Co-production of biopolymers and quinone via no-sugar fermentation--a case by Methylobacterium sp. XJLM

Purpose To explore a competitive PHB producing fermentation process, this study evaluated the potential for Methylobacterium sp. XJLW to produce simultaneously PHB and coenzyme Q 10 (CoQ 10 ) using cheap and abundant methanol as sole carbon and energy source. Methods The metabolic pathways of PHB and CoQ 10 biosynthesis in XJLW strain were rst mined based on the genomic and comparative transcriptomics information. Then, Real-time uorescence quantitative PCR (RT-qPCR) was employed for comparing the expression level of important genes involved in PHB and CoQ10 synthesis pathways response to methanol and glucose. Transmission electron microscope (TEM), gas chromatography/mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), Fourier transformation infrared spectrum (FT-IR), and liquid chromatography/mass spectrometry (LC-MS) methods, were used to elucidate the yield and structure of PHB and CoQ 10 , respectively. PHB and CoQ 10 productivity of XJLW strain were evaluated in asks for medium optimization, and in a 5-L bioreactor for methanol fed-batch strategy according to dissolved oxygen (DO) and pH control. Results Comparative genomics and transcriptomics analysis showed that the PHB and CoQ 10 biosynthesis pathways coexist in XJLW strain, and the transcription level of key genes in both pathways response to methanol was signicantly higher than that response to glucose. Correspondingly, strain XJLW can produce PHB and CoQ 10 simultaneously with higher yield using cheap and abundant methanol than using glucose as sole carbon and energy source. The isolated products showed the structure characteristics same to that of standard PHB and CoQ 10 . The optimal medium and cultural conditions for PHB and CoQ 10 co-production by XJLW strain was in M3 medium containing 1% (v/v) of methanol, 0.5 g/L of ammonium sulfate, 0.1% (v/v) of Tween 80, and 1.0 g/L of sodium chloride, under 30°C and pH 7.0. In a 5-L bioreactor coupled with methanol fed-batch process, a maximum DCW value (46.31 g/L) with the highest yields of PHB and CoQ 10 , reaching 6.94 g/L and 22.28 mg/L, respectively. Conclusion Methylobacterium sp. XJLW is potential for eciently co-producing PHB and CoQ 10 employing methanol as sole carbon and energy source. However, it is still necessary to further optimize fermentation process, and genetically modify strain pathway, for enhanced production of PHB and CoQ 10 simultaneously by XJLW. It also suggests a potential strategy to develop eciently co-producing other high value metabolites using methanol-based bio-process.


Introduction
Nowadays, along with the increasing demands for polymer plastics, which can be widely used from product packing and daily tools to equipment parts and construction sectors, the growing serious petroleum-based plastic pollution has drawn more attractive attentions due to its less biodegradation property (Cardoso et al. 2020;Mostafa et al. 2020). In order to solve this global circumstance, many scientists have put great efforts on biodegradable polymers production. For showing similar thermoplastic, elastomeric and other physical-chemical properties to conventional plastics, polydroxyalkanoates (PHAs) are regarded as the most potential substituent, which can be completely degraded to CO 2 and H 2 O (Sukruansuwan 2018;Mostafa et al. 2020). However, the high cost of PHAs production from costly substrates has seriously limited the utilization of PHAs in commercial elds, which forces scientists to explore alternative approaches to produce it at a lower price (Parveez et al. 2015). The production costs of PHAs depend on many factors including strains, substrates, cultivation conditions, extraction and puri cation processes (Gamez-Perez et al. 2020). Carbon source is regarded as the major factor that accounts for 70-80% of the total expenses of PHAs (Mohandas et al. 2017), because PHAs are usually synthesized under a speci c condition of limitation of nutrients and excess of carbon source (Cardoso et al. 2020). Thus, development of a PHAs producing process with a cheap and renewable substrate is still necessary. As one of the common industrial by-products and a cheaper and renewable chemical feedstock, methanol has been widely used as carbon and energy source in methylotrophs fermentation processes for value-added chemicals production (Zaldivar Carrillo et al. 2018;Zhang et al. 2019). Hence, methanol-based fermentation for PHAs production is still a highly promising process without sugar consumption and carbon emissions.
