Advances in the Coenzyme Q10 Biosynthesis Pathway in Rhodobacter sphaeroides and the Enha

Kuo TANG　Zhiping ZHAO

Abstract Coenzyme Q10is widely used in food， cosmetics and pharmaceuticals， possessing a broad market. Rhodobacter sphaeroides is enriched in natural coenzyme Q10and is becoming an important microorganism for producing natural coenzyme Q10. The paper reviewed the biosynthesis pathways of coenzyme Q10in R. sphaeroides and the advances in enhancement of coenzyme Q10production in R. sphaeroides based on metabolic engineering.

Key words Rhodobacter sphaeroides; Coenzyme Q10; Biosynthesis; Metabolic engineering

Coenzyme Q10， also called as ubiquinone， is chemically known as 2，3dimethoxy5methyl6decaprenylbenzoquinone. It is a natural liposoluble polyenoic quinone compound with the functions of resisting lipid peroxidation， removing superoxides， etc.[1-2]， widely used in medicine， health care products and cosmetics[3]. At present， the production methods of coenzyme Q10mainly include animal and plant tissue extraction， chemical synthesis and microbial fermentation[4-5]. The production of coenzyme Q10by microbial fermentation has the advantages of relatively cheap and abundant raw materials， simple separation and purification and high biological activity[6-7]， which has become the most promising production method[8-9]. Rhodobacter sphaeroides is one of the most important microorganisms producing natural coenzyme Q10[4，10]. This paper focused on the coenzyme Q10biosynthesis pathway in R. sphaeroides and the advances in the improvement of coenzyme Q10production in R. sphaeroides based on metabolic engineering pathway.

Coenzyme Q10Biosynthesis Pathway of R. sphaeroides

In the 1980s， Olson and Rudney[11]revealed the biosynthetic route of coenzyme Q10， pointing out that phydroxybenzoic acid is an important precursor for the synthesis of coenzyme Q10， and found that polyisoprene is a precursor for the synthesis of carotenoids and coenzyme Q10. Reducing the synthesis of carotenoids may increase the biosynthesis of coenzyme Q10. In 1994， Grǜnler[12]further improved the coenzyme Q10biosynthesis pathway. Afterwards， Megathanthan[13]and Kawamukai[14]elaborated the biosynthetic pathway of coenzyme Q10. The biosynthesis of coenzyme Q10consists of three parts： methylerythritol 4phosphate （MEP） pathway （side chain synthesis）， shikimate pathway and ubiquinone modification.

MEP pathway

The biosynthesis of side chains is an important step in the synthesis of terpenoids in organisms. The side chain synthesis pathway in eukaryotes provides the precursors dimethylallyl pyrophosphate （DMAPP） and isopentenyl pyrophosphate （IPP） through mevalonate pathway （MVA）， while in prokaryotes， IPP and DMAPP are usually provided by the 2methylDerythritol 4phosphatepathway.

Most bacteria producing coenzyme Q10possess the nonmevalonate pathway， i.e.， the EMP pathway （Fig. 1）， which synthesizes IPP and DMAPP through eight consecutive enzymatic reactions[16]. ① DOXP is produced by the action of 1deoxydxylulose5phosphate synthase （DXS） using the glycolysis products pyruvic acid and glyceraldehyde3phosphate as substrates. ② DOXP generates MEP under the catalysis of 1deoxyDxylulose5phosphate reductoisomerase （DXR）. ③ MEP synthesizes 4diphosphocytidyl2CmethylDerythritol （CDPME） under the catalysis of MEP cytidylyltransferase （IspD）. ④ CDPME produces 4（cytidine5diphospho）2CmethylDerythritol （CDPMEP） under the catalysis of 4（cytidine5diphospho）2CmethylDerythritol kinase （IspE）. ⑤ CDPMEP produces DMAPP and IPP sequentially through 2CmethylDerythritol 2，4cyclodiphosphate synthase （IspF）， 4hydroxy3methylbut2enyl diphosphate synthase （IspG） and 4hydroxy3methylbut2enyl diphosphate reductase （IspH）. ⑥ Isopentenyl pyrophosphate isomerase （IDI） catalyzes the mutual conversion of IPP and DMAPP. ⑦ IPP and DMAPP form farnesol pyrophosphate under the catalysis of farnesol pyrophosphate synthase （CrtE） at a ratio of 1∶1. ⑧ IPP is sequentially added to farnesol pyrophosphate with the catalysis of CrtE enzyme and decaprenyl diphosphate synthase （DPS） until a certain length of polyisoprenyl diphosphate is formed. The length of polyisoprenyl diphosphate can determine the type of coenzyme Q; and in animal and plant cells and certain microbial cells， the chain can reach 50 carbon atoms， i.e.， decaprenyl diphosphate， corresponding to coenzyme Q10. The key enzymes involved in the side chain synthesis pathway are DXS， DXR， IDI， IspD and IspF[17-18]. In addition， DPS determines the length of the coenzyme Q10chain， so in some noncoenzyme Q10producing microorganisms， DPS is also a key gene for the synthesis of coenzyme Q10[19-20].

