

1 . 2 . 1 Biosynthesis of p-Hydroxybenzoic acid (PHB)

The synthesis of PHB, an important precursor in the aromatic ring synthesis pathway of coenzyme Q10, has been of great interest. Meganathan has investigated the synthesis of PHB from E . coli and S. cerevisa mutants. Meganathan studied the synthesis pathway of coenzyme Q10 in two mutant strains of E. coli and S. cerevisa, ubi and coq, and found that their in vivo pathways for aromatic ring synthesis are slightly different. E . E. coli cleaves the mangiferolic acid pathway (a pathway for the metabolism of sugars to aromatic amino acids, which is widely found in plants and microorganisms) to produce PHB by using a branching acid cleavage enzyme encoded by the ubic gene. S. cerevisa, on the other hand, can cleave branching acid to produce PHB by using a branching acid cleavage enzyme encoded by the S. cerevisa mutant. S. cerevisa, in addition to cleavage of branching acids, can also obtain PHB by a series of deamination, acylation, and deacylation of tyrosine [7].

1 . 2 . 2 Aromatic Ring Modifications  

Meganathan's study also found that the synthetic pathway starting with PHB involves a total of 7 enzymes in an 8-step reaction (see Figure 2). The PHB-poly-2-methylbutene(2)-yltransferase encoded by ubi A/ coq 2 transfers the poly-2-methylbutene(2)-yl side chain to the aromatic ring, and the enzyme has strong substrate specificity. The subsequent metabolic pathway varies from organism to organism.

In S . cerevisa, hydroxylation, methylation and then decarboxylation take place, whereas in E. coli, decarboxylation, hydroxylation and methylation take place first, followed by methylation. In S. cerevisa, hydroxylation, methylation and then decarboxylation take place, whereas in E. coli, decarboxylation, hydroxylation and then methylation take place. In the synthetic pathway, O-transmethylases are encoded by ubi G/coq 3, C-transmethylases by ubi E/coq 5, decarboxylases by ubiD/ubiX, and monooxygenases by ubiB and ubi E/coq 5, respectively. In some reports of plant and animal genes used to synthesize coenzyme Q10, all of these genes are found in plant and animal genes as homologs. However, the ubi F/coq 7 gene, which also encodes a monooxygenase, has not yet been found in plant or animal genes [8].

Figure 2 E . coli and S . cereuisiae in vivo coenzyme Q10 aromatic ring synthesis pathways [6]

1 . 3 Biosynthetic Pathways for Poly(2-Methylbutene(2)-Based Side Chains

1 . 3 . 1Side-Chain Precursors

3.1 Side-chain precursor synthesis In the synthesis of poly(2-methylbutene(2)-based) side chains, two isomeric precursors, DMAPP and IPP, are required, which are obtained via the mevalonate pathway (MVA) in yeast and the 2-C- methyl- D-erythritol 4-phosphate pathway (MEP) in bacteria [9]. methyl- D-erythritol 4-phosphate (MEP) in bacteria [9].

1 . 3 . 1 . 1 MVA Pathway  

The synthesis of IPP and DMAPP in fungi and yeast is shown in Fig. 3. The starting precursor in the synthesis pathway is acetyl coenzyme A, which first interchanges with its own ethyl group and the carbomethyl group of another acetyl coenzyme A to form bis(acetyl coenzyme A), which in turn combines with the other molecule to form 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA). HMG-CoA undergoes a two-step reduction reaction to form mevalonate. Mevalonate is converted to mevalonate pyrophosphate by a two-step phosphorylation process. After dehydration and decarboxylation of mevalonate pyrophosphate, IPP is formed, and IPP and DMAPP are interchanged by IPP isomerase [10].

1 . 3 . 1 . 2 MEP Pathway  

The starting precursors for the synthesis of IPP and DMAPP in bacteria are derived from two intermediate products of glycolysis: propionic acid and 3-phospho-D-glyceraldehyde, the synthesis pathway of which is shown in Fig. 4. First, the product of the decarboxylation of propionic acid combines with D-phospho-D-glyceraldehyde to form 5-phospho1-deoxyxylated xyloglucans (DXP) via the action of isp D-gene-coded D-xyloglucosan synthesis enzyme. DXP is further reduced to MEP, which combines with 1 molecule of CTP to form 2-methyl 4-cytidine diphosphate-D-erythritol via the action of 2-methyl 4-cytidine diphosphate-D-erythritol synthetase coded for by the isp D gene. The substance is further phosphorylated to produce 2-methyl-4-cytidine diphosphate-D-erythritol.

