Endocytosing Escherichia coli as a Whole

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Endocytosing Escherichia coli as a whole-cell biocatalyst of fatty acids Jonghyeok Shin, Jiwon Yu, Myungseo Park, Chakhee Kim, Hooyeon Kim, Yunjeong Park, Choongjin Ban, Seydametova Emine, Young-Ha Song, Chul-Soo Shin, Kyung Hwun Chung, Ji-Min Woo, Hyunwoo Chung, Jin-Byung Park, and Dae-Hyuk Kweon ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00519 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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ACS Synthetic Biology

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Endocytosing Escherichia coli as a whole-cell biocatalyst of fatty acids

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Jonghyeok Shin,† Jiwon Yu,† Myungseo Park,† Chakhee Kim,† Hooyeon Kim,† Yunjeong Park,†

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Choongjin Ban,†, ¶ Emine Seydametova,† Young-Ha Song,‡ Chul-Soo Shin,‡ Kyung Hwun

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Chung,§ Ji-Min Woo,‖ Hyunwoo Chung,‖ Jin-Byung Park,*,‖ Dae-Hyuk Kweon*, †, ¶, #

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†Department

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Bioengineering, Sungkyunkwan University, Suwon 16419, Republic of Korea

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‡AP

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§Electron

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South Korea

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‖Department

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Republic of Korea

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¶Biomedical

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Korea

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#Center

of Biotechnology and Bioengineering, College of Biotechnology and

Technology, 147 Gwanggyo-ro, Yeongtong-gu, Suwon 16229, Republic of Korea Microscope Facility, Dental Research Institute, Seoul National University, Seoul,

of Food Science and Engineering, Ewha Womans University, Seoul 03760,

Institute for Convergence, Sungkyunkwan University, Suwon 16419, Republic of

for Biologics, Sungkyunkwan University, Suwon 16419, Republic of Korea

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1 Environment ACS Paragon Plus

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Whole cell biocatalysts

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can be used to convert fatty acids into

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various value-added products. However,

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fatty acid transport across cellular

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membranes into the cytosol of microbial

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cells limits substrate availability and

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impairs membrane integrity, which in

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turn decreases cell viability and

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bioconversion activity. Because these problems are associated with the mechanism of fatty acid

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transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was

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generated to promote substrate endocytosis. Caveolin-1 (CAV1) expression in Escherichia coli

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increased both the fatty acid transport rate and intracellular fatty acid concentrations via

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endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated

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substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in

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dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction

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systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-

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hydroxyoctadec-9-enoic acid) to (Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that

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CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of

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hydrophobic substrates.

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KEYWORDS: Caveolin-1, Heterologous caveolae, Endocytosis, Biotransformation, Fatty acid

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INTRODUCTION

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Plant oils and fatty acids are some of the most abundant natural resources. Most of them are used

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as cooking oils but only a small proportion is used for biodiesel and biochemical production.1-2

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Currently, genetically engineered microorganisms are being strategically utilized to create value-

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added products with diverse biological and chemical functions from plant oils and fatty acids.3-8

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Biotransformation of hydrophobic substrates by whole-cell biocatalysts tends to be limited by

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low productivity and yield due to various reasons. First, hydrophobic substrates are often toxic to

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microorganisms.9-13 In particular, fatty acids act as toxins by damaging cell membranes and

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decreasing intracellular pH. Several attempts have been made to improve membrane integrity for

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better bioconversion efficiency.14-17 The second inhibitory factor is limitation in transport of fatty

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acids.18-19 Substrates should be delivered to the cytoplasm to initiate biotransformation in a

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whole-cell catalyst across two intrinsic barriers in E. coli: the outer membrane and the inner

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membrane. Larger hydrophobic substrates are restrained by hydrophilic lipopolysaccharides on

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the outer membrane of gram-negative bacteria,20-25 whereas small hydrophobic substrates readily

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diffuse into the cell. Surfactants and organic solvents may be used to increase the membrane

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permeability, thereby facilitating intracellular uptake of hydrophobic substrates.22, 26-27 However,

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membrane-permeabilizing agents may adversely affect cell membrane integrity, leading to

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reduced cell viability and metabolic activity.28-31

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As cytotoxicity and reduced metabolic activity are closely associated with the mass transfer of

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substrates across cell membranes, an elegant solution to this problem involves the use of

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transporters. Long-chain fatty acids are transported to the periplasmic space via the transporter

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FadL in E. coli.32-35 As the periplasmic space is slightly acidic, long-chain fatty acids become

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protonated, enabling partitioning to the cytoplasmic face of the inner membrane via a flip-flop



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mechanism.36 FadD, an inner membrane-associated long-chain acyl-CoA synthetase, renders the

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transport unidirectional via fatty acid esterification.32 Indeed, fatty acid bioconversion increases

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when FadL is expressed in E. coli.35 However, FadL overexpression can be toxic to cells and

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thus lead to reduction of bioconversion activity.35 In addition, AlkL, the outer membrane protein

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of Pseudomonas putida GPo1, which improves hydrophobic substrate uptake in E. coli,37 enables

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efficient -oxyfunctionalization of fatty acid methyl esters only after fine-tuning the AlkL

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expression level.38 It is likely that although a specialized transporter can reduce the cytotoxicity

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derived from passive diffusion to a certain extent, there is a trade-off between substrate uptake

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and metabolic activity. Thus, a more desirable transport system for hydrophobic substrates

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should facilitate intracellular substrate uptake without sacrificing the biotransformation activity.

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In this study, we developed a recombinant E. coli strain that facilitates the biotransformation of

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fatty acids and plant oils (e.g., ricinoleic acid (Figure 1), 10-hydroxyoctadecanoic acid

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(Supporting Information, Figure S1), and castor oil (Figure S2)) by internalizing substrates via

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endocytosis. Owing to endocytosis in E. coli, fatty acid-induced cytotoxicity was alleviated,

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whereas intracellular fatty acid concentration was maintained rather high, resulting in efficient

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biocatalysis of the fatty acids and plant oils into value-added products.