Shikimate pathway

The shikimate pathway is an important metabolic pathway for the synthesis of aromatic amino acids， folic acid， ubiquinone and chorismate in bacteria， fungi， alga and higher plants[21]. The ubiquinone synthesis pathway of coenzyme Q10means the shikimate pathway， which is a key pathway for the synthesis of aromatic amino acids by using chorismate. Since its metabolic intermediate is chorismate， this pathway also becomes the shikimate pathway. The shikimate pathway uses erythrose4phosphate and phosphoenolpyruvate as initial substrates， and then catalyzes the formation of shikimate by multiple enzymes such as AroF， AroB， AroD， AroE， AroL， AroA and AroC， respectively[22]; and the shikimate is further catalyzed by UbiC to synthesize phydroxybenzoic acid （pHBA） which is the precursor substance of coenzyme Q10mother nucleus. pHBA is the first direct precursor in the coenzyme Q10biosynthetic pathway for prenylation and ring modification. UbiC is a key enzyme that limits the rate of reaction across the shikimate pathway[23]. Studies have shown that under the premise that bacteria do not supplement exogenous aromatic compounds， the growth of bacteria will be inhibited after the pathway is blocked， indicating that this pathway plays a very important role in maintaining bacterial life activities[24-25].

Ubiquinone modification

In the ubiquinone modification pathway （also known as the ubiquinone pathway）， polyisoprenyl diphosphate produced in the side chain synthesis pathway and phydroxybenzoic acid produced in the shikimate pathway are condensed under the catalysis of membrane bound enzyme and phydroxybenzoate prenyltransferase （UbiA）， forming 3polypentenyl4hydroxybenzoic acid[26-27]; and coenzyme Q10is then produced through decarboxylation， trihydroxylation and trimethylation under the action of several quinone ringmodifying enzymes such as UbiD/X， UbiB， UbiG， UbiH， UbiE and UbiF[28]. All methyl groups in the reaction are derived from Sadenosylmethionine， and all oxygen is derived from molecular oxygen （O2）. In addition， studies have shown that UbiA， UbiE and UbiG are the three key enzymes for coenzyme Q10biosynthesis[29-31].

Kuo TANG et al. Advances in the Coenzyme Q10Biosynthesis Pathway in Rhodobacter sphaeroides and the Enhancement of Coenzyme Q10Production Based on Metabolic Engineering

Improvement of Coenzyme Q10Production in R. sphaeroides Based on Metabolic Engineering

At present， most of the coenzyme Q10in the international market is produced by microbial fermentation[32]， but coenzyme Q10is relatively low in natural cells. The use of molecular biology techniques to identify key enzyme genes in the coenzyme Q10biosynthesis pathway and the increase in the yield of coenzyme Q10by metabolic engineering are currently hot topics.

