Squalene Biosynthesis in Engineered Escherichia Coli

Abstract Squalene is a natural triterpenoid that is important in medicine, and daily-use chemical and nutraceutical industries. The demand for squalene remains high and constant. Shark liver oil is the source of the compound; however, the source is unsustainable due to the protection of the gradually extinct animal. This study aimed to construct an engineered Escherichia coli strain to produce squalene in a green and sustainable manner. In this study, the activity of several squalene synthases from different species was screened. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used for chemical structure identification. The mevalonate pathway (MVA) was constructed into BL21(DE3) to enhance the supply of farnesyl diphosphate to obtain squalene at a concentration of 69.3 mg/L. IspH was overexpressed in BL21(DE3) to decrease squalene production due to the accumulation of dimethylallyl diphosphate. By balancing the overexpression of ispH and ispG , the production of squalene increased to 298.3 mg/L. The genes of the MVA pathway were further separated upstream and downstream, and constructed into two plasmids with different origins to balance the metabolic flux. By optimizing the expression strength of genes in MVA and 2-C-methyl-d-erythritol-4-phosphate pathway, an engineered strain was finally obtained with squalene production of 974.3 mg/L. By replacing chassis cell BL21(DE3) with XL1-Blue, squalene production was further increased to 1,274 mg/L.


Introduction
Squalene is a natural triterpenoid that acts as an important precursor in the biosynthesis of sterols, cholesterol, and terpenoids, 1 and plays a broad role in reducing cancer risk, 2,3 medication usage, 3,4 anti-infection, 4,5 and moisture retention, 5 suggesting its wide application in daily-use chemical, medical, and nutraceutical sectors.Squalene is abundant in the liver of deep-sea sharks, 1,6 but access to this source is limited due to animal protection. 7Squalene can also be produced by genetically modified plants and microorganisms, 8 but these methods are still not widely used due to low yield and high cost. 6,7,9n nature, squalene is synthesized from the two pathways: 2-C-methyl-d-erythritol-4-phosphate pathway (MEP pathway, mainly presents in eubacteria and plant plastids) and mevalonate pathway (MVA pathway, mainly presents in plant and animals) (►Fig.1). 10,11In Escherichia coli, farnesyl diphosphate (FPP), a precursor substance, already exists, and squalene can be synthesized from two molecules of FPP head-to-head by the addition of squalene synthase (SQS).Escherichia coli does not consume squalene, yet produce and store squalene reliably.Thus, E. coli may be an excellent genetically engineered host for squalene production. 2,5n the past, Furubayashi et al proved that the expression of human SQSs is a predominant strategy for the production of squalene in E. coli, 12 Sun et al introduced the entire MVA pathway to enhance the production of squalene in E. coli, 13 and Katabami et al took the same measure and introduced the MVA pathway to increase the production of squalene to 230 mg/L, 9 Xu et al increased the production of squalene in E. coli by enhancing NADPH supply, and achieved net increment of 21-folds compared to the origin strain. 14However, whether co-expressing the MEP and MVA pathways and increasing metabolic flow 15 would further improve the production of squalene remained largely unknown.
It is also favorable to increase the expression of ratelimiting enzymes and rate-limiting substrates, 5,16 and replace the chassis cell 17,18 to enhance the production of squalene.In this study, the activity of several SQSs from different species was screened.The MVA pathway was constructed into BL21(DE3) to enhance the supply of FPP, isopentenyl pyrophosphate (IPP), and dimethylallyl diphosphate (DMAPP).In addition, we tried to overexpress different combinations of idi, ispA, dxs, ispG, and ispH to further enhance FPP supply.Genes of the MVA pathway were all on plasmid p35151, and separated into two plasmid backbones with different copy numbers to balance the metabolic flux.Chassis cells were also changed to improve the production of squalene.The study constructed an engineered E. coli to achieve a biosynthetic pathway to obtain squalene efficiently.