In the presence of 2-methyl-D-erythritol-2,4-cyclic pyrophosphate synthase encoded by isp F, 2-methyl-2-phosphate-4-cytidine diphosphate-D-erythritol molecules are degraded from one molecule of CMP to 2-methyl-D-erythritol-2,4-cyclic pyrophosphate. The enzymes and genes involved in the metabolism of cyclic pyrophosphate to IPP and DMAPP need to be further investigated. IPP and DMAPP are mutated by the action of idi genes encoding IPP isoenzymes [11].

Figure 3 Fungal and yeast endosomal side chain precursor synthesis pathways [10]

1 . 3 . 2 Side-chain Synthesis

In the side-chain biosynthesis pathway (see Figure 5), the primer DMAPP is first synthesized with IPP by GPP synthase to synthesize C10 glucocorticoid pyrophosphate (GPP). GPP and one molecule of IPP are catalyzed by FPP synthase to synthesize a C15 farnesyl pyrophosphate (FPP). FPP combines with IPP to form a C20 geranylgeranyl pyrophosphate (GGPP) via GGPP synthetase. After that, a series of side chains of coenzyme Q are synthesized by a series of isopentenyl pyrophosphate synthetases [12].

Side chain lengths of the coenzyme Q series are usually greater than C25, while those less than C25 are rarely the side chains of the natural coenzyme Q series. kainous found that FPP synthetase and GGPP synthetase have two aspartic acid-rich regions, and that the five-position aspartic acid in the first region plays an important role in the length of the side chains, and that precursors of different origins have certain effects on the lengths of the side chains. The precursors from different sources have a certain effect on the length of the side chains. E . E. coli can synthesize the precursors obtained in the MEP pathway into a series of side chains from Q7 to Q10 through a series of isopentenyl pyrophosphate synthases encoded by its isp A and isp B genes. S . cereui-sa can synthesize the precursors in the MVA pathway into a series of side chains from Q5 to Q10 [13].

Figure 5 E . coli and S . cerevisiae E . coli and S . cerevisiae in vivo coenzyme Q10 side chain synthesis pathway [7] Fig 5 Pathway f0r the bi0synthesis 0f p0lyprenyl diph0sphate in E . coli and S . coli and S . cerevisiae S. cerevisiae

2 Optimization of Coenzyme Q10 Fermentation Process

2 . 1 Production Strains

Currently, the number of reported CoQ10 strains is mainly concentrated in Pseudomonas, Saccharomyces, Photosynthesizing Bacteria and Paracoccidioides, etc. The distribution of CoQ10 in microorganisms is heterogeneous. The distribution of coenzyme Q10 in microorganisms is heterogeneous, with low production in cells that do not require oxygen respiration, such as Gram-positive bacteria, and relatively high production in Gram-negative cells. With the further clarification of the pathway of coenzyme Q10 synthesis in organisms and the application of enzyme engineering and genetic engineering, the synthesis of coenzyme Q10 by genetically modified bacterial strains has also been reported in recent years. Y0shida [14] obtained the mutant strain AU-55 by mutagenesis of Rhodococcus globulus and rationalization screening, and the highest yield of 180 mg/L was achieved by shake flask fermentation culture.

2 . 2 Optimization of Medium Composition

2 . 2 . 1 Carbon Sources  

A poly-2-methylbutene(2)-based side chain is required for the synthesis of coenzyme Q10.Tanaka et al. [13] used shake flasks to cultivate the strain C . T .  Tanaka et al. [13] used shake flask culture of strain C. T. PK-233, with C10~C13 hydrocarbons as carbon sources, and incubated the strain at 30 ℃ for 45 h. The coenzyme Q10 in the culture solution was 4.66 mg/L. Coenzyme Q10 was obtained from the culture solution at a concentration of 4.66 mg/L.

2 .2 . 2 Nitrogen Sources  

Wu Zufang et al. [15] used different nitrogen sources, such as fermentation ointment, peptone, corn syrup, ammonium chloride, ammonium sulfate and ammonium nitrate, to ferment Rhizobium radiobacter WsH2601 in shake flasks, and the results showed that organic nitrogen was more effective than inorganic nitrogen. When 16 g/L peptone was used as the nitrogen source, the synthesis of coenzyme Q10 was maximized.

2 .2 . 3 Metal Ions  

A study by ACI (Japan) showed that metal ions, especially Mg2+, Fe2+ and Mn2+, have a beneficial effect on the fermentation of R. sphaeroides to produce Coenzyme Q10. sphaeroides fermentation to produce Coenzyme Q10. The addition of 12.2 mm0l/L Mgso2 + and Mn2 + to the culture medium promoted the fermentation of Coenzyme Q10. 2 mm0l/L Mgso4、1 . 12.2 mm0l/L Mgso4, 1.8 mm0l/L Feso4, 0.9 mm0l/L Mnso4 The addition of 12.2 mm0l/L Mgso4, 1.8 mm0l/L Feso4, and 0.9 mm0l/L Mnso4 in the culture medium increased the Coenzyme Q10 production from 2.0 mg/g to 8.0 mg/g. 0 mg/g to 8 . 9 ~ 9 . 6 mg/g [2].

