Lycopene production from glucose, fatty acid and waste cooking oil by metabolically engineered Escherichia coli

Journal Pre-proof Lycopene Production from Glucose, Fatty acid and Waste Cooking Oil by Metabolically Engineered Escherichia coli Na Liu (Investigation) (Writing - original draft) (Writing - review and editing), Bo Liu (Investigation) (Writing - original draft) (Writing review and editing), Gaoyan Wang (Writing - review and editing), Ya-Hue Valerie Soong (Writing - review and editing), Yong Tao (Conceptualization) (Methodology), Weifeng Liu (Supervision) (Writing - review and editing), Dongming Xie (Supervision) (Conceptualization) (Methodology) (Writing - review and editing)

PII:

S1369-703X(20)30003-6

DOI:

https://doi.org/10.1016/j.bej.2020.107488

Reference:

BEJ 107488

To appear in:

Biochemical Engineering Journal

Received Date:

24 September 2019

Revised Date:

26 December 2019

Accepted Date:

3 January 2020

Please cite this article as: Liu N, Liu B, Wang G, Soong Y-HueV, Tao Y, Liu W, Xie D, Lycopene Production from Glucose, Fatty acid and Waste Cooking Oil by Metabolically Engineered Escherichia coli, Biochemical Engineering Journal (2020), doi: https://doi.org/10.1016/j.bej.2020.107488

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Lycopene Production from Glucose, Fatty acid and Waste Cooking Oil by Metabolically Engineered Escherichia coli

Na Liu*1, Bo Liu*2,3, Gaoyan Wang2, Ya-Hue Valerie Soong1, Yong Tao2,3, Weifeng

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Liu**2 [email protected], Dongming Xie**1 [email protected]

Department of Chemical Engineering, University of Massachusetts Lowell, One

CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State

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University Avenue, Lowell, MA 01854, USA.

Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy

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of Sciences, NO.1 Beichen West Road, Chaoyang District, Beijing 100101,

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P.R.China.

University of Chinese Academy of Sciences, NO.19A Yuquan Road, Shijingshan

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Destrict, Beijing100049, P.R.China

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These two authors contributed equally.

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Corresponding authors: Dongming Xie (Tel: +1-978-934-3159; ); Weifeng Liu (Tel:

+86-10-6480-7798; ).

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Graphical abstract

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Highlights:

Biosynthesis of lycopene from metabolically engineered Escherichia coli

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Capable of using free fatty acids or waste cooking oil to improve lycopene yield

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Co-feeding with glucose and waste cooking oil to produce 2.7 g/L lycopene in 40 h

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Lycopene content in biomass (94 mg/g) is among the highest levels reported so far

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Abstract Lycopene is an antioxidant that can be used as functional food or a high-value product

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for nutraceutical, pharmaceutical, and cosmetic applications. In this paper, the mevalonate and lycopene synthesis pathways were first built in Escherichia coli to

produce lycopene from hydrophilic substrates such as glucose and glycerol. Further

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introducing the fatty acid transport system in the E. coli strain led to improved

synthesis of lycopene from hydrophobic substrates such as fatty acids. In 1-L fed-

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batch bioreactors, the engineered E. coli FA03-PM produced 2.7 g/L lycopene within

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40 hours in fermentation co-fed with glucose and hydrolyzed waste cooking oil (WCO). This is the first study that reports high lycopene production by fermentation

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with fatty acids or waste cooking oils. In addition, the lycopene content in biomass

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reached 94 mg/g, which is among the highest levels reported so far in engineered E. coli for lycopene production. The research results will also help build a new

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biomanufacturing platform for making more high-value products from the common agriculture commodities, oils and fats, or their wastes.

Keywords: lycopene, metabolic engineering, Escherichia coli, fatty acid, waste cooking oil, fed-batch fermentation. 3

1. Introduction Lycopene is an unsaturated acyclic carotenoid with 11 conjugated double bonds. Due to the special molecular structure, its antioxidant capacity is 100 times higher than that of α-tocopherol [1]. Lycopene imparts series of potential health benefits as a biological antioxidant [2]. Epidemiological studies have shown that lycopene is

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associated with a decreased risk of oxidative stress, cardiovascular disease, hypertension, cancers and diabetes [3]. Consequently, lycopene is widely used in the

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pharmaceutical, functional food, and cosmetic industries [4].

Lycopene is produced by direct plant extraction, chemical synthesis, or microbial

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fermentation. In nature, lycopene is abundantly found in red-colored fruits and

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vegetables such as tomato, watermelon, pink grapefruit, apricots, pink guava and papaya [3]. As a major source of lycopene, tomatoes contain lycopene ranges from 30

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to 140 μg/g [5]. Currently, lycopene from plants does not meet the market needs due to the low content of lycopene in plants and high-cost of extraction process. Chemical

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synthesis for lycopene production is complex and involves using hazardous materials

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[6]. Recent developments in metabolic engineering and synthetic biology have allowed the cost-effective production of lycopene by microorganisms [4,5,7-9]. Microbial fermentation is viewed as a potential solution to meet the high demand for lycopene [10-12]. In the microbial process, lycopene synthesis starts with the formation of the universal five-carbon (C5) isoprenoid precursors isopentenyl

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diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are then condensed to form farnesyl pyrophosphate (FPP, C15) catalyzed by FPP synthase, encoded by ispA in E. coli, after which FPP is converted by geranylgeranyl diphosphate (GGPP) synthase, phytoene synthase, and phytoene desaturase, encoded by crtE, crtB, and crtI, respectively, to successively form GGPP (C20), colorless phytoene (C40) and finally the red-colored lycopene, which have high commercial

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values as nutraceuticals and cosmetic products [13-16]. There are two known

pathways for the biosynthesis of the two isoprenoid precursors IPP and DMAPP: the

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methylerythritol phosphate (MEP) pathway initiated from pyruvate, and

glyceraldehyde 3-phosphate (G3P) and the mevalonate (MVA) pathway using acetyl-

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CoA as precursor [17]. Escherichia coli possesses native MEP pathway for the

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biosynthesis of isoprenoids. Because the native MEP pathway is not sufficient for lycopene production, the supply of IPP and DMAPP should be enhanced by either

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improving MEP pathway or introducing heterologous MVA pathway during

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metabolic engineering study [18-25].