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RESULTS AND DISCUSSION

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Formation of caveolae in E. coli cytoplasm. Expression of human CAV1 in E. coli induces the

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formation of heterologous caveolae in the cytoplasmic membrane.39 The recombinant E. coli can

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incorporate the periplasmic medium and provide additional membranes for transmembrane

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protein incorporation.39-40 We hypothesized that endocytosis in E. coli may constitute a general


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uptake system for biotransformation of substrates, as eukaryotic endocytosis is a form of active

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transport for incorporating substances from the surrounding medium.

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To test the potential advantages of endocytosing E. coli as a whole-cell biocatalyst for fatty acid

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biotransformation (Figure 2a), heterologous caveolae formation was induced in E. coli by

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expressing CAV1. Two plasmids, pT7-His6-CAV1 and pT7-GST-CAV1, were constructed

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(Figure 2b), and CAV1 expression in E. coli was confirmed using western blot analysis with an

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anti-CAV1 antibody (Figure 2c). Although CAV1 is a membrane-bound protein, some CAV1 was

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present in the soluble fraction, suggesting that some caveolae were detached from the

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cytoplasmic membranes. When the E. coli cells were sectioned and observed using electron

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microscopy, a number of circular caveolae were detected near the cytoplasmic membrane

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(Figure 2d). Soluble caveolae were purified using Ni-NTA or GST-agarose affinity

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chromatography after cell lysis by mild sonication. The presence of CAV1 in the purified

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caveolae was confirmed again using western blotting with an anti-CAV1 antibody (Figure 2e).

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The average diameters of purified caveolae measured using dynamic light scattering

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spectroscopy were 120 nm and 200 nm for His6-caveolae and GST-caveolae, respectively

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(Figure 2f). The size variation between the two caveolae appeared to be due to dimerization of

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the GST protein 41-42. When purified caveolae were treated with Triton X-100 (TX-100), the

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caveola size decreased to 10–20 nm because the detergent disrupted the caveola membranes. It

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seems that some micelles were formed after the caveolae were broken by the detergent

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(Supporting Information, Figure S3a, b). Some aggregation and small disrupted particles were

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observed when proteinase K (PK) was added to degrade CAV1 on the caveolae (Supporting

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Information, Figure S3c, d). In conclusion, caveolae were successfully formed in E. coli



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expressing CAV1 fusion proteins, with a fraction of the caveolae separated from the cytoplasmic

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membrane (Figure 2a).

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Endocytosis of fatty acids. To investigate endocytosis during caveolae formation in E. coli,

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cells containing pT7-His6-CAV1 or pT7-GST-CAV1 were treated with 10 mM 5-6-

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carboxyfluorescein and 0.5 mM isopropyl -D-1-thiogalactopyranoside (IPTG). After incubation

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at 25°C for 3 h, free dyes were washed with tris-buffered saline (TBS), and cellular uptake of the

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fluorescent dyes was analyzed using fluorescence microscopy (Figure 3a and Supporting

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Information, Figure S4) and flow cytometry (Figure 3b). Both experiments clearly showed that

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compared to the control cells, caveolae-forming cells readily incorporated the fluorescent dyes

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supplemented in the medium. To investigate whether the endocytosing E. coli could import

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extracellular fatty acids, cells expressing CAV1 fusion proteins were treated with various

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concentrations of ricinoleic acid (RA) (Figure 1, 1). After incubation at 37°C for 1 h with

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shaking at 250 rpm, extracellular ricinoleic acid was washed with phosphate-buffered saline

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(PBS) and intracellular RA was extracted using ethyl acetate. The extracted RA was analyzed

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using gas chromatography. The amount of RA transported into the cells was proportional to the

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RA concentration in the medium. Compared to control cells, the intracellular RA concentration

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was increased by 33–114% when caveola formation was induced (Figure 3c). For instance,

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CAV1-expressing cells incorporated 2-fold more intracellular RA than control cells when 30 mM

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RA was supplemented in the medium. Furthermore, endocytosing cells incorporated RA faster

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than wild type cells, with a 33–50% higher uptake rate (Figure 3d). Finally, cell-tolerance to the

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fatty acid RA was measured. After treating cells with 20 mM RA for 3 h, colony-forming units

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were determined to compare fatty acid tolerance among different cell types. Only 45% of control

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cells were viable after RA treatment, whereas cells expressing His6-CAV1 or GST-CAV1 were


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more viable than the control cells (Figure 3e), and cells expressing GST-CAV1 retained almost

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100% viability after RA treatment. In conclusion, the caveolae-forming E. coli incorporated

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more RA than the control cells at an increased uptake rate, and fatty acid toxicity was mitigated,

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leading to maintenance of cell viability.

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Constitutive expression of CAV1. Plasmids that constitutively express His6-CAV1 and GST-

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CAV1 were constructed using the J23100 promoter (Figure 4a). The 5'-untranslated region (5'-

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UTR) sequences were designed using UTR designer43-44 to generate variants with varying

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expression levels (Figure 4b). Four plasmids pCW-His6-CAV1, pCS-His6-CAV1, pCW-GST-

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CAV1, and pCS-GST-CAV1, were designed using the UTR designer to strongly (pCS) or weakly

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(pCW) express His6- or GST-tagged CAV1 (Figure 4a). Western blotting with an anti-CAV1-

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antibody showed that cells expressing pCW-His6-CAV1 and pCS-His6-CAV1 showed distinct

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weak and strong CAV1 expression, respectively, whereas cells expressing pCW-GST-CAV1 and

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pCS-GST-CAV1 showed similar expression levels (Figure 4c). Caveolae were formed even when

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CAV1 was constitutively and weakly expressed (Figure 4d).

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Constitutive expression of CAV1 did not inhibit cell growth regardless of expression level in rich

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medium (Supporting Information, Figure S5). However, constitutive and strong expression of

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CAV1 from pCS-His6-CAV1 in Riesenberg medium dramatically impeded cell growth, whereas

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expression of pCW-His6-CAV1 did not (Figure 4e). Constitutive expression systems increased

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the RA uptake rate by 10–45%, which is comparable to that in inducible expression systems

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(Figure 4f). Intracellular RA concentration increased proportionally to the supplemented RA

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concentration regardless of CAV1 expression (Figure 4g). Consistent with the results obtained

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using inducible systems, E. coli strains constitutively forming caveolae incorporated more RA

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inside cells than that observed in control cells. Specifically, the intracellular concentration of RA



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containing pCS-His6-CAV1 was almost four-fold higher than that of the control cells when 30

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mM RA was supplemented in the medium (Figure 4g).