Strains, Plasmids, Reagents, and Shake Flask Cultivation
The bacterial strains and plasmids used in this study are listed in ►Table 1. DE3 (E.coli) and XL1-Blue (E.coli) were used as the host for metabolic engineering.DH5α (E.coli) was used in plasmids amplification.Plasmid p35151, pETDuet-1, pUC19m, and pBBR1MCS-2 were kept in our laboratory and used as the backbone for other plasmids' construction.
The standard of squalene was purchased from Sigma-Aldrich (Shanghai, China).Restriction enzymes, PrimeSTAR Max DNA Polymerase, and In-Fusion HD Cloning Plus were purchased from Takara (Dalian, China).Gel extraction kit, PCR purification kit, and plasmid purification kit were purchased from Generay (Shanghai, China).
Strains for plasmid amplification were cultured at 37°C in LB medium (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl) for 12 hours on a rotary shaker (230 rpm).Engineered strains for squalene production were cultured at 37°C in TB medium (12 g/L peptone, 24 g/L yeast extract, 4 ml/L glycerol, 0.17 mol/L KH 2 PO 4 , 0.73 mol/L K 2 HPO 4 ) for approximately 3 hours on a rotary shaker (230 rpm).When the OD 600 of cultures reached 0.6, isopropyl-1-thio-β-D-galactopyranoside (IPTG; 0.1 mmol/L) was added to induce the expression of genes under the control of IPTG-inducible promoters.Engineered strains were grown for a further 48 hours at 37°C on a rotary shaker (180 rpm) before harvesting and analyzing for squalene content.According to different plasmids containing various engineered strains, ampicillin (100 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (25 μg/mL) were selected and added to the medium LB and TB.

Plasmid Construction
The primers used in this study are listed in ►Table 2. All primers were synthesized by GENEWIZ Inc. (Suzhou China).General molecular manipulations were performed according to standard protocols. 19T-KN, pET-NN, pET-thN, and pET-YN Plasmids pET-KN, pET-NN, pET-thN, and pET-YN were constructed from pETDuet-1, which contains ColE1 origin, ampicillin resistance gene, and T7 promoter.
The sequence of ispH (UniProt ID: P62623) was synthesized and constructed into plasmid pET-IAY by GENEWIZ Inc.
Table 2 The primers used in this study

Carotenoid Pigment Analysis
Carotenoid pigment analysis was performed according to a reported study. 20Each strain was harvested from a 10 mL culture medium into a 50 mL centrifugal cube.After centrifuging at 8,000 rpm for 15 minutes and discarding the supernatants, bacterial pellets were obtained.Then, 0.5 to 1 mL of acetone was added to each tube, and immediately vortexed the tubes for 1 minute to extract the carotenoids.The mixture was centrifuged at 10,000 rpm for 30 minutes to obtain acetone extracts.The absorbance at 470 nm was analyzed by using Ultrospec 2100 pro (GE, Boston, United States).The pigmentation level of each culture was determined from the absorbance of the resulting extract by using the molar adsorption coefficients of carotenoids (470 nm, 147,000 M À1 cm À1 ).

Identification and Quantification of Squalene
The identification and quantification of squalene was conducted according to a reported study. 21Each strain was harvested from a 20 mL culture medium and added into a 50 mL centrifugal cube.The suspension was centrifuged at 8,000 rpm for 5 minutes.Discard the supernatants and obtain the bacterial pellets.To each tube, add 2 mL NaCl (1% w/w) and invert five times to mix gently.Add acetone (5 mL), invert five times, and disrupt the mixtures by ultrasonication for 30 minutes.Add hexane (2 mL) and invert five times.The resulting mixtures stood for 5 minutes at room temperature, then centrifuged at 8,000 rpm for 5 minutes to obtain the hexane extracts, which were evaporated under reduced pressure.The residue was dissolved in acetonitrile (300 μL) and filtered through a 0.25 μm filter for gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) analysis.Squalene purchased from Aladdin; China was used as standard.
For squalene quantitative analysis, acetonitrile extract (20 μL) was loaded onto an HPLC system (Agilent 1260 infinity, California, United States) with UV detection at 210 nm.For chromatographic separation, an XB-C18 column (250 mm Â 4.6 mm, 5 μm) was used.The mobile phase consisted of 2% water and 98% acetonitrile.The flow rate was 1.0 mL/min and the column was held at 40°C during the separation.The peak area was converted into squalene concentration according to a standard curve plotted with a set of known concentrations of squalene.