In this paper, we pay particular attention to MVA pathway because it can be more

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efficient without a need for complex native regulation (Fig.1). Furthermore, acetylCoA, the precursor of MVA pathway, can be supplied from various kinds of biomass feedstocks. Fatty acids (FAs) are promising alternative feedstock to replace glucose. FAs can be obtained from various cheap feedstocks, such as palm FA distillate (PFAD) and waste cooking oil. So far almost all the literatures use glucose as the 5

substrate to produce lycopene [27-30]. In the present study, we also investigated the production of lycopene from fatty acids. Fatty acids could provide acetyl-CoA precursor for MVA pathway without carbon loss. Furthermore, it is also a source to form lipid bodies in E. coli to serve as a lycopene storage pool. Lipid bodies can be formed in E. coli cells [45], and the accumulation can be improved by importing extracellular fatty acids. However, most microbial strains cannot efficiently utilize

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fatty acids. The metabolic pathways of fatty acids utilization in E. coli must be systematically engineered to utilize fatty acids efficiently [31].

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In addition to introducing MVA pathway and the corresponding biosynthesis genes,

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fatty acid transportation system and metabolic pathway are also constructed in E. coli to achieve the bioconversion of fatty acids into lycopene. Specifically, the fadR

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repressing the fad (fadL, fadD, fadE, fadBA and fadJI) regulon was deleted, and fadD

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responsible for transport and further consumption of exogenous long-chain fatty acid (LCFA) was overexpressed [15]. The biosynthetic pathway of lycopene in the fatty

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acid-utilization E. coli (FA03-PM) is shown in Fig.1.

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After the E. coli strains were constructed, fed-batch fermentation experiments were conducted to test the strain’s capability of lycopene production under high celldensity condition, compare the use of glucose, fatty acids and hydrolyzed wasted cooking oil for lycopene production, and further improve lycopene production by optimizing fermentation conditions.

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2. Materials and Methods 2.1 Strains

Both E. coli strains R122 and FA03-PM were constructed for biosynthesis of lycopene. R122 uses glucose and FA03-PM uses either glucose, fatty acids, or a combination of both for lycopene production. The E. coli strains DH5α and

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BW25113/F′ were used as host strains for plasmid construction and lycopene biosynthesis, respectively. The detailed illustration of strain designation was

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described in the recent study [32]. All strains and plasmids used are listed in Table 1.

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The following genes were used in this study: pmk: phosphomevalonate kinase gene (GenBank accession number NM001182727.1); mvd: diphosphomevalonate

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decarboxylase gene (GenBank accession number NM001183220.1); mvaE2: acetoacetyl-CoA thiolase/3-hydroxy-3-methylglutaryl-CoA reductase gene (GenBank

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accession number AF290092.1); mvaS: hydroxymethylglutaryl-CoA synthase gene

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(GenBank accession number AF290092.1); mvaK: mevalonate kinase gene (GenBank accession number MH084473.1); crtE: geranylgeranyl diphosphate synthase

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gene (GenBank accession number HE663493.1); crtB: phytoene synthase gene (GenBank accession number M90698.1); crtI: phytoene desaturase gene (GenBank accession number M90698.1); idi: IPP isomerase gene (GenBank accession number NM001183931.1); fadR: fatty acid degradation regulator

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gene (GenBank accession number NP415705.1); fadD: long-chain-fatty-acid-CoA ligase gene (GenBank accession number NP416319.1).

2.2 Test tube bioconversion for lycopene production

To evaluate lycopene production capacitiy of each strains, test tube bioconversion assay was carried out. Cells were collected after being cultured for 16 hrs in auto-

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induction medium [33]. The collected cells were suspended in 5 mL (OD600=30) reaction mixtures containing M9 FA medium or M9 glucose medium [34]. The

2.3 Flask Batch Culture Protocol

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bioconversion reactions were performed at 37 °C in a 200-rpm shaker for 8 hrs.

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Seed Culture: The seed vails of strains stored at -80℃ were thawed for 15 min at

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room temperature. The seed culture inocula was prepared by transferring 1 mL vial solution to a 250-mL flask containing 30 mL seed culture medium, which consisted of

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Sigma yeast extract (YE) (5.0 g/L), BactoTM peptone (PPT) (10.0 g/L), NaCl (10.0

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g/L), streptomycin (50.0 μg/mL), and kanamycin (50.0 μg/mL). The seed cells were grown in flask for 4-5 h at 37℃, 280 rpm in a New Brunswick G25 Shaker Incubator until an OD600 of 1-3 was reached.