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Fatty acid cytotoxicity was also relieved in the constitutive caveolae-forming E. coli. When

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colony-forming units were measured after the cells were treated with 5 mM nonanoic acid (NA)

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or 20 mM RA, the caveolae-forming cells were more tolerant to the toxic fatty acids, whereas

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~50% of the control cells were dead (Figure 4h). This was also confirmed by cell growth curves

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in the presence of NA or RA (Figure 4i). Cell growth was inhibited by NA and RA. However,

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this inhibition was dramatically mitigated by constitutive CAV1 expression (Figure 4i). As

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slowly growing or non-growing cells are less sensitive to toxic chemicals including antibiotics

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one of the possible scenarios is that the tolerance was simply a result of growth inhibition by the

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formation of micro-compartments. However, cells that constitutively express CAV1 grew well in

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rich media, similar to the WT cells (Supporting Information, Figure S5). Moreover, the cells

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containing pCW-His6-CAV1 showed increased tolerance (Figure 4h) to RA and NA although

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cell growth was not retarded in R medium (Figure 4e). The caveolae-forming cells not only

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survived the toxic fatty acid treatment (Figure 4h), but also grew better than WT cells in the

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presence of toxic fatty acids (Figure 4i). These results suggest that the caveolae-forming E. coli

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did not merely maintain cell viability due to slowed growth. In conclusion, E. coli that

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constitutively formed caveolae rapidly transported more fatty acids into the cells compared to

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control cells while alleviating fatty acid-induced cytotoxicity.

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Biotransformation of RA is enhanced by endocytosing E. coli. (Z)-11-(Heptanoyloxy) undec-

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9-enoic acid (Figure 1, 3), denoted as ‘Ester’ in this paper, is a versatile platform chemical that

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can be enzymatically converted into medium chain fatty acids (e.g., n-heptanoic acid and 11-

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hydroxyundec-9-enoic acid, 1,11-undecanedioic acid, and 11-aminoundecanoic acid3-6).


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Thereby, biotransformation of RA into the Ester (3) was used as a model reaction to demonstrate

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the versatility of endocytosing E. coli as a whole-cell biocatalyst.

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The plasmid pAPTm-E6BVMOopt/ADH, which constitutively expresses the ADH of

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Micrococcus luteus and the BVMO of Pseudomonas putida KT2440,4, 45 was introduced together

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with either of the CAV1-encoding plasmids. GST-CAV1 and His6-CAV1 were expressed in the

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presence of pAPTm-E6BVMOopt/ADH (Supporting Information, Figure S6). Endocytosing cells

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(containing pT7-His6-CAV1 or pT7-GST-CAV1) and control cells showed similar results in

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biotransformation efficiency when 15 mM RA was used as substrate (data not shown). Hence,

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the biotransformation activity of the endocytosing E. coli cells was investigated in a cell-recycle

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reaction system. The recombinant E. coli cells, which had been used for RA conversion, were

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used again for the next round of biotransformation (Figure 5a, b). The toxic reactants, including

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the Ester product (3), which may inhibit cofactor-dependent whole-cell biotransformation,3, 12, 46

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were extracted and removed from the reaction medium using adsorbent resins. The Ester

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formation profile in the first round of RA conversion was not significantly different between the

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control E. coli (Figure 5a) and the caveolae-forming E. coli (Figure 5b). However, the Ester

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formation rate of the caveolae-forming E. coli was maintained in the second round of

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biotransformation, whereas the rate was reduced by ~50% in the control cells. The final Ester

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concentration from the second biotransformation by the caveolae-forming E. coli reached 9.6

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mM in the reaction medium, which was approximately 2-fold higher than that with the control E.

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coli biotransformation. This result suggests that the caveolae-forming E. coli was more stable

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and active in terms of RA conversion than the control E. coli.

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Conversion of RA at high cell density. Next, biotransformations of RA at higher concentrations

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were investigated. When 30 mM RA was contained in the reaction medium, the control cells



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allowed to produce 7 mM Ester, whereas the caveolae-forming cells produced 13–17 mM target

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product (Supporting Information, Figure S7). Although the caveolae-forming cells showed

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approximately 2-fold greater biotransformation efficiency compared to that of the control cells,

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the conversion remained below 60% in all cases. The low value could be due to low cell density

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(OD600 nm of 8, where OD600 nm ~ 0.31 g DCW/L) in the reaction medium. Hence, we next

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performed biotransformation of RA in a fermenter after high cell density cultivation. RA

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biotransformation was initiated by adding 150 mM RA into the culture medium when the OD600

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nm

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the end of the reaction, the caveolae-forming E. coli produced approximately 50 mM Ester,

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which was almost 1.6-fold higher compared to the control cells. In addition, repeated fed-batch

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reaction type biotransformations were performed by feeding 100 mM RA every 6 h (Figure 6c

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and d). In the first reaction, 100 mM RA was converted to almost 90 mM Ester both in the

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control and caveolae-forming cells. However, the Ester production by the control cells was

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dramatically decreased when 100 mM RA was fed for the second reaction, whereas the Ester

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production by the caveolae-forming cells was rather maintained during the second reaction,

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resulting in a production of the Ester to 90 mM (Figure 6d). These results suggest that the

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caveolae-forming E. coli was able to maintain their biotransformation activities throughout the

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reactions at high cell density.

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Biotransformation of 10-hydroxyoctadecanoic acid and castor oil by the endocytosing E.