Among all PHAs, polyhydroxybutyrate (PHB) is considered as the most competitive biopolymer because of its good biocompatibility, biodegradability, and similar properties to polypropylene (Parveez et al. 2015;Sharma 2019). Meanwhile, coenzyme Q 10 (CoQ 10 ) is the most valuable product among all natural quinone metabolites, and it is a good clinic biological drug for removing free radicals in the body, keeping biological membrane stable, anti-lipid peroxidation and strengthening the nonspeci c immune (Qiu et al. 2012;Lu et al. 2013). Thus, PHB and CoQ10 were selected as representatives of biopolymers and quinone metabolites, respectively, to evaluate the potential for their co-production via methanol-based process.
In our previous work, a new formaldehyde degradable methylotrophic bacterium was isolated and identi ed as Methylobacterium sp. XJLW (Qiu et al. 2014Shao et al. 2019. Its completed genome has been sequenced (Shao et al. 2019). Comparative genomic analysis exhibited XJLW contains both pathways of CoQ 10 and PHB biosynthesis ( Fig.1), suggesting the possibility to develop a new fermentation process to realize co-production of PHB and CoQ 10 with the abundant methanol as sole carbon source at the same time, which will provide a more economic process for PHB production.
In the present research, we aims to 1) verify the potential of PHB and CoQ 10 co-production by XJLW with different carbon sources, glucose and methanol; 2) elucidate the expression difference of the key genes in both pathways of PHB and CoQ 10 biosynthesis in XJLW response to methanol and glucose; 3) evaluate the effects of medium composition and cultivation conditions on PHB and CoQ 10 co-production in Erlenmeyer asks and in a 5-L stirred bioreactor employing methanol fed-batch strategy. This study will provide a new reference of strategy for improving value added products productivity with methanol-based fermentation process employing methylotrophs.

Materials And Methods
Chemicals PHB (purity above 95%, CAS No: 26063-00-3) and CoQ 10 (purity above 99.9%, CAS No: 303-98-0) were purchased from Sigma-Aldrich. Alcohol (HPLC grade, purity above 99.5%) was purchased from Tjshield ne chemicals Co., Ltd. (Tianjin, China). Other chemicals were analytical reagents purchased from local company. Initial pH of the above media was adjusted to 7.0 with 1 M NaOH. 1%(v/v) methanol was added to the two media used as sole carbon source after being autoclaved at 115°C for 30 minutes.

Culture Conditions
Fifty microliters suspension of frozen stock XJLW was inoculated into a 250 mL Erlenmeyer ask containing 50 mL medium M3, and incubated for 96 hours. Then 2 mL culture was inoculated into 250 mL Erlenmeyer asks containing 50 mL fermentation medium and incubated under 180 rpm and 30°C for 5 days.
Cell morphology observation via transmission electron microscope Cells in 1 mL culture broth was harvested by centrifuged at 8000 × g for 10 min at 4°C, and then were suspending in 4%(v/v) pre-cooled glutaraldehyde and immobilized for 1 h at 4°C. The ultrathin section of immobilized cell was observed under transmission electron microscope (HITACHI H-7650, Japan).
Physiology characteristics analysis combined with RNA-seq and RT-qPCR The cell growth and simultaneously production ability of PHB and CoQ 10 was detected in M3 medium supplemented with glucose and methanol respectively. Meanwhile, the cells were harvested for RNA-seq and RT-qPCR.

RNA-Seq data analysis
After culture in M3 containing methanol and glucose as carbon source, respectively, at 30℃ to log phase (OD 600 0.8), Methylobacterium sp. XJLW cells was harvested via centrifugation at 4000 rpm for 5 min under 4℃. Then, cells pellets were immediately mixed with RNA protect Bacteria Reagent (QIAGEN China Co. Ltd), and then stored at −80℃ for RNA extraction. A total amount of 1 μg quali ed RNA sample was used as input material for the library preparation. Library concentration was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary quantify. Insert size was assessed using the Agilent Bioanalyzer 2100 system, after the insert size consistent with expectations, quali ed fragment were accurately quanti ed using qPCR by Step One Plus Real-Time PCR system (ABI, USA). The raw reads were ltered by removing reads containing adaptors, ploy-N (i.e., unrecognized bases, reads with a recall ratio less than 5%) and low quality reads (the number of base≤10 and occupied less than 50% of the entire read) for subsequent analysis. Firstly, we used Tophat2 (Kim et al. 2013) to evaluate the sequencing data by comparison with the genomic sequences of reference strains. Based on the Tophat2 alignment results, Cu inks-2.2.1 was used to perform quantitative gene expression analysis. Gene expression is calculated as follows: FPKM (expected number of Fragments PerKilobase of transcript sequence per Millions of sequenced base pairs). In general, the screening criteria for signi cantly differentially expressed genes are: |log 2 fold-change|≥ 1 and p-value≤0.05. Scatter plot and volcano map are used to present the overall pro le of gene expression differences.