Flask Culture: One and half milliliter of the seed cultures from shake flasks were transferred to each 250-mL shake flask containing 50 mL fresh culture medium to

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initiate the flask culture. The flask culture medium contained peptone (10.0 g/L), Sigma yeast extract (5.0 g/L), MgSO4 (0.12 g/L), NaH2PO4 (0.35 g/L), KH2PO4 (0.34 g/L), NH4Cl (0.27 g/L), D-Glucose (0.50 g/L), glycerol (5 g/L) or glycerol (2.5 g/L) and oleic acid (OLA, 1.25 or 2.5 g/L), and trace metals I (100×) (1 mL/L). The trace metals I (100×) stock solution contained EDTA (840.0 mg/L), CuSO4∙5H2O (220.0 mg/L), MnCl2∙4H2O (1500.0 mg/L), CoCl2∙6H2O (250.0 mg/L), H3BO3 (300.0 mg/L),

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Na2MoO4∙H2O (250.0 mg/L), Zn(CH3COO)2 (1300.0 mg/L) and ammonium iron(III) citrate (10.0 g/L). The trace metals solution was filter-sterilized through 0.22 𝜇m

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sterile membrane and stored at 4 ℃. Glucose (0.5 g/L) in the culture medium was only used for cell growth, since a small amount of glucose may cause catabolite

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repression via PBAD promoter interaction. When glucose was almost depleted (OD600

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4-5), arabinose (200 g/L in stock solution) was added to a final concentration of 2 g/L in the flask medium to initiate the production phase. For the R122 strain, glycerol (5

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g/L) was used as the production substrate. For FA03-PM strain, the mixed substrate containing glycerol (2.5 or 5 g/L) and oleic acid (1.25 or 2.5 g/L) was used for

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lycopene production. The total carbon in each flask was kept at 2.15 ± 0.05 g C/L

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medium. All the substrates were added before inoculation. The surfactant of Brij-58 was also added to a final concentration 0.1% (v/v) when fatty acid was used. The flask culture cells were grown in flask for 30-50 h at 37℃, 280 rpm in a New Brunswick G25 Shaker Incubator.

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2.4 Fed-Batch Fermentation Protocol

Two-stage seed culture: The first-stage seed culture was carried out as introduced in the flask culture protocol. When the first stage culture reached OD600 3-4, 1 mL of the seed culture was transferred to a 250-mL flask containing 30 mL fresh seed culture medium to grow for 3~4 h at 37℃, 280 rpm in a New Brunswick G25 Shaker

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Incubator until an OD600 of 1.5~2.5 was reached. The second-stage seed culture was used to inoculate the 1-L fermentor at 5% (v/v).

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Fed-Batch Fermentation: The second-stage flask seed culture (30 mL, OD = 1.5~2.5) was transferred to a 1-L fermentor (Biostat B-DCU, Sartorius, Germany) to initiate

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the fermentation (t = 0 h). The initial fermentation medium was 600 mL and

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contained citric acid (1.7 g/L), MgSO4 (0.60 g/L), KH2PO4 (14.0 g/L), (NH4)2HPO4 (4.0 g/L), D-Glucose (20.0 g/L), trace metals I (100×) (10 mL/L), and antifoam (Poly

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(propylene glycol) monobutyl) (Sigma, America) (1.0 mL/L). The dissolved oxygen (measured by pO2) level of the fermentation experiments was set at 30% of air

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saturation by cascade controls of agitation speed between 500 and 1200 rpm, gas flow

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rate between 0.3 lpm and 0.6 lpm, and pure O2 enrichment (if needed). The temperature was maintained at 37℃ throughout the run. The pH value was maintained at 7.0 throughout the run by feeding ammonium solution (28-30%). Carbon source (glucose, glycerol, and/or oleic acid/hydrolyzed waste cooking oil)

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feeding commenced when initial glucose was depleted, which was indicated by rapid increases in pO2 levels and decreases in agitation speed.

For R122 strain, glucose (600 g/L) was used as the feeding substrate. For FA03-PM strain, single substrate glucose (600 g/L) or oleic acid, dual substrates with both glucose and pure oleic acid or glucose and hydrolyzed waste cooking oil were used

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for feeding. The waste cooking oil (french-fried canola oil obtained from a local restaurant) was hydrolyzed to free fatty acids by mixing with isometric pH 7.2 sodium phosphate buffer (0.1 M), which contains 1% MP Biomedicals lipase (w/v), at 37 ℃

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for 48 h. After 48 h, around 63% of oil was converted to free fatty acids, and the oil

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and fatty acids layer was collected by centrifugation. In addition, a pre-feed solution containing MgSO4 (1M) (3 mL) and trace metals II (100x) (6 mL) was added to the

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fermentor when feeding started. The trace metals II (100×) stock solution contained EDTA (1300 mg/L), CuSO4∙5H2O (370.0 mg/L), MnCl2∙4H2O (2350.0 mg/L),

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CoCl2∙6H2O (400.0 mg/L), H3BO3 (500.0 mg/L), Na2MoO4∙H2O (400.0 mg/L),

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Zn(CH3COO)2 (1600.0 mg/L) and ammonium iron(III) citrate (4.0 g/L). The trace metals solution was filter-sterilized through 0.22 𝜇m sterile membrane and stored at 4

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℃.

Glucose concentrations were maintained at limited levels (< 0.1 g/L) during the fedbatch fermentation by using the pre-set feeding profile. For a glucose fed-batch fermentation with a maximum cell density between 30-40 g/L, the following glucose

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feeding profile was typically used: 6 g/L/h, 0 h (time of initiating the feeding); 7.2 g/L/h, 2 h; 10 g/L/h, 4 h; 11.3 g/L/h, 6 h; 12 g/L/h, 8 h; 14 g/L/h, 10 h. For feeding fatty acid or a mixed substrate, the total carbon fed during the fed-batch fermentation experiments were kept the same as for glucose feeding. The feeding profile may be adjusted based on off-line measurements of cell densities and residual glucose concentrations. To induce lycopene biosynthesis, arabinose (300 g/L in stock

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solution) was added to a final concentration of 2 g/L in the medium at the late stage of

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the exponential growth phase (OD600 = 60-70).