Results
Squalene production of engineered strains in this study is listed in ►Table 3.

Screening of Highly Active Squalene Synthase
In this study, SQSs from different species were employed for the heterologous production of squalene in E. coli. 9CrtN was used to convert squalene into carotenoids with yellow pigment. 12lasmid p35151, containing genes of the MVA pathway, was introduced into BL21(DE3).Plasmids pET-KN, pET-NN, pET-thN, and pET-YN were introduced separately into an engineered strain containing p35151, yielding strains N1, N2, N3, and N4 (►Fig.2).
After a 48-hour culture, the carotenoid pigment of each strain showed that N4 had a higher carotenoid production, indicating that YSS had a higher activity.
Strain N4 was cultured at 30°C and 37°C, respectively, and the carotenoid production was compared.The result showed that YSS performed much better at 37°C (►Fig. 2).

SQS Functional Expression
Plasmids p35151 and pET-YSS were both introduced into BL21(DE3), yielding strain H1 (►Fig. 3).After cultured for 48 hours with 0.1 mmol/L IPTG induction, the production of squalene reached 18.9 mg/L.The result from GC-MS analysis showed that the structure of squalene produced by strain H1 was consistent with the standard (►Fig.4).

Increase in the Supply of Precursor FPP
Plasmids p35151 and pET-IAY were both introduced into BL21(DE3), yielding strain H2 (►Fig. 3).Strain H2 overexpressed idi and ispA based on strain H1, and the production of squalene reached 69.3 mg/L.

