Increased Production of Coenzyme Q10 from Genetic Engineered Rhodobacter sphaeroides Overexpressing UbiG

The aim of this study was to increase the Coenzyme Q10 (CoQ10) yield from Rhodobacter sphaeroides via genetic engineering pathway. CoQ10 plays important roles in many biological processes and has been proven to be effective in the treatment of many diseases. In the present study, the ubiG gene located in CoQ10 biosynthesis pathway was effectively overexpressed in Rb. sphaeroides to increase CoQ10 production. The growth of host cells was slightly influenced by overexpressing ubiG. The crude CoQ10 production was enhanced by 58.31% compared to that from the control. The ubiG mRNA level was significantly increased compared to the wild type harboring empty vector as measured by qRT-PCR. Moreover, the crude CoQ10 exhibited strong anti-oxidant activity as measured in vivo by zone of inhibition assay.


INTRODUCTION
CoQ10 (2,3-dimethoxyl, 5-methyl, 6-decaisoprene parabenzoquinone) is a lipid-soluble material widespread in prokaryotes and eukaryotes, which could be used in the treatment of many diseases. It has been proposed that CoQ10 could effectively protect rat cardiomyocytes against cisplatin-induced cardiotoxicity via attenuating oxidative stress (Zhao, 2019). Yousef and co-workers suggest that CoQ10 has beneficial effects against neuronal damage induced by lead acetate (PbAc) through its antioxidant, anti-inflammatory, antiapoptotic and neuromodulatory activities (Yousef et al., 2019). Jahangard and co-workers suggest that CoQ10 is considered a safe and effective strategy for treatment of patients with Bipolar disorder during their depressive phase (Jahangard et al., 2019). Treatment by CoQ10 will reduce p53, Puma and Bax mRNA expression levels and increase Bcl-2 mRNA expression levels and thus mitigates ionizing radiation-induced testicular damage through inhibition of oxidative stress and mitochondriamediated apoptotic cell death (Said et al., 2019). Moreover, CoQ10 possesses strong anti-oxidant capacity and thus can protect phosphate, lips, proteins and DNA (Cluis et al., 2007;Kaci et al., 2018;Rizvi et al., 2015).
Production of adequate and low cost CoQ10 is required because of its applications in many fields related to people's health. Currently, CoQ10 is normally produced by three approaches including chemical synthesis, semi-chemical synthesis and microbial fermentation. Compared to other two ways, microbial fermentation is becoming more and more popular. Rb. sphaeroides is considered a promising microorganism for producing natural functional CoQ10 (Zahiri et al., 2006;Zhu et al., 2017). The whole genome of this bacterium has been completely sequenced. The biosynthesis pathway for the formation of CoQ10 in Rb. sphaeroides includes three pathways, the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) pathway, the shikimate pathway and the Quinine Modification Pathway (QMP). UbiG is an oxygenmethyltransferase, participating in two steps for the synthesis of CoQ10 in Rb. sphaeroides. UbiG will catalyzes 2-Decaprenyl-6-hydroxyphenol into 2-Decaprenyl-6-methoxyphenol and catalyzes the formation of CoQ10 from 2-Decaprenyl-3-methyl-5hydroxy-6-methoxy-1,4-benzoquinone, which is the last step for the biosyntheisi of CoQ10 in Rb. sphaeroides (Lu et al., 2015).
Rb. sphaeroides is considered an excellent model for studying photosynthesis and membrane development (Kiley and Kaplan, 1987). LH1 is one of the most important photosynthetic apparatus encoded by puf operon (Hu et al., 2002). puf operon promoter is normally regulated by oxygen tension and light intensity (Hu, et al., 2010). A powerful promoter and optimal growth conditions are very important for largest production of CoQ10 in genetic engineered Rb. sphaeroides.
Up to date, enhanced production of CoQ10 from Rb. sphaeroides by overexpression of ubiG under puf operon promoter and micro-aerobic growth conditions has not been reported. In the present study, the ubiG was overexpressed in Rb. sphaeroides initiated by puf operon promoter. The production of CoQ10 from the genetic engineered strain was enhanced by over 58%, which was increased much higher than reported literature (Lu et al., 2015). The present study will promote the application of Rb. sphaeroides for large scale production of functional CoQ10.

MATERIALS AND METHODS
Bacterial strains and growth conditions: Rb. sphaeroides strains were grown at 30C in malate minimal medium (Remes et al., 2014). Growth under micro-aerobic conditions was performed as described in our previous study (Hu et al., 2010). E. coli strains were cultivated vigorously in flasks at 37C in Luria-Bertani medium. Antibiotics were added to the growth media at the following concentrations when necessary: 200 μg/mL ampicillin, 20 μg/mL tetracycline for E. coli and 1.5 μg/mL tetracycline for Rb. sphaeroides.

Construction of genetic engineered Rb. sphaeorides:
The constructed plasmid pRKubiG was transferred into Rb. sphaeroides 2.4.1 by using the E. coli S17-1 as the donor as described in the previous study (Hu et al., 2010).
Production of crude CoQ10 from the genetic engineered strain: Colonies of the conjugant were selected and cultivated under micro-aerobic conditions in the dark at 30°C until OD660 reached approximately 0.6. Pre-cultures were respectively inoculated into 100-mL flasks containing malate minimal media with 1.5 µg/mL tetracycline at the ratio of 1% and grown under micro-aerobic conditions in the dark at 30°C for 48 h. Crude CoQ10 was extracted and quantified as described by Chen et al. (2006), respectively.
Quantitative RT-PCR: Total RNA was isolated from cell cultures by using the Tiangen Bacteria RNA Isolation Kit (#DPN430) according to the manufacturer's instructions. mRNA from genetic engineered strain and wild type strain harboring empty vector was considered sample mRNA and control mRNA, respectively. To further confirm the absence of DNA, PCR was performed targeting gloB (RSP_0799). qRT-PCR was performed as described previously (Remes et al., 2014) in a Bio-Rad CFX96 Real Time system. Primers used for qRT-PCR were ubiG-real-F (5'-GCAAAGCTCCATGC CGAG-3') and ubiG-real-R (5'-GTCGAGCAGATCATCAGG-3'). Relative mRNA expression levels were normalized to the reference gene rpoZ (Zeller et al., 2007) according to the formula given by Pfaffl (2001).
Zone of inhibition assay: Zone of inhibition assay was performed as described in our previous study . Filters soaked with 5 µL of 700 mM H2O2 were placed on the top of the plates.