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coli. Bioconversion of a saturated fatty acid and a plant oil was conducted to confirm whether the

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caveolae-forming E. coli-based biocatalyst can be applied to biotransformation of other

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substrates. First, castor oil biotransformation (Supporting Information, Figure S2) was carried

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out in the culture medium of recombinant E. coli cells by adding 5 g/L castor oil, 0.1% (v/v)

of cells reached 100 (Figure 6a and b). While the control E. coli has produced 30 mM Ester at


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Tween80, and 20 U/mL lipase from Thermomyces lanuginose. The castor oil was completely

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hydrolyzed within 1 h, generating glycerol and fatty acids (e.g., RA, octadecanoic acid,

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hexadecenoic acid). Glycerol was consumed as a carbon source whereas RA was converted to n-

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heptanoic acid (Figure S2, S9) and 11-hydroxyundec-9-enoic acid (S10) via the Ester (S8) by the

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recombinant cells and the added lipase (Figure S2 and Figure 7a, b). Notably, the toxic reaction

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intermediate (i.e., 12-ketooctadec-9-enoic acid (S7)) was accumulated to approximately 1.5 mM

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in the reaction medium when E. coli BL21 (DE3) pAPTm-E6BVMOopt/ADH was used as the

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biocatalyst (Figure 7a). On the other hand, the intermediate concentration remained below 0.5

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mM with the caveolae-forming E. coli-based biocatalysts (Figure 7b). The concentration of n-

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heptanoic acid and 11-hydroxyundec-9-enoic acid was increased up to 10.3 mM, which was

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approximately 60% higher as compared to the concentration reached by E. coli BL21 (DE3)

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pAPTm-E6BVMOopt/ADH.

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When 10-hydroxyoctadecanoic acid (S2), which had been produced from oleic acid (S1)

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(Supporting Information, Figure S1) as previously described 35, 47, was used as the reaction

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substrate instead of RA, the whole-cell biotransformation profiles appeared similar to each other

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(Figure 7c, d). However, the toxic reaction intermediate (i.e., 10-ketooctadecanoic acid (S3)) was

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accumulated in the culture medium and reached 0.8 mM after 9 h with E. coli BL21 (DE3)

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pAPTm-E6BVMOopt/ADH. In contrast, the reaction intermediate concentration remained below

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0.3 mM when the endocytosing E. coli was used as a biocatalyst (Figure 7d). Moreover, co-

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expression of CAV1 allowed the recombinant E. coli-based biocatalysts to produce the Ester (i.e.,

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9-(nonanoyloxy)nonanoic acid (S4)) to a concentration of up to 2.6 mM, which was

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approximately 30% greater compared to the concentration reached with E. coli BL21 (DE3)

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pAptm-E6-BVMOopt/ADH. All the results indicated that co-expression of CAV1 allowed the



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recombinant E. coli-based biocatalysts to better maintain the fatty acid biotransformation

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especially with BVMO reaction activities under the process conditions.

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Fatty acid uptake by E. coli expressing CAV1 and/or FadL. FadL is the fatty acid transporter

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of the E. coli outer membrane. Controlled expression of FadL improves fatty acid

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biotransformation.35 Fatty acid uptake by cells expressing FadL, CAV1, or both proteins was

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compared (Supporting Information, Figure S9). While the cells were incubated with 20 mM RA

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for 60 min, the changes in intracellular RA concentration was measured. Fatty acid uptake was

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increased about 2-fold by expression of FadL or CAV1. However, we could not observe an

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additional increase in the fatty acid uptake by expressing both proteins. Although it is difficult to

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determine the rate-limiting step at present, it is likely that co-expression of both CAV1 and FadL

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needs to be optimized for synergetic uptake because overexpression of FadL is toxic to cells. The

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relationship between mass transfer through the outer membrane by FadL followed by

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endocytosis by caveolae deserves further investigation for maximized substrate uptake and

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biotransformation.

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Bioconversion of a substrate to a value-added product by a whole cell catalyst requires substrate

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uptake into the cytosol. Sugar transport varies depending on the host cell type; however, host

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cells can be engineered with specialized transporters to facilitate specific sugar uptake.48 Small

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apolar substrates are easily transported into cells via passive diffusion even though this results in

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cytotoxicity. However, large hydrophobic substrates such as long-chain fatty acids are not easily

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transported into cells as they are restrained by hydrophilic lipopolysaccharides on the outer

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membrane of biocatalysts.20-25 Organic acids, ionic liquids, and surfactants have been used to

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improve the hydrophobic substrate uptake by reducing the outer membrane permeability

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barrier.22, 26-27 However, these strategies severely reduce cell viability and metabolic activity.28-31


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Biocatalysts engineered to exhibit improved outer membrane integrity may show increased cell

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viability and productivity.14-17 Recombinant transporters that traverse the lipopolysaccharide

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layer can also be employed to improve the transport efficiency of hydrophobic molecules inside

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E. coli without significantly affecting the outer membrane integrity. For example, AlkL, an

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alkane transporter from Pseudomonas putida Gpo1, can transport aliphatic alkanes (C7-C16) as

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well as fatty acid methyl esters into E. coli cells.37, 49 Expression of FadL, a fatty acid transporter

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of the E. coli outer membrane, improves the biotransformation of various fatty acids.35 However,

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facilitated influx of fatty acids into the periplasm via transporters improves substrate uptake and

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amplifies reactant-mediated cytotoxicity as passive diffusion across the inner membrane directly

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reduces cell viability. These transporters have been shown to increase the biotransformation

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efficiency only when their expression levels are optimal because of a trade-off between cell

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viability and substrate uptake. Passive diffusion of hydrophobic substrates including fatty acids

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across cytoplasmic membranes is toxic to host cells as they damage the cell membrane and lower

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the pH inside the microorganism.9-13 Acidification of the cytosol promotes the beta-oxidation

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pathway of fatty acids,50 which reduces the product yield and the productivity of whole cell

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biocatalysts of fatty acids. However, the mechanism by which substrates are imported across the

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inner membrane without losing metabolic activity remains unknown. In the present study, we

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showed for the first time that an E. coli biocatalyst incorporates apolar substrates inside cells

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efficiently while maintaining biocatalytic activity when the cells are engineered to endocytose

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substrates.