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2.5 Analyses of Fermentation Samples

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Fermentation samples (~12 mL for each) were removed six times a day (3~10 h intervals). In general, 1.0 mL fresh broth sample was quickly frozen and stored at -

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20℃ for lycopene analysis, 0.1 mL was used for OD600 analysis, and 5 mL was used

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for determination of dry cell weight (DCW).

To measure DCWs, cells from 5 mL of culture were harvested by centrifugation at

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4,300 g for 5 min and washed three times with distilled water. The cells were dried for 12-24 h at 95℃ until a constant weight reached and weighed to calculate DCWs. The product lycopene was identified by LC-MS/MS (liquid chromatography–mass spectrometry) with a variable wavelength detector set to 474 nm [29]. And the lycopene production was quantified by using a spectrophotometric assay using 12

absorbance at 474 nm [28]. To determine the lycopene content of the cells, 100 μL of 1:100 diluted fermentation broth was harvested by centrifugation at 4,300 g for 5 min and washed once with water. The following lipid extraction method was adapted from Sun et al. [29]. The cells were suspended in acetone (1.0 mL) and incubated at 55 ℃ for 15 min in the dark. The samples were centrifuged at 4,300 g for 10 min, and the acetone supernatant containing lycopene was transferred to a clean tube. The

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lycopene content of the extracts was quantified by measuring the absorbance at 474 nm using a Genesys 20 spectrophotometer and calculated based on a lycopene

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standard curve (lycopene titer (the concentration of lycopene in fermentation broth)

g/L = A474 × dilution ratio × 4.5). The results were reported as the mean from two

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independent determinations. The standard deviations were in the range of 10% of the

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means.

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3. Results and Discussion 3.1 Construction of lycopene producing strain R122

In our previous work, an optimized lycopene-biosynthesis module has been developed using OLMA assembly method, and lycopene can be produced with a yield of 15.17 mg/g DCW in a MEP pathway-enhancement strain [35]. However, a foreign MVA

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pathway is necessary because only acetyl-CoA can be supplied from fatty acids. Thus, to develop the lycopene producing strain using MVA pathway, a MVA chassis E. coli

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strain was used. In our previous work, a foreign MVA pathway has been introduced

into E. coli BW25113 and optimized for the efficient production of isoprene [36]. The

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MVA pathway was divided into two parts (Fig. 2A): up module and down module.

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The up module includes genes mvaE, mvaS, and mvk. Down module includes genes pmk and mvd. Using this chassis strain, lycopene can be produced with a yield of 22.1

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mg/g DCW (M122 strain), but biomass was reduced. However, the lycopene production was very low when using native MEP pathway (W122) (Fig.2). To further

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improve lycopene production and relieve the suppression of cells growth, several

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modifications of MVA module were made. The up module was constructed into a low-copy-number plasmid pLB1s under the strong arabinose-inducible araBAD promoter (resulting pLE2SK). The down module was expressed under the constitutive 119 promoter and was inserted to the lpxM site in the chromosome. Then the resulting strain G01 was introduced the plasmid pLY122 which contains the lycopene

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biosynthesis genes cluster to generate the lycopene production strain R122. The lycopene production of strain R122 was first studied by using the test tube bioconversion assay. After bioconversion using glucose as substrate for 8 hrs, a specific lycopene production of 24.3 mg/g DCW was obtained in the E. coli biomass (Fig. 2C).

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3.2 Lycopene production of R122 using glucose as substrate

Lycopene production of R122 was further optimized using glucose as substrate at

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both flask and 1-L bioreactor scales. The flask culture was carried out with glucose

and glycerol as the carbon source. Arabinose (200 g/L in stock solution) was added as

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the induction agent at around 5 h (OD600 4~5) to a final concentration of 2 g/L in the

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medium. As shown in Fig. 3A and Table 2, a DCW of 3.0 g/L, a lycopene titer of 0.15 g/L and a specific titer (the concentration of lycopene in biomass) of 51.0 mg/g DCW

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were obtained.

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Cell growth and lycopene production of the R122 strain was further studied in a 1-L bioreactor with the fermentation medium (glucose was the only substrate) as

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described in the fed-batch fermentation protocol. Glucose feeding commenced when initial glucose was depleted at around 10 h. The lycopene fed-batch process was a typical two-phase process, which started with a growth phase for the first 20-24 h and then followed by a production phase for the rest of the run, in which lycopene production was initiated by adding arabinose at OD600~60 to a final concentration of 15

2 g/L in the medium. As shown in Fig. 3 (B-D) and Table 2, a DCW of 20.8 g/L, a specific titer of 14.7 mg/g DCW and a lycopene titer of 0.31 g/L were obtained at 40 h. Compared with the specific titer of 51.0 mg/g achieved in the flask culture with a rich medium containing 5.0 g/L yeast extract and 10.0 g/L peptone, the specific titer obtained in bioreactor was significantly lower. We believe that the tight control of glucose levels in the fed-batch fermentation is critical to lycopene production by the

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strain R122.

When glucose was used as the substrate, a major challenge was to maintain the high

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efficiency of the inducible araBAD promoter, which is sensitive to glucose

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metabolism related factors, such as cAMP level [37]. The promotor is typically repressed by high glucose levels. In shake flask experiments, the initial glucose (0.5

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g/L) was completely consumed and used for cell growth only. Subsequently, glycerol

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(2.5~5.0 g/L) was used as the carbon source to support additional cell growth and energy maintenance in the stationary phase. Then the promoter was turned on and

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lycopene could be more efficiently produced.