Data analysis:
All experiments were repeated for 3 times. Turkey test and GraphPad Prism software were used to analyze the data trend.

Construction of the expression vector:
The expression vector used in this study was constructed as shown in Fig. 1. UbiG is an important catalyzed enzyme in the ubiquinone pathway for the biosynthesis of CoQ10 in Rb. sphaeroides (Lu et al., 2015). It is an oxygen-methyltransferase, involved in catalyzing 2-Decaprenyl-6-hydroxyphenol into 2-Decaprenyl-6-methoxyphenol and catalyzing the 2-Decaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4 benzoquinone into CoQ10. puf operon is comprised of pufB, pufA, pufL, pufM and pufX, encoding the LH1 and reaction center in Rb. sphaeroides (Eisenhardt et al., 2018;Gong and Kaplan, 1996). The puf operon promoter initiates two transcripts, a 2.7-kb transcript for pufBALMX and a 0.5-kb transcript for pufBA (Gong et al., 1994). Under the micro-aerobic growth conditions or optimal light intensity, the puf operon promoter exhibits strongest activity. Moreover, the pRK415 vector  is a broad host range expression vector.  Construction of the genetic engineered strain overexpressing UbiG: The constructed expression vector was mobilized into Rb. sphaeroides by conjugation by using E. coli S17-1. The growth curve for the wild type, 2.4.1/pRKpuf and 2.4.1/pRKubiG were constructed to demonstrate whether the growth was influenced by overexpression of ubiG, as revealed in Fig. 2. It was obvious that overexpression of ubiG slightly influenced the growth of the host cells. In the first 20 h, growth rates for the three different strains were nearly the same since the growth curve was overlapped. However, at the stationary phase, significant difference between the wild type and 2.4.1/pRKubiG at the time point of 48 h was observed. Similarly, remarkable differences between 2.4.1/pRKpuf and 2.4.1/pRKubiG at the time points of 22 and 48 h were observed. Although it has been proposed that the CoQ10 plays very important roles in energy generation and many other processes, which are important for cell's survival (Zahiri et al., 2006). In the present study, production of CoQ10 possibly did not strongly affect the growth of the host cells.
The crude CoQ10 was extracted from the genetic engineered strain, as observed in Fig. 3. The yield of crude CoQ10 from wild type 2.4.1 and 2.4.1/pRKpuf was around 26.30 and 25.869 mg/L, respectively. However, the crude CoQ10 production from the genetic engineered 2.4.1/pRKubiG was 41.47 mg/L. Compared to the wild type, the CoQ10 production in 2.4.1/pRKubiG was increased by 58.31%, which was much higher than the rate described in reported study (Lu et al., 2015). It could be concluded that overexpression of the ubiG could significantly increase the crude CoQ10 production under micro-aerobic growth conditions initiated by the puf operon promoter.
Quantitative RT-PCR analysis for ubiG: qRT-PCR was employed to test the ubiG mRNA expression levels to further describe the reasons resulted in the enhancement of CoQ10 from the genetic engineered 2.4.1/pRKubiG, as seen in Fig. 4. As expected, the ubiG mRNA level was significantly upregulated, with log2 fold change of approximately 13.63. UbiG is an oxygen-methyltransferase, involved in catalyzing 2decaprenyl-6-hydroxyphenol into 2-decaprenyl-6methoxyphenol and catalyzing the 2-decaprenyl-3methyl-5-hydroxy-6-methoxy-1,4 benzoquinone into CoQ10 (Lu et al., 2015). The UbiG enzyme expression level should be upregulated because of the increased ubiG mRNA expression levels and crude CoQ10 production.
Anti-oxidant activity: From the zone of inhibition experiment, it could be concluded that the strain 2.4.1/pRKubiG possessed much higher anti-oxidant activity than that of the control strain 2.4.1/pRKpuf, as revealed in Fig. 5. The H2O2 is a normally used oxidant produced ˖OH radical by Fenton reaction (Fischbacher et al., 2017). Obviously, the size of zone of inhibition for 2.4.1/pRKubiG was much smaller than that of 2.4.1/pRKpuf, indicating that the strain 2.4.1/pRKubiG was less sensitive to H2O2 than that of the control strain 2.4.1/pRKpuf. The genetic engineered strain 2.4.1/pRKubiE was constructed in our previous study (Tang et al., 2019), which produced more CoQ10 than the present genetic engineered strain 2.4.1/pRKubiG and possessed a little bit smaller size of zone of inhibition. The zone of inhibition assay indicated that the crude CoQ10 was functional and thus possessed the potential for commercial utility in food, cosmetic and pharmaceutical industries after further purification.

CONCLUSION
In the present study, we constructed the genetic engineered Rb. sphaeroides strain 2.4.1/pRKubiG to increase the CoQ10 production. Production of the crude CoQ10 from 2.4.1/pRKubiG was increased by 58.31% and ubiG mRNA was significantly upregulated. Moreover, the genetic engineered strain 2.4.1/pRKubiG exhibited more stronger anti-oxidant activity because of the much more production of crude CoQ10.