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The endocytosing E. coli may find useful applications in addition to the biotransformation of

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various apolar substrates. First, hydrophobic products formed inside cells by metabolic

287

engineering deserve further investigation. Value-added products such as terpenoids, which are



288

versatile for cosmetics or pharmaceuticals 51, may damage the cell membrane causing leakage

289

and reduced productivity 52-53 because they are incorporated into the cell membrane during

290

formation. As caveolae-forming E. coli provide more space for membrane-anchoring products, it

291

may be expected that the product-induced cytotoxicity may be alleviated resulting in elevated

292

productivity. Second, the endocytosing E. coli may provide a chance to offer an efficient

293

bioremediation system. Because the environmental problems of crude oil spills have become

294

more serious, studies on the bioremediation of lipophilic hydrocarbons are being actively

295

conducted 54. Bacterial bioremediation of hydrocarbons using bacteria has great potential

296

compared to physical and chemical methods 55. However, microbial bioremediation is

297

insufficient to for areas contaminated at high levels because of cellular toxicity 56. Because the

298

caveolae-forming E. coli were more tolerant to toxic substrates and efficiently transported fatty

299

acids digested directly from oils into the cells (Figure 7), it is likely that the bacterial endocytosis

300

system may enlarge the toolbox available for the bioremediation of environmental pollution 56.

301

A central hydrophobic domain of CAV1 forms a hairpin structure within the membrane, exposing

302

both the N- and C-terminal domains to the cytoplasm. Both N- and C-terminal domains interact

303

to form homo-oligomers of CAV1. Oligomerized CAV1 molecules generate the proteinaceous

304

coat of caveolae. Heterologous caveolae formed inside E. coli are likely to be generated through

305

the same mechanism as in mammalian cells. Fatty acids transported to the periplasm may diffuse

306

to the small invaginated area called caveolae during this step. Perhaps, the dynamin-like activity

307

of an unknown protein in E. coli mediates the final budding of vesicles from the plasma

308

membrane, during which fatty acids in the periplasm are trapped in the endosome. Previous

309

analysis has shown that the formation of heterologous caveolae begins inside E. coli after only

310

30 min of inducing CAV1 expression 39. The E. coli cytoplasm can be filled with circular


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311

caveolae after 90 min of induction with an approximate half-life of 12 h. Thus, fatty acids

312

contained in the caveolae may move to the cytosol either (1) after breakdown of caveolae or (2)

313

via passive diffusion across the caveolae membrane. Although it is not clear which of the

314

pathways is dominant, both pathways likely contribute to the increased uptake and tolerance.

315

Passive diffusion across the caveolae membrane will be less toxic to the cells because it happens

316

outside the plasma membrane. It is probable that recycling of fatty acid-containing vesicles is

317

faster than that of the empty vesicles. Thus, we expect that inclusion of fatty acids in the

318

endosome and movement of fatty acids to the cytosol independent of the plasma membrane

319

constitutes the basic principles of increased tolerance at higher intracellular concentrations.

320 321

CONCLUSIONS

322

We used a eukaryotic transport system to induce bulk transport of fatty acids across the inner

323

membrane of E. coli and to overcome the cytotoxicity caused by passive diffusion. E. coli

324

expressing CAV1 formed caveola-like structures and endocytosed externally supplemented

325

substrates. The endocytosing cells rapidly incorporated more fatty acid substrates compared to

326

the control cells. Fatty acid cytotoxicity was alleviated by endocytosing the substrate, resulting in

327

improved biocatalyst performance. Thus, we propose that the endocytosing E. coli described in

328

the present study represents a versatile tool for whole cell biocatalysis of hydrophobic substrates.

329 330

MATERIALS AND METHODS

331

Chemicals and materials. Ricinoleic acid (RA, 12-hydroxyactadec-9-enoic acid) and N-methyl-

332

N-(trimethylsilyl) trifluoroacetamide (TMS) were purchased from Tokyo Chemical Company



333

(Tokyo, Japan). Antibiotics, isopropyl β-D-1-thiogalactopyranoside (IPTG), trace elements for

334

culture medium, and ethyl acetate were purchased from Sigma-Aldrich (ST. Louis, MO, USA).

335

The anti-CAV1 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).

336

Lipase from Thermomyces lanuginose was purchased from GenoFocus (Daejeon, Korea).

337

Plasmid construction. All cloning was performed using a ligation-independent cloning method

338

and T4 DNA polymerase (New England Biolabs, Ipswich, MA, USA). CAV1 was obtained from

339

Addgene (Genbank accession number 403980). The plasmid pT7-His6-CAV1 was constructed by

340

inserting CAV1 cDNA into pColAduet-1 (Merck Millipore, Billerica, MA, USA). The plasmid

341

pT7-GST-CAV1 was constructed by replacing the His6 tag of pT7-His6-CAV1 with glutathione S

342

transferase (GST). GST was amplified from pGex4T-1 (GE Life Sciences, Pittsburg, PA, USA).

343

Plasmids (pCW-His6-CAV1 and pCS-His6-CAV1) for constitutive expression of CAV1 were

344

constructed by replacing the T7 promoter of pT7-His6-CAV1 with the bacterial constitutive

345

promoter J23100 (5′-ttgacggctagctcagtcctaggtacagtgctagc-3′, identifier: BBa_J23100,

346

http://parts.igem.org/Promoters/Catalog/Anderson). Two types of 5′ UTRs were used to control

347

CAV1 expression: 5′-tactagggtaccagaaagaggagaaatactag-3′ for weak expression and 5′-

348

ttaactttaagaaggagatatacat-3′ for strong expression.

349

Purification of heterologous caveolae. E. coli cells containing pT7-His6-CAV1 or pT7-GST-

350

CAV1 were cultured in Terrific broth. Protein expression was induced by adding 0.5 mM IPTG

351

at 25°C. After overnight culture, E. coli cells were lysed by sonication (25% amplitude, cycles of

352

4 s ON and 4 s OFF for 90 s). Cell debris and the insoluble fraction were removed by

353

centrifugation at 10,000  g for 30 min. The supernatant was incubated with Ni-NTA agarose

354

(Qiagen, Valencia, CA, USA) or glutathione-agarose (Sigma-Aldrich, ST. Louis, MO, USA)

355

with rotation at 4°C. The unbound fraction was washed with 10 column volumes of PBS (137


Page 16 of 41

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356

mM NaCl, 2.7 mM KCl, 10 mM Na2SO4, 1.8 mM KH2PO4, pH 7.4). Caveolae were eluted with

357

PBS containing 150 mM imidazole or PBS containing 300 mM reduced glutathione. Eluted

358

caveolae were concentrated using Amicon ultra centrifugal filters with a 100 kDa molecular

359

weight cut-off (Merck Millipore, Billerica, MA, USA).