RNA extraction and quantitative RT-qPCR
The cells in the early exponential stage, cultured in M3 medium supplemented with glucose and methanol respectively, were centrifuged at 10000 × g for 5 min at 4 ℃. The total RNA was extracted by using RNA isolator (Vazyme Biotech Co., Ltd., Nanjing). And then, HiScript II Q RT SuperMix qPCR kit (Vazyme Biotech Co., Ltd., Nanjing) was used to develop reverse transcription reactions. The reaction buffer system of RT-qPCR was prepared with ChamQ SYBR qPCR Master Mix, and performing the quantitative PCR with Bio-Rad CFX real-time PCR system. The expression level of the 16S rRNA gene was used as internal references. Each reaction was repeated at least three times. The primers used for RT-qPCR are listed in Table 1.
Optimization of XJLW strain fermentation in shake ask level Firstly, to choose a better initial medium, the cell growth and biosynthesis of target products of XJLW strain cultivated in M3 and MSM were evaluated. A one-factor-at-a-time design was employed to analyze the effects of methanol concentration, ammonium sulfate concentration, fermentation temperature, initial pH of medium, different types of oxygen carriers and osmotic pressure regulated by adding different concentration of sodium chloride on XJLW strain growing and target metabolites biosynthesis.
Scale-up of XJLW strain fermentation system with fed-batch strategy After optimization of fermentation conditions in ask, a fed-batch fermentation was carried out in a 5 L stirred tank reactor (Biostat-Bplus-5L, B.Braun Germany) with a working volume of 3.0 L, at 30°C, 400 rpm and pH 5.5 (controlled using aqueous NH 4 OH solution), and with a dissolved oxygen concentration above 20% of air saturation. Firstly, the basal salts of optimal medium were dissolved in 2670 mL ddH 2 O and were autoclaved in the bioreactor. To start the fermentation, 30 mL methanol and 300 mL inoculum suspension were added to the bioreactor by peristaltic pump. Filter-sterilized air was the source of oxygen and was supplied at a ow rate of 3 vvm. After initial added methanol was completely exhausted implied by the dissolved oxygen level rising up to 100%, additional methanol (mixed with 1% trace elements solution) was pulse fed into the reactor regulated by the dissolved oxygen monitor to further increase the cell density. At the same time pH was also adjusted at a stable level of 5.7 by adding NH 4 OH solution which could supply nitrogen source simultaneously. If needed, increasing stirred speed strategy was also employed to increase dissolved oxygen level. The whole fermentation period was about 5 to 7 days.

Separation of CoQ 10 and PHB
After fermentation, cell biomass was separated by centrifugation at 8000 × g, 4°C for 10 min (Biofuge Stratos Sorvall, Thermo, Germany), then 20 mL alcohol was added to the pellets for suspending cells. Subsequently, the cell suspension was subjected to sonication in an ultrasonicator at 500 W for 12 minutes with a pulse of 15 seconds on and 10 seconds off. After cell disruption, the suspension was centrifuged at 8000 × g, 4°C for 10 min, and then the supernatant was sampled for CoQ 10 analysis, while the precipitation was sampled and kept in a 45°C oven to a constant weight before PHB extraction.
For PHB extraction, a certain amount of chloroform was added to the dry disruption cell for 1 h extraction at 60°C. Then, the PHB extract was separated by vacuum ltration and air dried as the crude PHB, which was further puri ed by adding acetone-methanol mixed liquor (volume ratio 7:2) and washing for twice to remove the pigment. The puri ed PHB was obtained after drying at 45°C.