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In the fed-batch bioreactors, glucose was the sole carbon source used. Too high glucose levels may repress the lycopene biosynthesis, but too low glucose levels may also limit cell growth and provide insufficient energy maintenance to support lycopene biosynthesis. In the 1-L fed-batch experiments, we used a pre-determined glucose feeding profile that was designed for E. coli fermentation with cell densities

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between 30-40 g/L, as described in the experimental protocols. Due to unknown reasons, strain R122 grew slower and only reached cell densities between 14-21 g/L. The unexpected lower cell densities caused slow-down consumption rates and slightly increased the glucose accumulation. The increased glucose accumulation due to slowdown cell growth repressed lycopene biosynthesis and led to poor specific lycopene titer (14 mg/g) in bioreactor as compared to what was achieved in shake flasks (51

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mg/g). A more tightly controlled, glucose-limiting feeding strategy based on real-

time cell densities should be developed in future so that the lycopene production by

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the R122 strain can be significantly improved under fed-batch bioreactor conditions.

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To accelerate cell growth and glucose consumption rates, 5 g/L yeast extract and 10.0 g/L peptone were added to the fermentation medium in the next experiment. As

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shown in Fig. 3 (B-D), supplementing with yeast extract and peptone in the medium

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was only able to improve the growth rate in the early stage, but failed to improve the overall cell density and lycopene production. Therefore, missing important nutrients

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from the yeast extract or peptone was not the major reason accounting for the low

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lycopene titer and productivity in fermentor for strain R122.

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3.3 Construction of fatty acid utilization strain FA03-PM for lycopene production

To obtain fatty-acids utilization strains for lycopene production, strain G01 was further modified by chromosomal deletion of the fadR gene, resulting in strain F01. Then fadD gene was further overexpressed under strong constitutive CPA1 promoter,

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resulting in strain F02. After introduction of pL122 into F01 and F02, lycopene production strains FA02-PM and FA03-PM were obtained, respectively. Hence, the difference between strains FA02-PM and FA03-PM is that the fadD gene in FA03-

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PM was overexpressed. Then lycopene production of FA02-PM and FA03-PM was

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determined using the fatty acid (oleic acid) as carbon resource during the test tube

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bioconversion assay.

The results (Fig. 2 - B) showed that FA02-PM and FA03-PM produced lycopene at

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specific titers of 29 mg/g and 33 mg/g DCW, respectively, from oleic acid (C18:1 FA), which were much higher than that of R122 (17.44 mg/g DCW) from glucose. At

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the same time, the specific titer of lycopene of both FA02-PM and FA03-PM from

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glucose were 24 mg/g DCW, similar to that of R122. The lycopene production for FA02-PM and FA03-PM from glucose was slightly lower than from the fatty acid, which needs to be further investigated under fed-batch bioreactor conditions.

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3.4 Lycopene production of FA03-PM from single substrates in 1-L bioreactors

3.4.1 Using oleic acid as the single substrate

The cell growth and lycopene production of strain FA03-PM in 1-L bioreactor were investigated by feeding with oleic acid (C18:1 FA) only. The minimal fermentation medium and process control conditions were kept the same as the fermentation for the

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R122 strain. The inducing reagent, arabinose (300 g/L in stock solution), was also

added at the late stage of the exponential growth phase (OD600 ~ 70). As shown in

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Fig. 4 and Table 2, a DCW of 30 g/L, a lycopene titer of 1.41 g/L, and a specific titer of 47 mg/g DCW were obtained at 40 h. Compared with the R122 strain under the

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same conditions but with glucose substrate, the DCW was 44% higher, and the

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lycopene titer and specific titer were also improved by 4 folds and 2 folds, respectively (Fig. 4). In addition to the possible glucose overfeeding in the R122

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fermentation, another possible reason for the improvement from FA03-PM with oleic acid was due to the non-repression effect of the fatty acid on the araBAD promoter,

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which can be easily subject to catabolite repression when glucose is present.

3.4.2 Using glucose as the single substrate

Strain FA03-PM was originally constructed for lycopene production from fatty acids. However, it is still interesting to compare the efficiency of using glucose and fatty acid as the carbon and energy source for biosynthesis of lycopene. The glucose fed19

batch fermentation procedure for FA03-PM was similar to that for strain R122, except that the feeding profile was more carefully adjusted. The adjustments were based on off-line measurements of cell density and glucose so that glucose was controlled at almost undetectable levels. To our surprise, the FA03-PM produced much more lycopene from glucose than from the oleic acid. As shown in Fig. 4 and Table 2, the DCW of FA03-PM reached 28 g/L with a specific lycopene titer of 84 mg/g, which

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led to a final lycopene titer of 2.37 g/L at 40 h. The specific titer for lycopene

production of FA03-PM using limited feeding of glucose was almost doubled as

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compared to the run using fatty acid only (47 mg/g). It was also 5 times higher than

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that of strain R122 using glucose (14 mg/g) in 1-L fed-batch fermentation.

It seemed that the glucose metabolism profile of FA03-PM was modified by deletion

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of fadR and/or enhancement of fadD. To verify this observation, the induced R122

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and FA03-PM cells were harvested and re-cultured in shake flasks to compare their glucose consumption rates. As shown in Fig. 4 D, the glucose consumption rate of

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FA03-PM was about 40% higher than that of R122. Meanwhile, Fig. 4 B shows that the specific lycopene titer of FA03-PM increased more rapidly and significantly after

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the inducer L-arabinose was added as compared to the fermentation with R122.