360

Analysis of fluorescence dye transport. The dye transport assay was performed according to

361

published methods.39-40 CAV1-expressing cells were cultured in Terrific broth at 37°C overnight.

362

Precultured cells (1%) were inoculated in Terrific broth and cultured at 37°C until the optical

363

density (OD) reached 0.3. Then, 5-6-carboxyfluorescein (10 mM) was added and cultured for an

364

additional 1 h. Next, 0.3 mM IPTG was added and incubated for 3 h at 25°C. Unencapsulated

365

residual dye was washed off with cold Tris-buffered saline (TBS, 50 mM Tris-HCl, 150 mM

366

NaCl, pH 7.4) until a transparent supernatant was obtained, and the cells were then observed

367

using a fluorescence microscope and flow cytometer.

368

Analysis of fatty acid transport. Cells were cultured in Riesenberg medium (4 g/L

369

(NH4)2HPO4, 13.5 g/L KH2PO4, 1.7 g/L citric acid, 1.4 g/L MgSO4, and 10 mL/L trace metal

370

solution [10 g/L FeSO4, 2.25 g/L ZnSO4, 1.0 g/L CuSO4, 0.5 g/L MnSO4, 0.23 g/L Na2B4O7, 2.0

371

g/L CaCl2, and 0.1 g/L (NH4)6Mo7O24])57. After 2 h of induction with 0.5 mM IPTG in the

372

stationary phase, the solution was titrated to pH 8.0 with NaOH. After incubating the cells with

373

RA, extracellular fatty acids were washed with PBS. Fatty acids transported into the cells were

374

extracted using ethyl acetate and analyzed using gas chromatography.

375

Whole-cell biotransformation. Bioconversion of RA (Figure 1), 10-hydroxyoctadecanoic acid

376

(Supporting Information, Figure S1), and castor oil (Figure S2)) was performed following

377

previously published methods.45, 57-59 The pAptm-E6BVMOopt/ADH vector45 and one of the



378

pACYC-based CAV1-encoding plasmids were used for simultaneously expressing ADH,

379

BVMO, and CAV1. Cells transformed with both plasmids were pre-cultured in the presence of 50

380

µg/mL kanamycin and 34 µg/mL chloramphenicol in Luria-Bertani (LB) broth. Cells that were

381

pre-cultured overnight were cultivated at 37°C in Riesenberg medium60 with 10 g/L glucose as a

382

carbon source until they reached the early exponential phase, after which the temperature was

383

lowered to 25°C. The cells were then cultured at this temperature until they reached the

384

stationary phase. Cells in the early stationary phase were titrated with NaOH, pH 8.0. After

385

titration, bioconversion was performed by adding 15 mM RA, 5 mM 10-hydroxyoctadecanoic

386

acid, or 5 g/L castor oil and 0.1% (v/v) Tween80 to cells in the stationary phase at 35°C with

387

shaking at 200 rpm (cell density: 3 g dry cells/L). When bioconversion of RA was performed

388

using pT7-GST-CAV1 or pT7-His6-CAV1, 0.3 mM IPTG was added 2 h before the reaction with

389

RA.

390

High density cell cultivation. High density cell cultivation was performed based on previous

391

experiments.45 Cells were cultured in Riesenberg medium at 30C in a laboratory-scale (5 L)

392

bioreactor. Cells were grown batch-wise until the initial 20 g/L glucose was consumed. When the

393

initial glucose was consumed and the pH exceeded 6.9, a mixture of glucose (600 g/L) and

394

MgSO47H2O (20 g/L) was added as an acid in the pH stat mode. pH was maintained at 6.9 while

395

28% ammonia solution was added as a base. Dissolved oxygen tension (DOT) was maintained at

396

40% by adding pure oxygen into the bioreactor during fed-batch cultivation. Bioconversion was

397

launched by supplying 100150 mM RA into the culture broth. During the bioconversion

398

process, glucose and pure oxygen supply was stopped, and the pH and temperature of the

399

bioreactor was maintained at pH 8.0 and 35C, respectively.45


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400

Biotransformation in a cell-recycle system. RA biotransformation using recombinant E. coli

401

was performed according to the methods described above. After approximately 90% of the initial

402

reaction substrate was depleted, the reactants and the product (Z)-11-(heptanoyloxy) undec-9-

403

enoic acid (Ester), were recovered by mixing with Diaion HP-20 resin (5%, w/v) for 1 h at 20°C

404

and 80 rpm. After separation of the resin, the recombinant E. coli culture broth was reused for

405

the next round of RA biotransformation. The second biotransformation was initiated by adding

406

15 mM RA to the reaction medium.

407

Analytical method. Concentrations of RA, the intermediate 12-ketooctadec-9-enoic acid, and

408

the final product (Z)-11-(heptanoyloxy) undec-9-enoic acid (Ester) were analyzed following

409

previously published protocols.4, 45 Two volumes of ethyl acetate were added to the reaction

410

medium. Methyl palmitate (0.5 g/L) was used as the internal standard. After vortexing, the

411

organic solvent layer was separated by centrifugation and reacted with TMS. The TMS

412

derivatives of fatty acids were separated using a non-polar capillary column (length 30 m,

413

thickness 25 m, HP-5MS, Agilent Technologies, Palo Alto, CA, USA) and analyzed using gas

414

chromatography (Younglin, Ansan, Korea). The temperature of the column was changed from

415

90°C to 255°C at a rate of 5°C/min and was maintained at 255°C. The temperature of the injector

416

was 260°C, whereas that of the detector was 250°C.