Assays methods
Methanol was analyzed by gas chromatography (GC; Shimadzu-2010, Japan) equipped with ame ionization detector (FID) and elastic quartz capillary column (AT-FFAP). Chromatographic condition: injection temperature 200°C, detector temperature 250°C, temperature programming: keeping at 70°C for 4 min, then heating to 150°C at the speed of 50°C per min and keeping for 1 min. The carried gas is nitrogen and column ow was 3.0 mL/min, split ratio 10/L and a sampling quantity of 1 μL.
Cell biomass was measured by analyzing the optical density at 600 nm. Firstly 1 mL culture samples were centrifuged at 8000 × g for 10 min at 4°C; the cells were washed twice in distilled water, centrifuged at the same condition, and nally were diluted by adding distilled water to the linear concentration range according to the standard curve describing the tting relation between dry cell weigh (DCW) and absorbance at 600 nm (OD 600 ), OD 600 was tested and DCW would be calculated according to a standard curve of the relationship between optical density of cells and DCW of XJLW. Each sample was in triplicate.
PHB content analysis was according to Pal A's method (Pal et al. 2009). Firstly 10 mg PHB sample was turned into crotonic acid by treatment with 10 mL concentrated H 2 SO 4 in the boiling water bath for 30 min, then the tube was naturally cooled to room temperature, and the absorbance was tested under 235 nm by the UV-1800 spectrophotometer (Shimadzu, Japan) with concentrated H 2 SO 4 as the blank. The standard curve was drawn by the same method. The chemical structure of PHB was identi ed by gas chromatography-mass spectrometric (GC-MS), nuclear magnetic resonace (NMR) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy, respectively.
To nd the polymer composition, the puri ed PHB was dissolved in chloroform (5 mg PHB mL -1 ), one microliter of which was injected into a GC-MS instrument (Agilent Technologies 7890A GC System, America; Bruker esquire 6000 MS instrument, German). The column and temperature pro le used for GC analysis were as follows: capillary column (HP5MS), 30m ×0.25mm, lm thickness 0.25μm; injection temperature 250°C, ion source temperature 200°C, and transfer line temperature 275°C; oven temperature programming: initially at 60°C, then heating to 250°C at the speed of 20°C per min and keeping for 15.5 min. The carried gas is helium and column ow was 40 cm/s. Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded by using a Anance III spectrometer (Bruker, Switzerland) at 400 MHz and 100 MHz, respectively, at the following experimental conditions: 0.5% (w/v) polymer sample was dissolved in spectrochem grade deuterochloroform (CDCl 3 ) and tetramethylsilane (TMS) was used as an internal reference. The chemical shift scale was in parts per million (ppm).
For FT-IR analysis, 2 mg polymer sample was thoroughly mixed with 100 mg spectroscopic grade KBr with the help of mortar and pestle. From this mixture 15 mg was used for making KBr pellets. The pellets were kept in an oven at 100°C for 4h to remove atmospheric moisture from the sample. The IR spectrum of the polymer sample was recorded with a Nicolet 6700 FT-IR spectrophotometer (Thermo, America) in the range 4000-600 cm -1 .

Statistical analyses
The mean and standard deviation were calculated from at least three independent experiments in triplicate.

Results
Strain XJLW can produce PHB and CoQ 10 simultaneously Transmission electron microscope observation results (Fig. 2) showed that there were many white particles with high refraction inside strain XJLW cells, occupying nearly half or more space. It suggested high content of PHAs inside the XJLW cells.