There are also three other possible reasons that account for higher specific lycopene titer from glucose than from oleic acid for strain FA03-PM in the fed-batch experiments. First, the catabolism of fatty acids in the current E. coli strain is still not

20

efficient enough to provide energy maintenance and to support lycopene biosynthesis as compared to the glucose metabolism. The fatty acid transport and catabolism via βoxidation may need to be further improved in strain FA03-PM (Fig. 1). Second, the efficiency of mixing and mass transfer of extracellular fatty acid in the bioreactor may need to be improved [38]. Since fatty acid is insoluble in aqueous medium and lighter than water, it tends to stay in the top region of the bioreactor. Optimization of

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bioreactor design and operation should be considered in the future to facilitate the

extracellular mixing and mass transfer to improve the overall lycopene biosynthesis

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from fatty acids. Third, oleic acid (C18:1 FA) was selected as the fatty acid example in the experiments due to availability and low melting point (easy to be fed as liquid

re

at room temperature), but it may not be the most efficient fatty acid substrate for

na

lP

biosynthesis of lycopene.

3.5 Lycopene production of FA03-PM from mixed substrate in 1-L bioreactors

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Although currently the strain FA03-PM produced more lycopene from glucose than

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from the C18:1 fatty acid, it has been reported in many literatures that increasing lipid levels in cells can help solubilize the produced hydrophobic carotenoids in the intracellular lipid bodies so that the overall production can be improved [39]. Therefore, we further investigated the strategy of using a mixed substrate containing both glucose and fatty acids (or hydrolyzed oils) in the fermentation.

21

3.5.1 Using a mixed substrate containing glucose and oleic acid

In this fermentation experiment, pure oleic acid and 600 g/L glucose were co-fed into the fermentor at 1:1 carbon ratio after the initial glucose (20 g/L) was consumed. As shown in Fig. 5 and Table 2, the DCW increased from 28 g/L to 32 g/L. It seemed that the fatty acid in the mixed substrate indeed improved the cells’ tolerance of

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lycopene, and thus led to a slightly higher biomass yield. However, the current strain and process were still not optimized for fatty acid uptake and bioconversion, which

led to a slight decrease in specific lycopene titer from 84 mg/g to 77 mg/g though the

-p

overall titer was slightly improved from 2.37 g/L to 2.46 g/L (Table 2). Compared

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with the fermentation that was fed with only glucose, there was no significant improvement in the overall lycopene production by using a glucose/oleic acid

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fed with only oleic acid.

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mixture, but the results were much better than what were achieved in the fermentation

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3.5.2 Using a mixed substrate containing glucose, oleic acid and yeast extract

In our previous study [34] it was found that the fatty acid uptake might be the limiting

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step during the fatty acid utilization which then limits the accumulation of intracellular lipid levels. Addition of organic nitrogen sources such as yeast extract during the transformation phase could significantly promote the uptake of fatty acid [34]. Therefore, in our most recent fed-batch experiments, 15 g/L yeast extract was added to the glucose feed solution (600 g/L glucose + 15 g/L yeast extract). The new 22

glucose feed solution and the oleic acid were co-fed at 1:1 carbon ratio into the 1-L bioreactor for lycopene production. As shown in Fig.6 and Table 2, feeding yeast extract improved the specific titer from 77 mg/g to 86 mg/g while the DCW remained the same. This also led to a higher lycopene titer of 2.74 g/L, about 12% improvement over the previous run without yeast extract.

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3.5.3 Using a mixed substrate containing glucose, waste cooking oil and yeast extract

Since feeding a pure fatty acid such as oleic acid is not economically attractive to a

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commercial production process, the hydrolyzed waste cooking oil was used to replace the oleic acid to co-feed with glucose and yeast extract into the fermentor. It was

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found that the cell density slightly decreased, but specific titer of lycopene in biomass

lP

(Fig. 5 and Table 2) was enhanced. After 40 h fermentation, a DCW of 28 g/L, a lycopene titer of 2.65 g/L and a specific titer of 94 mg/g DCW were obtained. No

na

significant inhibition in cell growth and lycopene production was observed when

ur

WCO substrate was used.

So far the highest lycopene production levels among all published literatures were

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achieved by Sun et al. [29], who reported a lycopene titer of 3.5 g/L, a specific titer of 51 mg/g, and a volumetric productivity of 0.032 g/L/h. Our engineered E. coli strain FA03-PM demonstrated significantly higher specific lycopene titers (up to 94 mg/g) and volumetric productivities (~0.070 g/L/h) under 1-L fermentation conditions, but

23

the titers (~2.7 g/L) will need to be improved in future by increasing cell densities under further optimized fed-batch fermentation conditions.

In addition, we observed that the fermentation run with the waste cooking oil had much less foaming compared with other runs with a pure fatty acid. This is because the residual triglycerides in the waste cooking oil can partially serve as an antifoam.

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The use of waste cooking oil not only reduces the raw material cost for lycopene production, but also makes the fermentation process more controllable and scalable.

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The overall progress of lycopene production by the engineered E. coli strain was

summarized in Fig. 6. An 8-fold improvement of lycopene production was achieved

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by introducing the fatty acid metabolism pathway and the fermentation optimization.

lP

Currently, sugars, especially glucose, are the dominant carbon sources or substrates used for the biomanufacturing of fuels, chemicals, industrial enzymes,

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biopharmaceuticals and many other high-value products. At the same time, the global

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production of plant oils from oil crops (palm, soybean, and etc.) and rendered animal fats are more than 200 million tons each year, which is more than the total annual

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production of sugars [40]. Oils and fats are mainly used for food and feed application and generates low or limited economical values. In addition, nearly million tons of waste cooking oils and fats are being generated every year during the food applications, which are typically used for biodiesel [41, 42], bioplastics, or other chemical productions [43]. At the same time, a significant portion of waste oils and 24

fats are released to the environment without appropriate treatments and causes serious pollutions [44]. To significantly improve the economical values of original or wasted oils and fats, we have investigated and demonstrated the feasibility of using fatty acid, especially from the waste cooking oil, as the major or partial substrate for lycopene production. Our research covered both strain and fermentation engineering studies,

products from any hydrophobic substrates in future.