417

Sample preparation for transmission electron microscopy (TEM) analyses. TEM analysis

418

was performed following a previously established procedure. 61-62 Briefly, samples were first

419

fixed with 4% paraformaldehyde, washed in PBS, and post-fixed in 1% osmium tetroxide in 0.1

420

M PBS. The specimens were dehydrated using a 70% to 100% graded ethanol series, exchanged

421

with propylene oxide, and embedded in a mixture of Epon 812 and araldite. Ultrathin sections

422

(70 nm) were cut using a Leica EM UC6 ultramicrotome. A ribbon of serial ultrathin sections



423

from each sample was collected on copper grids and stained with uranyl acetate and lead citrate.

424

Serial fields were photographed at 30,000100,000× magnification using a ZEOL EM-

425

1200EX11 electron microscope operated at 80 kV.

426

Statistics. No statistical method was used to define the sample size in advance. The experiment

427

and analysis did not involve randomization or blinding. Error bars represent the standard error of

428

the mean from at least three independent replicates. Graph Pad Prism 5 (Graph Pad Software

429

Inc., San Diego, CA) was used for statistical analysis of the results. To determine the statistical

430

significance of comparisons, two-tailed t-tests were used. The p-value was considered significant

431

at less than 0.05.

432 433

ASSOCIATED CONTENT

434

Supporting Information

435

Table S1. Primers used in this study. Figure S1. Designed biotransformation pathway 2. Figure

436

S2. Designed biotransformation pathway 1. Figure S3. Dynamic light scattering analysis of

437

purified caveolae treated with 0.1% (w/v) Triton X-100 (TX-100) or 2 g/mL Proteinase K (PK).

438

Figure S4. Bright field images of cells (same as shown in Figure 3a) treated with 5-6-

439

carboxyfluorescein. Figure S5. Growth of the caveolae-forming cells in Terrific broth. Figure S6.

440

SDS-PAGE and western blot analysis of ADH, BVMO, and CAV1 co-expressed in E. coli BL21

441

(DE3) cells. Figure S7. Fatty acid biotransformation by caveolae-forming E. coli BL21 (DE3)

442

cells. Figure S8. EM image of E. coli BL21 (DE3). Figure S9. Fatty acid uptake by FadL and/or

443

CAV1 expression.

444


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445

AUTHOR INFORMATION

446

Corresponding Authors

447

*E-mail:

448

Author Contribution

449

J.S., J.-B.P., and D.-H.K. designed research. J.S., Y.-H.S., K.H.C., J.-M.W., and H.C. performed

450

the experiments. J.S., J.-B.P., D.-H.K., J.Y., M.P., C.K., H.K., Y.P., C.B, and E.S. analyzed the

451

data. J.S., J.-M.W., J.-B.P., and D.-H.K. wrote the manuscript.

452

Conflicts of Interest

453

The authors declare no competing financial interests.

[email protected] (J.-B.P.), E-mail: [email protected] (D.-H.K.)

454 455

ACKNOWLEDGMENTS

456

This research was supported by the Basic Science Research Program through the National

457

Research Foundation of Korea (NRF), funded by the Ministry of Education

458

(2017R1A6A1A03015642 and NRF-2017R1A2B2008211), the Advanced Biomass R & D

459

Center (ABC) of Korea (2011-0031359), and the R&D Program of MOTIE/KEIT (10044604).

460 461

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Page 30 of 41

Table 1. Bacterial strains and plasmids used in this study Strain or plasmid

Relevant characteristic(s)

References or sources

BL21 (DE3)

F- ompT lon hsdSB(r-B,m-B) gal dcm

New England Biolabs

Top 10

F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG

Invitrogen

pT7-His6-CAV1

T7 promoter/Express His6-Caveolin1

In this study

pT7-His6-CAV1

T7 promoter/Express GST-Caveolin1

In this study

pCW-His6-CAV1

J23100 constitutive promoter/Express His6Caveolin1/Weak RBS

In this study

pCS-His6-CAV1

J23100 constitutive promoter/Express His6Caveolin1/Strong RBS

In this study

pCW-GST-CAV1

J23100 constitutive promoter/Express GSTCaveolin1/Weak RBS

In this study

pCS-GST-CAV1

J23100 constitutive promoter/Express GSTCaveolin1/Strong RBS

In this study

pAptmE6BVMOopt/ADH

J23100 constitutive promoter/Express ADH (from M. luteus), E6BVMOopt (from P. putida KT2440)/Strong RBS

Seo et al.45

E. coli

Plasmids

635


Page 31 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


636

Figure legends

637

Figure 1. Designed biotransformation pathway. Ricinoleic acid (1) is converted into (Z)-11-

638

(heptanoyloxy)undec-9-enoic acid (3) by the multi-step enzyme cascade, which was constructed

639

according to our previous study. 4

640 641

Figure 2. Formation of caveolae in E. coli cytoplasm upon CAV1 expression. (a) Schematic of

642

an endocytosing E. coli. Long-chain fatty acids pass the outer membrane of E. coli through FadL

643

and cross the plasma membrane using a flip-flop mechanism. Fatty acids in the periplasm of E.

644

coli are transported into the cytoplasm by endocytosis in CAV1-expressing cells. (b) Plasmids for

645

CAV1 expression in E. coli. His6- and GST-tags were fused to the N-terminus of CAV1 for

646

purification and detection. (c) Expression of CAV1 in E. coli identified using an anti-CAV1

647

antibody. Arrows indicate CAV1 fusion proteins. T: total cell lysates; S: soluble fraction of cell

648

lysates. (d) EM images of caveolae-forming E. coli. Arrows indicate caveolae formed inside the

649

cells. Enlargement of a section is shown on the right. Scale bar: 100 nm. (e) Western blot

650

analysis of purified caveolae using an anti-CAV1 antibody. (f) Dynamic light scattering analysis

651

of purified caveolae.

652 653

Figure 3. Endocytosis of fatty acids. (a) Encapsulation of 5-6-carboxyfluorescein in E. coli.