After isolation and puri cation, the exact structure of PHAs from XJLW was identi ed via GC-MS, NMR and IR analysis methods, respectively. Fig. S1A shows the GC spectra of PHAs extracts of XJLW strain and the 7.59 min peak is corresponded to the hydrolyzed product of PHB according to standards. In order to obtain an exact structure of this polyester, a further MS analysis of the 7.59 min peak fragment was carried out and the spectra were shown in Fig. S1(B). The 101.0 m/z molecular fragment was identical to the 3-hydroxybutyrate, while the molecular fragments of 85.0 m/z represented butyrate. The 1 H-and 13 C-NMR spectra of PHB standards and PHAs produced by strain XJLW are shown in Fig. S2. The 1 H-NMR spectra show the presence of three signals in both spectra of the two polymer samples, which are corresponded to the methyl group (CH 3 at 1.28 ppm), methylene group (CH 2 at 2.61 ppm) and methine group (CH at 5.26 ppm), respectively (Fig. S2A). The methyl group (CH 3 ), methylene group (CH 2 ), methyne group (CH), and carbonyl group (C=O) are found at 19.8, 40.8, 67.6 and 169.2 ppm respectively (Fig.   S2B). The chemical shifts of both 1 H-and 13 C-NMR of PHAs from XJLW are in good agreement with the data of PHB standards. IR spectra of PHB standards and PHAs from XJLW are shown in Fig. S3. It shows mainly two intense absorption bands at about 1280-1291 cm -1 , 1725 cm -1 and 2925-2978 cm -1 corresponding to C-O, C=O and C-H stretching groups respectively. The 3436.8 cm -1 absorption band indicates a small number of O-H existing in PHAs from XJLW and PHB standards referring to the terminal hydroxyl. Meanwhile, the great similarity of IR spectra characteristic indicates chemical group composition in PHAs from XJLW is the same to that of PHB standards. All the above evidences demonstrate PHB should be produced by XJLW strain.
LC-MS results of CoQ 10 standard and the sample extracted from strain XJLW cells were shown in Fig.S4.
It was found that the peak of CoQ 10 in sample appeared at the retention time same to that of CoQ 10 standard. Although the target peak area of sample looked lower than that of other unidenti ed peaks, the mass-to-charge ratio of CoQ 10 sample extracted from XJLW strain exhibited a molecular peak (m/s, 885.6) same to that of CoQ 10 standard. The result suggested that the strain XJLW has the ability of CoQ 10 biosynthesis. However, further puri cation of the sample CoQ 10 and enhanced production of CoQ 10 in the strain XJLW are required in future research.
Higher biomass, PHB and CoQ10 yield in M3 with methanol than with glucose As shown in Fig.3, XJLW exhibited much higher biomass and yield of both PHB and CoQ 10 when incubated in M3 medium supplemented with methanol than glucose as sole carbon source, respectively. It is interesting that the expression level of some genes coding the key enzymes in the pathway of PHB and CoQ 10 biosynthesis of stain XJLW in methanol medium was also signi cantly higher than that in glucose medium (Fig. 4). The expression level of much more genes was also compared based on the RNA-seq results ( Table 2, 3). Besides, the data of quantitative RT-qPCR of selected genes involved in PHB synthesis pathway indicated that PHB may be synthesized by different pathways or be regulated by different isoenzymes under different substrates or different cultivating condition. In the RT-qPCR analysis, we chose phaC-3 encoding poly(R)-hydroxyalkanoic acid synthase (class III) for analysis, results showed that phaC-3 was signi cantly up regulated by methanol, which was identi ed with RNA-seq results. However, phaC-1 catalyzing the same step in the pathway was down regulated by methanol, indicating different isoenzymes were regulated by different factors. Meanwhile, totally 5 acat genes, 3 paaH genes, 2 fadN genes and 2 phaZ genes were found in PHB synthesis pathway in XJLW showing different responses to methanol (Table 3), which indicated that there was a more complex regulation system in XJLW responsible for PHB production. From genomic data mining, we also found no gene encoding hydroxybutyrate-dimer hydrolase (EC: 3.1.1.22) and hydroxymethylglutaryl-CoA synthase (EC:2.3.3.10) existing in XJLW strain, suggesting PHB were mainly synthesized through FadJ catalyzed branch pathway. Besides, in CoQ 10 synthetic pathway of XJLW, we also found no gene encoding decaprenyldiphosphate synthase (EC: 2.5.1.91) existed in the genomic data, but the LC-MS had strickly veri ed the product of CoQ10 from this strain. So, it is very possible that there is another new branch pathway or unannotated gene responsible for decaprenyl-diphosphate, an important precursor of CoQ 10 , biosynthesis in XJLW.
Effects of medium composition and cultivation conditions on cell growth, PHB and CoQ 10 productivity in ask level Both medium M3 and MSM are recommended as suitable medium for Methylotrophs strain cultivating (Bourque et al. 1995) with methanol as sole carbon and energy source. Thus, the growth behaviors of XJLW in M3 and MSM were evaluated in asks. The results (Fig. 6A) showed that M3 medium exhibited more superiority for cell growth than MSM and 5 d was the best harvest time with maximum dry cell density. Meanwhile, the ability of PHB and CoQ 10 production by XJLW in M3 and MSM was also evaluated respectively. The results (Fig. 6B) also showed that XJLW exhibited better PHB and CoQ 10 biosynthesis capacity in medium M3 than in MSM. M3 was then selected as initial medium for the optimization of XJLW fermentation in the following experiments.