Improvement of coenzyme Q10production by the MEP pathway of recombinant R. sphaeroides

The side chain of coenzyme Q10belongs to terpenes， and the pathways for synthesizing terpenes in vivo mainly include the MVA pathway and MEP pathway. The synthesis of terpenes in bacteria is often enhanced by expressing key enzymes in the MEP pathway and heterologously expressing key enzymes in the MVA pathway. Overexpression of key enzymes in the MEP pathway in coenzyme Q10containing microorganisms is effective in increasing the supply of side chains. Lu et al.[33-34]overexpressed endogenous dxa， dxr， idi， ispD， ispF， ispG and other genes in R. sphaeroides， which greatly increased the supply of DMAPP. However， the accumulation of intracellular DMAPP will in turn lead to a decrease in the production of coenzyme Q10. To further elucidate this problem， the authors constructed a feedback control system through LacIq regulatory proteins and the introduction of RBS with different intensities to regulate the supply intensity of the side chain precursors， resulting in the condition that the production of coenzyme Q10increased from the original 50 mg/L to 80 mg/L.

In the side chain biosynthesis process， different lengths of polyisoprenyl diphosphate can be utilized by other branching pathways for the synthesis of hemiterpene， monoterpenes， polyterpenes and their derivatives. For instance， DMAPP is used for the synthesis of isoprene under the catalysis of isoprene synthase[18]. The geranylgeranyl pyrophosphate （GGPP） is used to synthesize the substrate for the synthesis of carotenoids， quinones， gibberellins， and various other isoprenoid compounds. GGPP enters the carotenoid biosynthesis pathway under the catalysis of CrtB， and can enter the biosynthesis pathway of chlorophyll under the catalysis of BChG[35]. GPP and PHBA produce geranylphydroxybenzoic acid （GBA） under the catalysis of GPT enzyme[36].   The synthetic pathways of these terpenoids and their derivatives can be used as competitive pathways for coenzyme Q10biosynthesis. At present， there are few reports on blocking the synthesis of carotenoids to increase the production of coenzyme Q10in R. sphaeroides， and no relationship has been found between the biosynthesis of other branched compounds such as monoterpenes， sesquiterpenes， triterpenoids and isoprenyl proteins and the synthesis of coenzyme Q10， which will be one of the important directions for improving the production of coenzyme Q10in R. sphaeroides in the future.

Improvement of coenzyme Q10production by the shikimate pathway of recombinant R. sphaeroides

The supply of isoprene side chains is closely related to the production of coenzyme Q10. The aromatic precursor substance phydroxybenzoicacid is the mother nucleus of coenzyme Q10synthesis. Therefore， the synthesis of phydroxybenzoic acid will increase the yield of coenzyme Q10. In 2016， Yu et al.[37]published a patent， i.e.， a R. sphaeroides strain and preparation method and  application thereof， which introduces a phydroxybenzoic acid translocator pcaK gene derived from Acinetobacter calcoaceticus into R. sphaeroides. The recombinant R. sphaeroides obtains increased ability to take up phydroxybenzoic acid from the outside of the cell， which in turn significantly increases the yield of its coenzyme Q10. In 2017， Qi et al.[38]heterologously expressed such three membrane transporters as A. calcoaceticus AcPcaK， Klebsiella pneumoniae KpPcaK and Corynebacterium glutamicum CgPcaK from different organisms in R. sphaeroides GY2， respectively， which increased the uptake of extracellular 4hydroxybenzoic acid. They also studied the transport efficiency and consumption of 4HBA and the ability of the resulting recombinant R. sphaeroides GY2 to produce coenzyme Q10. The external production of 4HBA effectively increased the yields of RSCgPcaK and RSKpPcaK coenzyme Q10， reaching 17.45 and 18.06 mg/g DCW， respectively， which were 18.05% and 20.82% higher than that of R. sphaeroides.