654

Scale bar: 50 µm. (b) Flow cytometry histogram of cells treated with the fluorescent dye. Black,

655

E. coli BL21 (DE3); gray, E. coli BL21 (DE3) pT7-His6-CAV1; white, E. coli BL21 (DE3) pT7-

656

GST-CAV1. (c) Relative intracellular ricinoleic acid (RA) concentration after supplementation

657

with indicated concentrations of RA in the medium. Intracellular RA concentration of BL21 31 Environment ACS Paragon Plus


658

(DE3) after 15 mM RA supplementation was used to normalize other treatment groups. (d) RA

659

uptake rate at 15 mM RA concentration in the medium. Intracellular RA concentrations were

660

normalized to those of the control cells after 60 min of the reaction. (e) Mitigation of fatty acid

661

cytotoxicity. The survival fraction was calculated by dividing the number of colony forming

662

units after 20 mM RA treatment for 3 h by that in the untreated control. Data represent mean

663

values and error bars represent standard deviation. P values were calculated by two-tailed t tests.

664

*p < 0.05, **p < 0.01, ***p < 0.001.

665 666

Figure 4. Constitutive CAV1 expression in E. coli. (a) Plasmids constructed for constitutive

667

expression of CAV1. Designed 5′-untranslated region (5′-UTR) is indicated on the right side of

668

the plasmid maps. (b) Translation efficiency of CAV1 in E. coli estimated using UTR designer.

669

(c) Expression analysis of constitutively expressed CAV1 using an anti-CAV1 antibody. T: total

670

cell lysates; S: soluble fraction of cell lysates. (d) Formation of caveolae in E. coli constitutively

671

expressing CAV1. (e) Growth curves of constitutively caveolae-forming E. coli cells in synthetic

672

Riesenberg medium. (f) Ricinoleic acid (RA) uptake rate at 15 mM medium concentration.

673

Intracellular RA concentrations were normalized to those in the control cells after 60 min of

674

reaction. (g) Relative intracellular RA concentration after supplementing the medium with the

675

indicated concentrations of RA. Intracellular RA concentration of BL21 (DE3) after

676

supplementation with 15 mM RA was used to normalize other treatment groups. (h) Mitigation

677

of fatty acid cytotoxicity. Survival fraction was calculated by dividing the number of colony

678

forming units after 20 mM RA (or 5 mM NA) treatment for 3 h by that in the untreated control.

679

Survival fraction of the fatty acid-treated cells. (i) Growth inhibition by fatty acids (5 mM NA

680

and 20 mM RA) was partially rescued by caveolae formation. Data represent mean values and


Page 32 of 41

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681

error bars represent standard deviation. P values were calculated by two-tailed t-tests. *p < 0.05,

682

**p < 0.01, ***p < 0.001.

683 684

Figure 5. Whole-cell biotransformation of RA 1. Cell-recycled biotransformation of 15 mM RA

685

by (a) E. coli pAptm-E6-BVMOopt/ADH and (b) endocytosing E. coli pAptm-E6-

686

BVMOopt/ADH/pCW-His6-CAV1. The cell density was 2.2 g dry cells/L. Symbols indicate

687

concentrations of RA (1, ●), 12-keooctadec-9-enoic acid (2, ■), and Ester (3, ▲).

688 689

Figure 6. Whole-cell biotransformation of RA 2. Time course of the biotransformation of RA to

690

the Ester at a high cell density by (a, c) E. coli BL21 (DE3) pAptm-E6-BVMOopt/ADH and (b, d)

691

endocytosing E. coli BL21 (DE3) pAptm-E6-BVMOopt/ADH/pCW-His6-CAV1. (a, b)

692

Biotransformation of 150 mM RA. (c, d) Three repeated biotransformations of 100 mM RA.

693

Symbols indicate concentrations of RA (1, ●), 12-keooctadec-9-enoic acid (2, ■), and Ester (3,

694

▲).

695 696

Figure 7. Whole-cell biotransformation of castor oil and 10-hydroxyoctadecanoic acid. Time

697

course of the biotransformation of (a, b) castor oil into n-heptanoic acid and 11-hydroxyundec-9-

698

enoic acid (Supporting Information, Figure S1) and of (c, d) 10-hydroxyoctadecanoic acid into 9-

699

(nonanoyloxy)nonanoic acid (Figure S1) by (a, c) E. coli BL21 (DE3) pAptm-E6-

700

BVMOopt/ADH and (b, d) endocytosing E. coli BL21 (DE3) pAptm-E6-BVMOopt/ADH/pCW-

701

His6-CAV1. Biotransformation was initiated by adding 5 g/L castor oil, 0.1% (v/v) Tween80,



702

and 20 U/mL lipase or 5 mM 10-hydroxyoctadecanoic acid into the culture broth. Symbols in

703

Fig. 6a indicate the concentrations of castor oil (▽), RA (●), 12-keooctadec-9-enoic acid (■),

704

Ester (▲), and 11-hydroxyundec-9-enoic acid (▼). Symbols in Fig. 6b indicate the

705

concentrations of 10-hydroxyoctadecanoic acid (○), 10-ketooctadecanoic acid (□), 9-

706

(nonanoyloxy)nonanoic acid (◆).

707 708

ABBREVIATIONS

709

CAV1, caveolin-1; RA, ricinoleic acid, 12-hydroxyoctadec-9-enoic acid; Ester, (Z)-11-

710

(heptanoyloxy) undec-9-enoic acid; TMS, N-Methyl-N-(trimethylsilyl) trifluoroacetamide;

711

IPTG, isopropyl β-D-1-thiogalactopyranoside; ADH, alcohol dehydrogenase; BVMO, Baeyer-

712

Villiger-monooxygenases; GST, glutathione S-transferase; PK, proteinase K; UTR, untranslated

713

region; NA, nonanoic acid

714


Page 34 of 41

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Figure 1 E. coli whole cell biocatalyst

OH

OH

O OH

5

O OH

5

1

1 Ricinoleic acid (RA)

NAD+

Alcohol dehydrogenase NADH

O

O OH

5

2 NADPH

O2

Baeyer-Villiger monooxygenase NADP+

H2O

O 5 O

O

O

O

OH

3

5 O

OH

3

(Z)-11-(heptanoyloxy)undec-9-enoic acid (Ester)

ACS Paragon Plus Environment

ACS Synthetic Biology 1 Figure 2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54


Page 36 of 41

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3


Figure 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54



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5


ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 Figure 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

6


Page 40 of 41

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7


Endocytosing Escherichia coli as a Whole

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