As medium components, carbon source and nitrogen source play the signi cant role in the fermentation productivity according to previous report (Wei et al. 2012;Mozumder et al. 2014). Thus, the effect of carbon and nitrogen sources is also very necessary to be evaluated for the optimization of XJLW fermentation process. In the previous publications, methanol and ammonium sulfate had been approved to be the suitable carbon and nitrogen source for Methylobacterium (Bourque et al. 1995;Yezza et al. 2006). Therefore, the effect of different concentrations of methanol (Fig. 7A) and ammonium sulfate (Fig. 7B) on PHB and CoQ 10 productivity of strain XJLW was evaluated respectively in the present study.
It was found that 1% (v/v) methanol led to maximal CoQ 10 concentration of 1.26 mg L -1 while the optimal biomass and PHB concentration was obtained under 1.5% (v/v) methanol. The phenomenon may result from the different biosynthesis pathway of CoQ 10 and PHB. In order to avoid cell intoxication caused by high methanol concentration, 1% (v/v) methanol was selected as the optimal carbon source concentration in further research. It was also found that 0.5 g L -1 of ammonium sulfate resulted in the highest yield of biomass, PHB and CoQ 10 . Therefore, 0.5 g L -1 of ammonium sulfate was selected as the optimal nitrogen source concentrate in the following experiments.
Besides medium components, cultural conditions such as culture temperature and initial pH also play important role in microbial fermentation. Thus, the effect of culture temperature and initial pH on XJLW fermentation was then evaluated in ask. The results ( Fig.7C and Fig.7D) showed that the best cultural temperature is 30 °C, and the optimal initial pH is 7.0. As fermentation broth may turn to lower pH caused by carbon metabolism of XJLW, feeding ammonium hydroxide to neutralize the excess formic acid derived from methanol metabolism is very important. Thus, the optimal initial pH and cultural temperature were selected as 7.0 and 30 °C respectively.
Due to the poor solubility of oxygen in aqueous medium, the dissolved oxygen (DO) supply is another key factor affecting the productivity in aerobic fermentation process, and one of the most effective strategies for improving oxygen mass transfer e ciency is adding oxygen carrier to the aerobic fermentation system (Lai et al. 2002;Xia 2013;Vieira et al. 2015). In this study, three different oxygen carriers were chosen to enhancing the oxygen supply, including two different surfactants (Triton X-100 and Tween 80) and hydrogen dioxide. Compared with the control group, 0.1% (v/v) of different oxygen carrier was added to the XJLW strain fermentation system respectively. The results (Fig.7E) showed that Tween 80 exhibits positive effects especially in the level of CoQ 10 and PHB biosynthesis, meanwhile the productivities of Triton X-100 group and hydrogen dioxide group were both lower than the control group. Perhaps excessive emulsi cation of Triton X-100 and denaturation of membrane protein caused by hydrogen dioxide can both inhibit normal metabolism of XJLW strain. Tween-80, a non-ionic surfactant, could improve the cell membrane permeability and the spec c surface area of oxygen at appropriate concentration, so it may also exhibit positive promotion for intracellular metabolites biosynthesis. According to these data, 0.1% (v/v) of Tween 80 was chosen as the best oxygen carrier in the following research.
As an important environmental factor, osmotic pressure may affect the mass transfer and the accumulation level of metabolites in many microorganisms (Xu et al. 2013;Mozumder et al. 2015), so the effects of ostomic pressure on XJLW metabolism were discussed through adding different concentration of sodium chloride. The results (Fig.7F) showed that the group adding 1.0 g L -1 of sodium chloride exhibited the highest cell yield and target products concentration, so this regulation strategy was chosen in the subsequent research.
Based on the above, the optimal medium and cultural conditions for CoQ 10 and PHB coproduction through XJLW strain fermentation were M3 medium containing 1% (v/v) of methanol, 0.5 g L -1 of ammonium sulfate, 0.1% (v/v) of Tween 80, and 1.0 g L -1 of sodium chloride under the fermentation temperature and initial medium pH of 30°C and 7.0, respectively.