At present， there are many reports on the use of metabolic engineering to improve the synthesis of endogenous phydroxybenzoic acid. Does increasing the synthesis of endogenous phydroxybenzoicacid increase the yield of coenzyme Q10. Lu et al.[39]explored R. sphaeroides on the basis of different ability to synthesize coenzyme Q10， and pointing out that the production of phydroxybenzoicacid in R. sphaeroides is not the bottleneck of coenzyme Q10synthesis. However， there are also reports showing that in the case of simultaneous increase of side chain precursors， increasing the amount of phydroxybenzoic acid synthesis increases the synthesis of E. coli coenzyme Q10[19]. pHydroxybenzoate destroys the cell membrane of microorganisms， causing denaturation of intracellular proteins and loss of respiratory enzyme system activity[40]. In addition， there are few studies on the ubiquinone synthesis pathway， and many links need to be explored and clarified. At present， whether the failure in intime translocation of phydroxybenzoicacid synthesized at an improved level causes unbalance in metabolic pathway， poisoning of cells and inhibition on bacterial growth has not been elucidated. There are few reports on the relationship between the amount of phydroxybenzoic acid and coenzyme Q10produced by R. sphaeroides， which needs to be confirmed in the future. The modification of ubiquinone synthesis pathway is an important direction for current and future research.

Improvement of coenzyme Q10production by the ubiquinone modification pathway of recombinant R. sphaeroides

The reactions of the ubiquinone modification pathway are catalyzed by enzymes encoded by the ubi series of genes， and the order of reactions is somewhat different in eukaryotes and prokaryotes[15]. In 2014， Yu et al.[41]of Zhejiang University disclosed the construction method of engineering bacteria producing coenzyme Q10， the engineering bacteria and application thereof. They overexpressed ubiG by constructing a recombinant plasmid expression vector of R. sphaeroides， which increased the yield of coenzyme Q10by more than 30% through the regulation of the aromatic ring modification pathway in R. sphaeroides. Lu et al.[30]overexpressed the R. sphaeroides methyltransferase UbiG， and increased the yield of coenzyme Q10from 47.6 mg/L to 65.8 mg/L， confirming that UbiG is the ratelimiting enzyme for the synthesis of coenzyme Q10. To further investigate the effects of various catalytic enzymes in the ubiquinone modification pathway on the synthesis of coenzyme Q10， Lu et al.[31]performed induced expression and constitutive expression of ubi genes such as ubiA， ubiB， ubiD， ubiX， ubiDX， ubiE， ubiF， ubiH， etc.， which in turn illustrated that UbiE is also the ratelimiting enzyme for the synthesis of coenzyme Q10. Coenzyme Q10production reached 108 mg/L by fusion expression of UbiG and UbiE and localization to the cell membrane with PufX. In 2016， Chen et al.[42]disclosed a genetically engineered strain and its application in the production of coenzyme Q10. By coexpressing the ratelimiting enzymes UbiE， UbiG and UbiF in the coenzyme Q10synthetic pathway of R. sphaeroides， coenzyme Q10production was increased by more than 20%. In 2017， Zhao et al.[43]disclosed a method for constructing a plasmid expression vector for increasing the coenzyme Q10production in R. sphaeroides， which overexpressed such three important catalytic enzyme genes as ubiG， ubiE and dxsA in the coenzyme Q10synthetic pathway of R. sphaeroides， resulting in an increase of coenzyme Q10yield by nearly 80%.

Improvement of coenzyme Q10production in R. sphaeroides by blocking or weakening the competition pathways