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and the results will provide guidance for biomanufacturing of many other high-value

In terms of the strain engineering, we focus on using E. coli as the host for lycopene

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production though there have been many literatures reporting to use other

re

microorganisms such as Corynebacterium glutamicum, Pichia pastoris, Yarrowia lipolytica, Blakeslea trispora, and Saccharomyces cerevisiae for production of

lP

carotenoids at high titers. E. coli may not be referenced as the best host strain for

na

carotenoid production by other researchers, but it still remains to be a very competitive option [7-9]. We chose E. coli in this study based on the overall cost

ur

analysis. The fermentation cost for a specific product not only depends on the product titer, but also depends on the raw material cost, the conversion yield, and the

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productivity. Many other microbial strains such as the oleaginous yeast Yarrowia lipolytica have been reported to produce carotenoids at high titers [39]. However, those strains usually require much longer fermentation time (a week or longer) to achieve high titers, which leads to relatively lower productivities. Here we chose E. coli strains to significantly shorten the fermentation time (40 hours or less) and 25

achieve high productivities with comparable titers. Using waste cooking oil to partially or completely replace glucose not only improves the carotenoid biosynthesis efficiency, but also further reduces the fermentation cost contributed by the raw materials. We believe our results may help to pave the way for future’s large scale production of many other high-value products derived from the MEP and MVA

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pathways by using metabolically engineered E. coli strains.

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4. Conclusion

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In this study, Escherichia coli was metabolically engineered to produce lycopene from both hydrophilic and hydrophobic substrates. After being introduced the fatty

lP

acid transport system and the pathways for MVA and lycopene synthesis, the new strain FA03-PM demonstrated production of lycopene from glucose, fatty acids, waste

na

cooking oils, and different combinations of them. In 1-L fed-batch fermentation

ur

within 40 h, FA03-PM produced 2.7 g/L lycopene with 94 mg/g in the E. coli biomass. The lycopene content in biomass is among the highest levels reported so far

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in all engineered bacteria for lycopene production. This study provides an economically attractive strategy for biomanufacturing high-value products from the common agriculture commodities, oils and fats, or their wastes.

26

Credit Author Statement

Na Liu: Investigation – Fermentation Engineering; Writing- Original Draft, Reviewing and Editing

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Bo Liu: Investigation – Strain Engineering; Writing- Original Draft, Reviewing and Editing

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Ya-Hue Valerie Soong: Reviewing and Editing

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Gaoyan Wang: Reviewing and Editing

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Yong Tao: Conceptualization; Methodology

Weifeng Liu: Supervision; Writing – Reviewing and Editing

Jo

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and Editing

na

Dongming Xie: Supervision; Conceptualization; Methodology; Writing- Reviewing

Declaration of Competing Interest None. 27

Acknowledgements The research at UML was supported by the UML New Faculty Start-Up funding. The authors would like to thank the Massachusetts Biomanufacturing Center at UML for providing the facilities for fermentation experiments. The research at CAS did not

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receive any specific grant from funding agencies in the public, commercial, or not-

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na

lP

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for-profit sectors.

28

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in expression of fatty acid transport genes, PLoS One, 7 (2012) e46275.

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[40] L. Honig (2018) November Crop Production USDA https://www.nass.usda.gov/Newsroom/Executive_Briefings/2018/11-08-

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2018.pdf

[41] W. Du, W. Li, T. Sun, X. Chen, D. Liu, Perspectives for biotechnological production of biodiesel and impacts, Appl Microbiol Biotechnol, 79 (2008) 331337. [42] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biodiesel production with immobilized lipase: A review, Biotechnol Adv, 28 (2010) 628-634. 33

[43] J.H. Song, C.O. Jeon, M.H. Choi, S.C. Yoon, W. Park, Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2, J of Microbiol Biotechnol, 18 (2008) 1408-1415. [44] A.A. Refaat, Different techniques for the production of biodiesel from waste vegetable oil, Int. J. Environ. Sci. Tech., 7 (2010) 183-213. [45] L. Xu, L. Wang, X.-R. Zhou, W.-C. Chen, S. Singh, Z. Hu, F.-H. Huang, X. Wan, Stepwise metabolic engineering of Escherichia coli to produce

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triacylglycerol rich in medium-chain fatty acids. Biotechnol. Biofuels, 11 (2018)

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177. doi:10.1186/s13068-018-1177-x.