XJLW fermentation in a 5 L fermenter
The cultural medium components and fermentation conditions in shake ask fermentation experiments are easy to control and regulate without complicated operation. However, there are also many great di culties in dealing with the problems occurring in shake ask level. These problems include but not limited to higher resistance in mass transfer and heat transfer, lower oxygen transfer e ciency, unpractical on line regulation and feeding strategies. Thus, it is very necessary to explore the metabolic behavior of strain XJLW in a 5 L stirred tank reactor. Base on the above results, a methanol feeding strategy coupled with pH and dissolved oxygen (DO) controlling was employed in a 5 L stirred tank reactor for a high-density fermentation. During the whole cultivation period, DO, stir speed, and pH were captured by on line monitors, and the acquisition curves were shown in Fig. 8A. Meanwhile, the changes of methanol concentration, biomass, PHB and CoQ 10 productivity during the whole process were shown in Fig. 8B. During the rst 36 h, the consumption of methanol added before fermentation was speeded up gradually until DO rebounding to 100%, meaning that there was no methanol enough for cell growth in the medium. From then on, methanol was fed at a pulsed pace to ensure su cient carbon source in the fermentation system without toxicity caused by excessive methanol. With cell density increasing, the limited dissolved oxygen became another key factor affecting cell growth. Thus, stir speed also gradually increased to ensure the DO level between 10% and 50%. During the whole fed-batch process, pH of broth was controlled at 5.7 approximately rather than 7.0, for excessive ammonium hydroxide used for adjusting pH may inhibit PHB accumulation according to previous report (Pieja et al. 2012). After 106 h when methanol accumulation occurred, methanol feeding ceased, and DO quickly rose up to 100%, indicating the respiration intensity of XJLW cells weakened sharply with little methanol consumption in the nal period. Thus, it was time for the harvest.
It was also found that low content of PHB and CoQ 10 were detected during the rst 36 h, suggesting initial adding methanol was almost completely exhausted for cell respiration and growth. Later, along with feeding substrates, concentration of biomass, PHB and CoQ 10 increased in the same trend, implying both of PHB and CoQ 10 were biosynthesized associated with cell growth. During the whole process, the total exhausted methanol volume is 830 mL, coupled with feeding 113.05 mL ammonium hydroxide.
Finally we got a maximum DCW value (46.31 g L -1 ), and the highest yields of PHB and CoQ 10 reached 6.94 g L -1 and 22.28 mg L -1 , respectively. Thus, the nal productivities of PHB and CoQ 10 in this fed-batch fermentation system reached 0.14 g g -1 of DCW and 0.48 mg g -1 of DCW, respectively. These results suggest that the feeding methanol coupled with DO controlled through adding ammonium hydroxide strategy should be an effective method to increase the cell density and productivities in XJLW submerged fermentation system.

Discussion
As carbon source storage in microbial cells, PHAs are usually synthesized and accumulated under imbalanced growth conditions by limiting a nutritional element, such as nitrogen, phosphate, or oxygen (Mozumder et al. 2014). PHAs could accumulate inside a membrane enclosed inclusion in many bacteria at a high content up to 80% of the dry cell weight (Khosravi-Darani et al. 2013). Thus, if a strain has the potential for PHAs production, there will be many polymer particles inside the cell suggesting PHAs existence. In this study, the cell morphology of Methylobacterium sp. XJLW under a transmission electron microscope (TEM) also showed a high content of polymer particles (Fig.1), which is similar to most PHAs producing strains.
For Methylotrophs cultivating with methanol as sole carbon and energy source, both medium M3 and MSM are recommended as suitable medium (Bourque et al. 1995). However, M3 medium exhibited superiority for strain XJLW cell growth than MSM. As medium components, carbon source and nitrogen source usually play the signi cant role in the fermentation productivity according to previous report (Wei et al. 2012;Mozumder et al. 2014). For Methylobacterium strains, methanol and ammonium sulfate had been approved to be the suitable carbon and nitrogen source (Bourque et al. 1995;Yezza et al. 2006). In the present study, a methanol utilized strain Methylobacterium sp. XJLW, which was isolated as formaldehyde degrading strain in our previous study (Qiu et al. 2014), also grows better in the M3 than in BSM containing methanol as sole carbon source (Fig. 5).