There are multiple competing branches in the R. sphaeroides coenzyme Q10biosynthesis pathway， such as the naphthoquinone pathway， the chlorophyll synthesis pathway， the carotenoid synthesis pathway， and the Ltyrosine pathway， while blocking or weakening one or more of the branches can increase the yield of coenzyme Q10， and can also effectively solve the feedback inhibition of nontarget products on the synthesis of regulatory enzymes in the common pathway[44-45]. Li et al.[46]made the isoprene precursor mainly used for the synthesis of coenzyme Q10by knocking out gene crtB， which encodes the phytoene synthase of R. sphaeroides， and then heterologously expressed Escherichia coli chorismate lyaseencoding gene ubiC and 4hydroxybenzoate transferaseencoding gene ubiA， which in turn increased the synthesis of 4hydroxybenzoic acid and the binding to polyisoprene， finally increasing the coenzyme Q10production of R. sphaeroides by 75.29%. Zhu et al.[47]studied the relationship between the synthesis of carotenoids and the production of coenzyme Q10， and inhibited the synthesis of carotenoids by deleting the carotenoid gene and overexpressing carotenoid synthesis  transcription repressor PpsR， respectively. However， the yield and biomass of coenzyme Q10were reduced to half compared with the wild type strain. In contrast， after ppsR overexpression， when carotenoid production decreased from 15.7 mg/L to 2.2 mg/L， coenzyme Q10yield and biomass increased by 28% and 34.2%， respectively. This phenomenon indicates that carotenoids can protect cells from damage by peroxidation and completely block the carotenoid synthesis pathway， or inhibit the growth and metabolism of R. sphaeroides[48-49]. To further enhance coenzyme Q10production， crtE was constitutively coexpressed with ppsR to improve the supply of GGPP， a key precursor of the isoprenoid side chain， resulting in a 47% increase in coenzyme Q10production. This result indicates that proper reduction of carotenoid synthesis rather than complete blockade can increase coenzyme Q10production in R. sphaeroides.

Li et al.[50]studied the relationship between R. sphaeroides cytochrome and serotonin Q10synthesis by gene knockout. Cytochrome can compete with the precursor farnesyl pyrophosphate of coenzyme Q10and plays a very important role in the growth and metabolism of bacteria. There is a certain relationship between cytochrome and coenzyme Q10synthesis. By knocking out two transcriptional regulation factors PrrA and PpsR that regulate cytochrome synthesis in R. sphaeroides， it was found that the reduction of cytochrome promotes the synthesis of coenzyme Q10. By knocking out the coding genes of several key enzymes responsible for the electron transmission in the respiratory chain， it was found that cytaa3 and ccoN genes mainly affect the growth of R. sphaeroides and the synthesis of coenzyme Q10at higher oxygen concentrations. In addition， the knockout of sdhB can slow the growth of bacteria and increase the content of coenzyme Q10.

GGPP enters the chlorophyll biosynthesis pathway under the catalysis of enzyme BChG[35]， which competes with the coenzyme Q10synthesis pathway for the precursor GGPP. In 2014， Yu et al.[51]disclosed the construction method of engineering bacteria producing coenzyme Q10， the engineering bacteria and application thereof. By knocking out the chlorophyll synthesis gene bchG of R. sphaeroides， the yield of coenzyme Q10was increased by about 15%. As can be seen from the coenzyme Q10synthesis pathway， many of the branching pathways compete for various precursors required for the synthesis of coenzyme Q10. At present， there are few reports on other competition pathways， and most of them are only to study a certain competition route. At the same time， weakening or blocking multiple competition pathways and synergistically regulating coenzyme Q10synthesis is one of the future research hotspots.

Improvement of coenzyme Q10production by recombining multiple synthetic pathways of coenzyme Q10

In order to further improve the yield of coenzyme Q10of R. sphaeroides， a plurality of synthetic pathways were modified and optimized to construct a highyield coenzyme Q10engineering strain. Huang et al.[52]effectively increased the yield of coenzyme Q10by maintaining the supply balance of pyruvate and glyceraldehyde3phosphate in the MEP pathway， the supply of NADPH and the metabolic balance in the synthesis of coenzyme Q10through gene knockout and integration， the copy number of artificial operon and promoter strength and optimization of medium composition and pH. At present， there are few reports on systematic optimization of R. sphaeroides. Lu et al.[31， 33]further comprehensively regulated the side chain synthesis pathway and the ubiquinone modification pathway. The MEP pathway was first controlled by a feedback regulatory system， while the overexpression of the ubiquinone pathway ubiG resulted in a coenzyme Q10yield of 94.34 mg/L. To further increase the yield of coenzyme Q10， ubiG and ubiE of ubiquinone pathway were subjected to fusion expression and the key enzymes in the ubiquinone modification pathway were localized to the cell membrane， resulting in a coenzyme Q10yield of 138 mg/L.