Discussion
Genes idi and ispA were the key enzymes to produce FPP, and overexpressing the two genes could increase squalene production in strain H1 (18.9 mg/L) and H2 (69.3 mg/L).In strain H3, ispH was overexpressed and contributed to a DMAPP accumulation in cells, which has been proven to be harmful to cell culture and can decrease terpenoid production in previous studies. 12,22,23In strain H3, squalene production was indeed decreased to 50.4 mg/L in comparison to strain H2 (69.3 mg/L), and this may be due to the accumulation of DMAPP.
The MEP pathway is naturally present in E. coli, and yields IPP 1.48-fold higher than the MVA pathway. 24,25Improving the metabolic flux of the MEP pathway may be helpful in squalene production.dxs was the key enzyme in the MEP pathway.By overexpressing dxs, the production of lycopene increased by 3.5-fold in engineered strain. 17Evidence suggests that Gene ispG favors the production of lycopene and isopentenol. 26,27Thus, in this study, dxs and ispG were both overexpressed in E. coli, as a result, squalene production in strain H4 (298.3 mg/L) was significantly increased in comparison to strain H2 (69.3 mg/L).
Overexpression of ispG leads to the accumulation of intermediate hydroxymethylbutenyl diphosphate, which seriously interferes with the synthesis machinery of nucleotide and protein in E. coli. 16Li et al found that balanced activation of ispG and ispH could push the carbon flux away from methylerythritol cyclodiphosphate and increase β-carotene and lycopene titer by 73 and 77%, respectively. 16Thus, in this study, ispG was constructed in plasmid pMEP-DG (low copy number, medium-strength promoter lac) and ispH was constructed into plasmid pET-HIAY (high copy number, strong promoter T7) to balance their expression; as a result, squalene production in strain H5 (472.3 mg/L) was elevated obviously in comparison to strain H4 (298.2 mg/L).
The MVA pathway could be divided into an upstream portion (MVU, contains atoB, hmgs, hmgr) and a downstream portion (MVD, contains mk, pmk, pmd, idi).Li et al showed that increasing the expression strength of MVD and decreasing the expression strength of MVU could contribute to the production of IPP and DMAPP. 28In this study, genes of MVU were constructed into the backbone of pBBR1MCS-1 with P15A origin ($10 copy in E. coli), generating plasmid pMVA1, while genes of MVD were constructed into the backbone of pBBR1MCS-2 with pBBR1 origin ($19 copy in E. coli), generating plasmid pMVA2.By replacing plasmids p35151 with pMVA1 and pMVA2, squalene production in strain H6 increased to 617.8 mg/L in comparison to strain H1 (18.9 mg/L), and in strain H7 increased to 974.3 mg/L in comparison to strain H2 (69.3 mg/L).
Katabami et al obtained 230 mg/L of squalene in XL1-Blue, which was higher than other engineered E. coli strains. 9,29Thus, we replaced the chassis cell from BL21 (DE3) to XL1-Blue, and encouragingly, the production of squalene reached 1,274 mg/L.
The other strategies that may increase the production of squalene were further discussed.Squalene is a nonpolar lipid and is stored in the cell membrane of E. coli.Therefore, enlarging the cell membrane area may be beneficial in the accumulation of squalene in E. coli. 30In addition, screening the key enzymes of MEP and MVA pathways of different species helps optimize the two pathways, and in turn, influences the subsequent production of squalene.Also, rational-design engineering could improve the biochemical properties of enzymes, including kinetic behaviors and substrate specificity, 31 and fusing upstream and downstream enzymes may make their activities more efficient. 32Besides, glycerol was a potential carbon source for the production of terpenoids in E. coli. 33Thus, constructing a glycerol-utilization pathway in E. coli by introducing genes like aldehyde reductase (alrd) and aldehyde dehydrogenase (aldH) may make it possible to improve the production of squalene and cut the cost. 34

Conclusion
This study demonstrated that the production of squalene could be improved by the co-expression of MVA and MEP pathways and the introduction of a highly active SQS.The production of squalene was also facilitated through the use of promotors of different strengths and the adjustment of the genes' copy number of key enzymes in the MEP and MVA pathways.In this study, based on XL1-Blue, a strain  with squalene production of 1274 mg/L was constructed.This study also presented several promising strategies to produce squalene in E. coli, helping squalene biosynthesis through a potential low-cost and highly effective production source.

Fig. 2
Fig. 2 Screening for SQS activity by colorimetric detection.(A) The SQS genes from different species and the crtN gene are cloned into plasmid pETDuet-1.(B) The colorimetric detection of squalene produced in the strain.(C) Plasmid p35151.SQS, squalene synthase.

Fig. 4
Fig. 4 HPLC and GC-MS analysis for squalene produced by strain H1. (A) Standard curve plotting to quantify squalene yield.(B) HPLC of squalene produced by strain H1.The peak of the sample overlaps with the standard.(C) GC-MS analysis of squalene produced by strain H1.GC-MS, gas chromatography-mass spectrometry; HPLC, high-performance liquid chromatography.

Fig. 5
Fig. 5 Optimization of MVA pathway.(A) The MVA pathway genes included in plasmid p35151 were separated into two parts and cloned in two plasmids.(B) Plasmids in strains H6 and H7.(C) Average yield of squalene in strains H1-H7.MVA, mevalonate pathway.

Fig. 6
Fig. 6 Effect of chassis cell on squalene production.(A) Plasmids used in XL1-Blue.(B) Plasmids in strains XH3, XH4, and XH5.(C) Average yield of squalene in strains constructed in this study.

Table 3
The squalene production of engineered strains in this study