In order to develop its potential applications in biotechnological industry, PHB and CoQ 10 were selected as representatives of biopolymers and quinone metabolites, respectively, to evaluate the potential for their co-production via methanol-based culture process of Methylobacterium sp. XJLW. An increasing number of PHB producing strains have been reported, including Methylobacterium extorquens (Ueda et al. 1992;Bourque et al. 1995), Paracoccus denitri cans (Ueda et al. 1992;Kalaiyezhini et al 2015), Alcaligenes latus (Yamane et al. 1996), Methylobacterium sp. ZP24 (Nath et al. 2008), Bacillus thuringiensis (Pal et al. 2009), Cupriavidus necator (Mozumder et al. 2015), Halomonas campaniensis (Chen Let al. 2019), Bacillus drentensis (Gamez-Perez et al. 2020). After process and culture conditions optimization, the yield of PHB has reached a high level more than 100 g L -1 PHB from methanol via high-cell-density fed-batch culture of methylotrophic bacteria (Ueda et al. 1992;Yamane et al. 1996). Based on the above, methylotrophic bacteria seem the potential industrial strains for PHB production via methanol-based biotechnology.
Meanwhile, CoQ 10 is another important compound which can be widely used as potent antioxidative dietary supplement in treating cardiovascular disease, cancer, periodontal disease, and hypertension acting (Hofer et al. 2010;Lu et al. 2013). There are also a number of strains capable of producing CoQ 10 .
However, no publication was found about CoQ 10 synthesis in methylotrophic bacteria. In this study, it was found that both metabolic pathways of PHB and CoQ 10 biosynthesis exist in XJLW strain based on the genomic and comparative transcriptomics information (Fig.1). RT-qPCR results also showed the transcription level of key genes in both pathways response to methanol was signi cantly higher than that response to glucose (Fig.4). Correspondingly, strain XJLW can produce PHB and CoQ 10 simultaneously with higher yield using methanol than using glucose as sole carbon and energy source (Fig.3). To our knowledge, it is the rst report on PHB and CoQ 10 production simultaneously by methylotrophic bacteria.
After optimization of medium composition and the culture conditions on PHB and CoQ 10 biosynthesis, a cell density of DCW 46.31 g L -1 with a PHB concentration of 6.94 g L -1 , and a CoQ 10 concentration of 22.28 mg L -1 were achieved in a 5 L bioreactor, which were 30 fold, 6 fold, and 17 fold higher than that in asks, respectively. Although the productivity of CoQ 10 was 0.48 mg g -1 of DCW, which was lower than that of previous reported strains such as Rhodobacter sphaeroides (2.01 mg g -1 of DCW ) (Kalaiyezhini 2015), the volumetric yield of 22.3 mg L -1 of XJLM was higher than that of several previous reported strains including the mutant strain of Rhodobacter sphaeroides (14.12 mg L -1 ) (Bule 2011), Paracoccus dinitri cans NRRL B-3785 (10.81 mg L -1 ) (Tian et al. 2010), and Sphingomonas sp. ZUTEO3 (1.14 mg L -1 ) (Zhong et al. 2009). Meanwhile, XJLM could accumulate PHB at the productivity level of 0.14 g g -1 of DCW. The PHB yield of XJLM was lower than several reported strains such as Methylobacterium extorquens DSMZ 1340 (0.62 g g -1 of DCW) (Mokhtari-Hosseini et al. 2009) and Methylobacterium extorquens ATCC 55366 (0.46 g g -1 of DCW) (Bourque et al. 1995), but the volumetric yield of PHB of XJLM in this study (6.94 g L -1 ) was higher than that of Methylobacterium sp. ZP24 (3.91 g L -1 ) (Nath et al. 2008).

Conclusions
In summary, it is feasible to develop a co-production process of two valuable metabolites by Methylobacterium sp. XJLW from methanol. However, it is still necessary to further optimize fermentation process, and genetically modify strain pathway, for enhanced production of PHB and CoQ 10 simultaneously by XJLW. This study also presented a potential strategy to develop e ciently coproducing other high value metabolites using methanol-based bio-process.

Declarations
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