34

Table 1. Strains and plasmids used in this study. Strain

Genotype

Source

E. coli BW25113/F′ rrnBT14 ΔlacZWJ16 hsdR514

CGSC*

ΔaraBADAH33 ΔrhaBADLD78 [F′ proAB lacIqZΔM15 Tn10 (Tetr)] E. coli BW25113/F’, ΔldhA::P119-pmk-mvd

G01

This study

ro of

(P119: 119 promoter [34]) F01

E. coli BW25113/F‫׳‬, ΔldhA::P119-pmk-mvd, ΔfadR

This study

F02

E. coli BW25113/F‫׳‬, ΔldhA::P119-pmk-mvd, ΔfadR,

This study

PCPA1-fadD

-p

(PCPA1: CPA1 promoter [34]) BW25113 carrying pLY122

This study

M122

BW25113 carrying pYESKs, pSKPMIc and pLY122

This study

R122

G01 carrying pLE2SK and pLY122

This study

FA03-PM

F01 carrying pLE2SK and pLY122

This study

Plasmid

Description

Source

pYESKs,

araBAD promoter, p15A origin, Strr, mvaE2-mvaS-

[36]

lP

re

W122

pSKPMIc

na

mvaK

araBAD promoter, pSC101 origin, Cmr, mvk-pmk-mvd-

[36]

ur

idi

pLE2SK

araBAD promoter, R6K origin, Strr, mvaE2-mvaS-

This study

Jo

mvaK

pLY122

Tac promoter, RSF1030 origin, Kanr, crtE-crtB-crtI-idi

[35], renamed from pLY116

*CGSC: Coli Genetic Stock Center.

35

Table 2. Summary of lycopene production by the metabolically engineered E. coli strain R122 and FA03-PM under shake-flask and bioreactor conditions. Substrate

DCW

Specific

Titer

(g/L)

titer (mg/g)

(g/L)

Shake flask

Glucose + Glycerol

3.0

51

0.15

Bioreactor

Glucose

21

14

0.30

Glucose + YE + PPT

14

7

0.10

Oleic acid

30

47

1.41

28

84

2.37

32

77

2.46

32

86

2.74

(1 L)

FA03-

Bioreactor

PM

(1 L)

Glucose

re

Glucose + oleic acid

lP

Glucose + oleic acid + YE

Jo

ur

na

Glucose + waste cooking oil + YE

36

28

ro of

R122

Experiment

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Strain

94

2.65

ro of -p re lP na ur Jo Fig. 1. An overview of the metabolic engineering strategy for E. coli to produce lycopene from glucose or fatty acids.

37

38

ro of

-p

re

lP

na

ur

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Fig. 2. A. Introduction of MVA pathway in E. coli R122 and FA03-PM strains. B. optimization of lycopene production strains. C. lycopene production of R122 and FA03-PM strains at test tube bioconversion level.

R122

200

Lycopene titer Specific titer

3

150

2

100

1

50

0 12

18 24 Time (h)

30

36

20 10 0 0

42

6

12

18

24

30

36

42

30

36

42

Time (h)

C 600

-p

D 40

Glc

Glc

Glc + YE + PPT

30

Glc + YE + PPT

re

400

Specific titer (mg/g)

6

Glc + YE + PPT

30

0 0

200

0 0

6

12

18

24

30

36

42

Jo

ur

na

Time (h)

lP

Lycopene titer (mg/L)

Glc

ro of

4

DCW (g/L)

B 40

250 DCW

DCW (g/L)

5

Lycopene titer (mg/L) Specific titer (mg/g DCW)

A

39

20

10

0 0

6

12

18

24

Time (h)

Fig. 3. Fermentation results of the glucose-utilization strain R122: (A) results in shake flask; (B)-(D) results of DCW, specific lycopene titer, and lycopene titer respectively in 1-L fed-batch bioreactor. Abbreviations: DCW, dry cells weight; Glc, glucose; YE,

B

40 R122, glc

DCW (g/L)

FA03-PM, glc

20

10

FA03-PM, OLA FA03-PM, glc

2000 1500 1000 500

0

0 6

12

18 24 Time (h)

C 100

30

36

42

0

FA03-PM, OLA FA03-PM, glc

na

60 40

0 0

ur

20

6

12

18

24

30

36

42

Glucose consumption rate (g/L/h)

R122, glc

lP

D

80

6

re

0

Specific titer (mg/g)

R122, glc

-p

FA03-PM, OLA

30

3000 2500

Lycopene titer (mg/L)

A

ro of

yeast extract; PPT, peptone.

Jo 40

18

24

30

Time (h)

3.5 3 2.5 2 1.5 1 0.5 0 R122

Time (h)

12

FA03-PM

36

42

Fig. 4. Comparison between fed-batch fermentation results of strain R122 and FA03PM using glucose, or oleic acid as substrate. (A) the DCW (g/L); (B) the specific titer (mg/g); (C) the lycopene titer (mg/L); (D) glucose consumption rate. Abbreviations:

A

50

ro of

DCW, dry cells weight; Glc, glucose; OLA, oleic acid.

OLA Glc Glc + OLA

-p

Glc + OLA + YE 30

Glc + WCO + YE

20

re

DCW (g/L)

40

0 0

6

12

18

lP

10

24

30

36

42

30

36

42

na

Time (h)

B 3500

OLA Glc Glc + OLA Glc + OLA + YE Glc + WCO + YE

ur

2500 2000 1500

Jo

Lycopene titer (mg/L)

3000

1000

500

0

0

6

12

18

24

Time (h)

41

C 120 OLA Glc

Specific titer (mg/g)

100

Glc + OLA 80

Glc + OLA + YE Glc + WCO + YE

60 40 20 0 0

6

12

18

24

30

36

42

ro of

Time (h)

Fig. 5. Comparison between fed-batch fermentation results of the strain FA03-PM

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using glucose, oleic acid, and various mixed substrates. A) the DCW (g/L); B) the

re

lycopene titer (mg/L); C) the specific titer (mg/g). Abbreviations: DCW, dry cells

Jo

ur

na

lP

weight; Glc, glucose; OLA, oleic acid; YE, yeast extract; WCO, waste cooking oil.

42

43

ro of

-p

re

lP

na

ur

Jo

Fig. 6. A schematic presentation of the strain and substrates used and the corresponding

Jo

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na

lP

re

-p

ro of

lycopene production achieved in this study.

44