2 . 2 . 4 Precursors  

Under certain conditions, the precursor can control the anabolic flow of the bacterium, thus increasing the yield of coenzyme Q10. shimizU et al. reported that propionic acid in the tricarboxylic acid cycle is the precursor of the side-chain 2-methylbutene(2)-based polymer in coenzyme Q10, and that 0.5 % of propionic acid was added to the medium, which resulted in a 52 mg/L yield of coenzyme Q10. Coenzyme Q10 production was 52 mg/L with the addition of 0.5 % propionic acid to the culture medium. Kawada et al. reported that the addition of isopentyl ethanol and tallow alcohol as precursors for side chain synthesis to the culture medium of P. schuy lkilliensis increased the Coenzyme Q10 content of the bacterium by about 20-60 %. Tanaka et al. used shake flasks to culture C. schuy lkilliensis. T . Tanaka et al. cultured strain C. T. PK-233 in shake flasks and found that the addition of p-hydroxybenzoic acid to the culture medium promoted the biosynthesis of coenzyme Q10 [16].

2 . 2 . 5 Adding Effectors  

Zhang Yanjing et al. [17] investigated the effects of the addition of soybean oil, soybean flour, carrot juice, tomato juice, tobacco leaf, β-carotene and orange peel juice on the production of coenzyme Q10 by yeast fermentation, and the results showed that the addition of the above mentioned effectors could increase the content of coenzyme Q10 in yeast.

2 . 3 Optimization of Culture Conditions

2 . 3 . 1Illumination

sasaki et al. cultured Pseudomonas aeruginosa in the absence of light, and the amount of coenzyme Q10 was 1.22 mg/g, while in the presence of light, the amount of coenzyme Q10 was 1.5 mg/g. In the absence of light, Pseudomonas aeruginosa was cultured with 1.22 mg/g of coenzyme Q10, whereas in the presence of light, the level of coenzyme Q10 was 1.98 mg/g [16]. Car and EXcell reported that the content of coenzyme Q10 in PsB was higher under anaerobic conditions in the light, but the yield dropped sharply once the culture was switched to the dark [2].

2 . 3 . 2 Dissolved Oxygen

The effect of dissolved oxygen on the fermentation of coenzyme Q10 varies greatly depending on the strain of the bacterium. Y0shida et al. [14] found that lowering the oxygen pressure increased the yield of coenzyme Q10 in the fermentation of Rhodococcus globulus KY-4113, and electron microscopic photographs of the cells showed that the inner membrane of the cells showed a multilayered structure in the presence of low oxygen. Wang Genhua et al. [18] found that the amount of dissolved oxygen in the shake flasks not only affected the growth of cells, but also coenzyme Q10 production. The higher the dissolved oxygen, the more developed the respiratory system of the cells and the higher the content of coenzyme Q10.

2 . 3 . 3 Inoculation Volume  

Increasing the inoculum rate can shorten the fermentation period and accelerate the growth rate of the bacteria, thus shortening the fermentation cycle. Wu Zufang et al. [15] showed that the highest yield of coenzyme Q10 was obtained at an inoculum level of 4% in a shake flask experiment with Rhizobium radiodurans (WSH2601) [15].

2 . 3 . 4 pH  

The initial pH value affects the rate of substrate utilization by the fungus and the structural state of the cell, which in turn affects the growth rate of the fungus and the metabolic products. Wang Genhua et al. [18] showed that Rhizobium leguminosarum produced more Coenzyme Q10 under acidic conditions than alkaline conditions in a shake flask experiment. The highest amount of intracellular coenzyme Q10 was found at pH 5.

3 Looking Forward

The development and utilization of coenzyme Q10 has become a hot research topic in recent years due to its wide range of applications and high market demand. The production of coenzyme Q10 by fermentation has many advantages, such as high product activity and unlimited raw material sources. However, the low level of fermentation and the complexity of the metabolites have resulted in high extraction costs. In order to improve the industrialization of the fermentation method, high-yielding strains can be selected by regulating the metabolic pathway of coenzyme Q10, and the fermentation level can be further improved through the improvement of the fermentation process. In addition, the study of a set of efficient separation and extraction routes is one of the effective ways to improve the industrialization of the fermentation process.

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