Improvement of coenzyme Q10production by increasing the supply of reducing power

In the cellular respiratory chain of R. sphaeroides， coenzyme Q10promotes the synthesis of ATP and provides energy for cell growth and product accumulation[53]. Studies have shown that the addition of respiratory inhibitors to the culture system can effectively inhibit the respiration of cells and inhibit the electron transfer in the respiratory chain[54]. In order to maintain normal cell growth， more coenzymes will be used to maintain normal electron transport， so the amount of intracellular coenzyme Q10will increase. Jiang et al.[55]screened strains with high coenzyme Q10yield by ion beam and ultraviolet compound mutagenesis with the structural analog of coenzyme Q10， vitamin K3， and the respiratory inhibitor Na2S as substrates， and finally increased the yield of coenzyme Q10by 58%. Coenzyme Q10has a certain relationship with aerobic respiration. Decreasing oxygen supply reduces the content of coenzyme Q10in R. sphaeroides， indicating that there should be a close relationship between redox respiratory chain （RRC） activity and coenzyme Q10biosynthesis. Zhang et al.[56]separately deleted the genes encoding the catalytic subunits of RRC component （sdhB， ccoN and cytaa3） in order to elucidate the effect of RRC activity on coenzyme Q10biosynthesis. It was found that sdhB deficiency significantly increased coenzyme Q10content under hypoxic conditions. To further explore the potential of sdhB deletion mutants in the actual production of coenzyme Q10， a twostep oxygen supply culture strategy was used， achieving a 41.1% increase in coenzyme Q10production compared with the wild type strain. The research provides a new solution to promote the production of coenzyme Q10through the regulation of RRC activity and reasonable culture scheme.

NADH and NADPH are essential cofactors for organisms and provide reducing power in the biosynthesis of many important compounds such as terpenoids， chiral alcohols， antibiotics， and the like. The physiological function of coenzyme Q10as an electron carrier indicates that it is related to the redox potential. Kim et al.[57]demonstrated a positive correlation between glyceraldehyde3phosphate dehydrogenase （GapA1） activity and NADH/NAD+ ratio with coenzyme Q10. Zhu et al.[10]increased the GapA1 overexpression in R. sphaeroides by increasing the ratio of NADH/NAD+ and simultaneously increased the yield of coenzyme Q10by 58%. The hemoglobin regulated by vgb gene in microorganisms can regulate cell growth and protein synthesis under microaerobic conditions[58]. Coexpression of gapA1 and vgb increased coenzyme Q10yield by 71%. When the gapA1 and vgb genes were coexpressed in the engineered RspMQd， the yield of coenzyme Q10reached 163.5 mg/L. The synergistic regulation of redox potential and oxygen uptake plays an important role in increasing the production of coenzyme Q10in R. sphaeroides. Improvement of coenzyme Q10production through coordinated regulation of redox potential and oxygen uptake is a research direction in the future.

Prospect

At present， the biosynthesis pathway of coenzyme Q10has been not fully elucidated. For example， how the shikimate pathway and the ubiquinone pathway are linked by enzymes such as AroF， AroB， AorD， and AorE？ In addition， metabolic engineering can significantly increase the production of coenzyme Q10produced by R. sphaeroides， but most of them focus on the overexpression of a single gene or multiple genes， and only a small part of the studies are about the relationship between the branching pathways and coenzyme Q10produced by R. sphaeroides.   From the entire metabolic network of R. sphaeroides， there are few reports on the comprehensive regulation of coenzyme Q10produced by R. sphaeroides. Comprehensively improving the capacity of R. sphaeroides to produce coenzyme Q10by weakening or blocking each competitive pathway， overexpressing single or coexpressing multiple key enzyme genes， regulating the respiratory chain and optimizing the fermentation process will be a new direction for the production of coenzyme Q10from R. sphaeroides to march toward industrial scale. The use of molecular biology techniques to elucidate some key enzyme genes in the metabolic pathway of coenzyme Q10of R. sphaeroides is also a future research direction.

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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU