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Letter 2

Improvement of squalene production from CO in Synechococcus elongatus PCC 7942 by metabolic engineering and scalable production in a photobioreactor Sun Young Choi, Jin-Young Wang, Ho Seok Kwak, Sun-Mi Lee, Youngsoon Um, Yunje Kim, Sang Jun Sim, Jong-il Choi, and Han Min Woo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00083 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 4, 2017

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ACS Synthetic Biology

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Letter

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Improvement of squalene production from CO2 in Synechococcus elongatus

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PCC 7942 by metabolic engineering and scalable production in a

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photobioreactor

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Sun Young Choi1,2, Jin-Young Wang1, Ho Seok Kwak3, Sun-Mi Lee1,2, Youngsoon Um1,

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Yunje Kim1, Sang Jun Sim2,3, Jong-il Choi4, Han Min Woo*,5

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1

Clean Energy Research Center, Korea Institute of Science and Technology, Hwarangro 14-

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gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea

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2

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and Biological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841,

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Republic of Korea

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3

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Seongbuk-gu, Seoul 02841, Republic of Korea

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4

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Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea

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Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea

Green School (Graduate School of Energy and Environment) and 3Department of Chemical

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

Department of Biotechnology and Bioengineering, Chonnam National University, 77

Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066

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*Corresponding author: E-mail address: [email protected]. (H.M. Woo). Phone: +82 31 290

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7808

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ABSTRACT

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The push-and-pull strategy for metabolic engineering was successfully demonstrated in

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Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene

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from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate

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synthase) of the methylerythritol phosphate pathway or the β-subunit of phycocyanin

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(CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed

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a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold

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compared to that of the control strain expressing SQS alone. To increase squalene production

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further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion

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protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of

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squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best

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squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization,

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which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell

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weight). Further optimization for photo-bioprocessing and strain development will promote

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the construction of a solar-to-chemical platform.

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KEYWORDS: Synechococcus elongatus PCC 7942, protein engineering, metabolic

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engineering, squalene, CO2 conversion, scalable production

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A Graphical Table of Contents entry

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Squalene (C30H50)1, a triterpene intermediate for sterol and hopanoid biosynthesis, is widely

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used in the food, personal care, and medical industries because of its various beneficial

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functions.2,3 Since the feedstock supply for squalene production is limited and unstable

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because of animal protection policies on the use of shark liver oil, and regional and seasonal

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variations of plant oils, synthetic squalene has also been commercialized by the biotech

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company Amyris, Inc. using an engineered yeast strain that is capable of converting sugar-

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based carbon sources to squalene.4 Recently, the metabolic engineering of cyanobacteria has

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shown the potential to overcome concerns related to greenhouse gas emissions and address

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the demand of sustainable renewable energy and chemicals.5,6 Thus, the sustainable

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production of squalene from CO2 is another promising platform that could lower the

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production costs and provide a continuous supply from CO2. However, the key to engineering

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a biosolar cell factory for the conversion of CO2 to squalene relies on achieving a high-level

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conversion yield and productivity.

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The push-and-pull strategy of metabolic engineering has been successfully applied for

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the production of isoprenoids7,8 and lipids9 in microbial hosts by supplying key intermediates

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and increasing the rate of formation of the final product. In cyanobacteria, expression of the

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fusion protein between the β-subunit of phycocyanin (CpcB) and phellandrene synthase

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(PHLS) has resolved the limitation of the β-phellandrene formation rate by increasing the

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fusion protein levels up to 20% of total protein.10 Subsequently, the β-phellandrene-

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producing strains were engineered to co-express mevalonate pathway enzymes to supply

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isopentenyl-diphosphate (IPP)/dimethylallyl-diphosphate (DMAPP) in Synechocystis sp.

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PCC 6803, producing 12.4 mg/g dry cell weight β-phellandrene.11 Similarly, the combined

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overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and 4-hydroxy-3-

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methylbut-2-enyl-diphosphate synthase (IspG) of Thermosynechococcus elongatus for 4 ACS Paragon Plus Environment

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optimization of the methylerythritol phosphate (MEP) pathway with overexpression of the

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fused enzyme of Saccharomyces cerevisiae IPP isomerase (IDI) with isoprene synthase (IspS)

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increased the DMAPP/IPP ratio as well as the rate of IPP conversion to isoprene, yielding

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1.26 g/L isoprene from CO2 in Synechococcus elongatus PCC 7942.12

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The production of squalene from CO2 in cyanobacteria was achieved through the

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metabolic engineering of S. elongatus PCC 7942 by overexpressing the MEP pathway genes

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(dxs and idi) and expressing a key gene (ispA, encoding farnesyl diphosphate synthase [IspA])

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in the OverMEP module, and by expressing the truncated squalene synthase (SQS) gene in

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the SQS (or terpene synthase [TPS]) module, yielding strain SeSC33S (Fig. 1A).13 To

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complete the push-and-pull strategy of metabolic engineering, we adopted a protein

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engineering strategy to construct a fusion protein of SQS for improvement of squalene

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production, based on the strain SeHL33 (a parental strain) using only the OverMEP module

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(Table 1).

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For squalene strain development, two fusion protein partners and three different flexible

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linkers were selected for the protein engineering of SQS. First, a key enzyme of the MEP

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pathway (IspA) was fused to the N-terminal of SQS via the peptide linkers (short linker [SF],

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long linker [LF], and long linker 2 [LF2]), since expression of the fusion protein12 of IDI and

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IspS via linkers was shown to improve isoprene production via artificial substrate channeling.

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Then, the fused SQS genes were inserted into SyneBrick vectors14 and the plasmids were

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transformed in strain SeHL33, yielding strains SeSC34S, SeSC35S, and SeSC36S.

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Confirmation of the transformants was established by polymerase chain reaction (PCR) (Fig.

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1A) using genomic DNAs and oligonucleotide primers (Table S2). As a result, the

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recombinant strains with the IspA-SQS fusion protein showed several growth defects

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compared to the control strain SeSC33S (with sole expression of SQS) (Fig. 1B). With

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respect to squalene production, strain SeSC34S showed higher squalene contents (5.71 ± 0.17 5 ACS Paragon Plus Environment


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mg/L/OD730) than SeSC33S (3.77 ± 0.09 mg/L/OD730). However, lower production levels

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were observed given that longer flexible linkers were used for establishing the fusion protein.

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The squalene production level in strain SeSC36S was even lower than that of the control. In

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the case of the strain expressing the fusion protein of IDI-IspS12, no differences in isoprene

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production and growth rate were observed, regardless of the length and composition of the

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linkers (flexible or helical linkers). Thus, expression of the IspA-SQS fusion protein may not

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have a negative influence on the growth rate or squalene contents in the cell. However, the

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structure of the IspA-SQS fusion protein due to the linkers used could influence the cellular

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activities given that the growth rate and squalene content were lower when a longer linker

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was used. A potential reason for this growth inhibition could be the accumulation of toxic

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farnesyl diphosphate (FPP) in association with the expression of IspA-SQS. Previously, the

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strain SeHL33 with the OverMEP module (expression of the dxs, idi, and ispA genes) alone

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also exhibited growth inhibition, and the growth rate was relieved when TPS was used to pull

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the accumulated FPP.13 In the present study, additional IspA activity from IspA-SQS

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expression could have increased the local concentrations of toxic FPP, which would inhibit

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cellular growth and reduce the squalene contents. Thus, the pull-and-push strategy for

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squalene production with expression of a direct fusion protein of IspA-SQS was not

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successful in this case.

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High-level expression of CpcB-PHLS has previously been achieved, which was reported

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to lead to 100-fold yield improvement.10 Thus, native CpcB1 (a homologous protein of CpcB

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of Synechocystis sp. PCC 6803) was fused to the N-terminal of SQS via the same peptide

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linkers described above (SF, LF, LF2). The cpcB1 gene, encoding the β-subunit of

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phycocyanin as an essential antenna for light harvesting, is highly expressed under the

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activity of the strong endogenous cpcB1 promoter in S. elongatus PCC 7942.10 Thus, the trc

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promoter of the SyneBrick vector was replaced with the strong endogenous cpcB1 promoter 6 ACS Paragon Plus Environment

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to express the CpcB1-SQS fusion protein. As a result, three different recombinant strains

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were constructed (SeSC37S, SeSC38S, and SeSC39S) (Fig. 1A). Unlike fusion IspA-SQS

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proteins, CpcB1-SQS fusion proteins did not cause severe growth defects. SeSC37S showed

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the least amount of growth defects compared to the control strain SeSC33S. Compared to the

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squalene production levels of SeSC33S (3.77 ± 0.09 mg/L/OD730), the SeSC375, SeSC386,

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and SeSC39S strains expressing the CpcB1-SQS fusion protein (SeSC37S, 7.15 ± 0.05

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mg/L/OD730; SeSC38S, 6.23 ± 0.16 mg/L/OD730; SeSC39S, 6.93 ± 0.31 mg/L/OD730) showed

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increased squalene production levels by 1.8-fold, 1.65-fold, and 1.8-fold, respectively (Fig.

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1B). There were no significant differences between the production levels, regardless of the

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length of the flexible linkers. Thus, SeSC37S with a fusion CpcB1-SF-SQS protein (7.16 ±

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0.05 mg/L/OD730) was chosen for further improvement of squalene production.

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Increasing the gene dosage of TPS that yields the final product and expressing it under a

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strong constitutive promoter has been shown to help enhance production levels.9,15,16 Thus,

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engineered strains (SeSC33SII, SeSC34SII, and SeSC35SII) with two-copy insertions of the

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SQS gene on the chromosome were constructed to investigate whether or not additional gene

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copies and replacement of the strong promoter could enhance the level of squalene

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production from CO2 (Fig. 2A). The squalene content of strain SeSC33SII harboring two

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gene copies (7.22 ± 0.17 mg/L/OD730) was 1.9-fold higher than that of the control strain

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SeSC33S harboring one gene copy (3.77 ± 0.09 mg/L/OD730) (Fig. 2B). When the SQS gene

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integrated at neutral site II (NSII) was replaced with the gene encoding a CpcB1-SF-SQS

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fusion protein, the production level of SeSC34SII was also increased by 2.3-fold (8.94 ± 0.46

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mg/L/OD730) compared to that of SeSC33S. Finally, the duplicated SQS gene strain with a

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fusion protein (SeSC35SII) resulted in a squalene content of 11.98 ± 0.49 mg/L/OD730, which

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represents an increase of 3.1-fold or 1.7-fold compared to the levels produced in strain

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SeSC33S or SeSC37S (6.94 ± 0.31 mg/L/OD730), respectively. Therefore, both strategies of 7 ACS Paragon Plus Environment


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increasing gene dosage and replacement of a strong promoter resulted in improvement of

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squalene production from CO2, which is consistent with the previous result of the high-level

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expression of the fusion CpcB-PHLS protein in Synechocystis sp. PCC 6803.10 High-level

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expression of the CpcB1-SF-SQS fusion protein could lead to high SQS activity for the

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formation of squalene. To date, the SeSC35SII strain has shown the highest production level

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of squalene from CO2 (11.98 ± 0.49 mg/L/OD730). Thus, the push-and-pull strategy with

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protein engineering was successful to construct squalene-producing cyanobacteria.

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For the scalable production of squalene, a 6-L CO2-fed bag-type photobioreactor17 that

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has been optimized for scale-up production (6 L×n) was used, and the strains SeSC35SII and

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SeSC33S (as a control) were cultivated with 5% CO2 under 100 µmol photons·m-2·s-1 (Fig.

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3A). As a result, both recombinant strains showed similar growth rates, although they

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exhibited slower growth than the wild-type. After isopropyl-β-D-1-thiogalactopyranoside

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(IPTG) induction at Day 3, both strains produced squalene at similar levels at Day 8. The

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final squalene content in SeSC35SII (5.60 ± 0.54 mg/L/OD730; equivalent to 54.3 mg

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squalene per g dry cell weight) was higher than that in SeSC33S (4.01 ± 0.46 mg/L/OD730;

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equivalent to 34.7 mg squalene per g dry cell weight) (Fig. 3B). To improve the production

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levels for scalable production, the light intensity was increased to 200 µmol photons·m-2·s-1

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to achieve better photosynthetic efficiency in the recombinant cyanobacteria. Both squalene-

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producing strains and the wild-type showed better cell growth than obtained under the

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previous culture condition. In addition, the strains SeSC35SII and SeSC33S produced 7.08 ±

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0.5 mg/L/OD730 (equivalent to 79.2 mg per g dry cell weight) and 4.79 ± 0.46 mg/L/OD730

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(equivalent to 57.5 mg per g dry cell weight) of squalene, respectively (Fig. 3C). Although

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the squalene content in the scalable photobioreactor was a bit lower than that in the test bottle

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culture, this is the first report to demonstrate the scale-up production of recombinant

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cyanobacteria to produce squalene from CO2 as a sole carbon source. Nevertheless, many 8 ACS Paragon Plus Environment

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factors still need to be considered for realizing the economically feasible mass production of

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squalene, including potential contamination18 in the absence of antibiotics and optimizing the

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photobioreactor operation and downstream processing conditions.

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In summary, the push-and-pull strategy of metabolic engineering for cyanobacteria was

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successfully applied for improvement of squalene production from CO2. Combined with

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MEP pathway engineering, high-level protein expression of a CpcB1-SF-SQS fusion protein

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led to improvement in squalene production by 2.3-fold. Subsequently, the engineered

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cyanobacteria were cultivated in a scalable photobioreactor for mass production. Further

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development of the photo-bioprocessing conditions with strain improvement will promote

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establishment of an engineered biosolar cell factory for industrial-scale CO2 conversion.

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METHODS

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Chemicals and reagents. All chemicals were purchased from Sigma-Aldrich (St. Louis, St.

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Louis, MO, USA) unless otherwise specified. Restriction enzymes, Phusion DNA polymerase,

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and ligases were purchased from Fermentas (Thermo Fisher Scientific Inc., Waltham, MA,

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USA).

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Plasmid and strain construction. The plasmids and strains used in this study are listed in

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Table 1. For fusion protein construction, SQS13,19 was modified to fuse with a protein partner

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(E. coli IspA13 or CpcB1) and a linker. The cpcB1 operon (the upstream 500 bp of SYNPCC

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7942_1047) and the cpcB1 (SYNPCC 7942_1047)-encoding DNA fragment were obtained

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by PCR with the genomic DNA of S. elongatus PCC 7942. Three different linkers were used:

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SF (amino acid sequence GGGS), LF (GSGGGGS), and LF2 [LS(GGGGS)4AAA].12,20 The

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DNA fragment containing either the ispA gene-linker (SF) or the cpcB1 gene-linker (SF) was

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inserted into the pSe2Bb1k-SQS plasmid (targeting NSII), yielding pSe2Bb1k-ispA-SF-SQS 9 ACS Paragon Plus Environment


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or pSe2k-cpcB1-SF-SQS, respectively. Similarly, each LF or LF2 linker was used to

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construct the fusion SQS plasmid (Table 1). The plasmids were constructed using the Gibson

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assembly cloning kit (E5510s, BioLabs, New England) with a pair of oligonucleotides (Table

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S1) and pSe1Bb1k-SQS. DNA sequences of the constructed plasmids were correctly verified

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by DNA sequencing. For the two-copy SQS gene expression, pSe3Bb1c-SQS and pSe3c-

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cpcB1-SF-SQS were constructed from pSe3Bb1c-eyfp, targeting NSIII.

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Transformation of S. elongatus PCC 7942 (SeHL3313, used as a parental strain for this

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work, and SeSC33S13 as the control) with the plasmid was performed as described

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previously21. In brief, the cyanobacterial strains were transformed by incubating cells at a

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mid-log phase (OD730 of 1–16) with 100 ng of plasmid DNA for 24 h in the dark. The mixed

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culture was then spread on BG-11 plates supplemented with appropriate antibiotics for

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selection of successful recombination. The strains were confirmed by genomic DNA PCR to

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verify chromosomal integration of the target DNAs into either the NSI, NSII, and/or NSIII

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sites (Fig. 1 and Fig. 2), and the DNA sequences were also correctly verified using a pair of

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oligonucleotides (Table S2). Recombinant strains are listed in Table 1 along with their

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genotypes.

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Cyanobacteria growth condition. For the production of squalene, S. elongatus PCC 7942

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and its derivatives were cultivated at 30°C in 100-mL cultures under continuous fluorescent

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light (100 µmol photons·m-2·s-1) in BG-11 medium supplemented with 10 mM MOPS (pH

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8.0).13,22,23 CO2 gas (5% v/v) was supplied at constant flow rate of 10 mL/min into the

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medium. Spectinomycin (10 µg/mL), kanamycin (5 µg/mL), and/or chloramphenicol 3

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(µg/mL) was added to the BG-11 medium for selection pressure where appropriate. For the

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induction, 1 mM IPTG was added to the culture medium at an OD730 of 1 after inoculation.

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No growth inhibition has been reported with 1 mM IPTG.14 10 ACS Paragon Plus Environment

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Gas chromatography-mass spectrometry (GC-MS) analysis for squalene quantification.

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For the quantification of squalene, the extraction method was performed as described

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previously.13 In brief, 50 mL of the culture was used for extraction. After centrifugation at

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3000 ×g for 10 min, cell pellets were re-suspended with 2 mL of a mixture of chloroform and

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methanol (1:2 ratio), and liquid-liquid extraction was conducted for 30 min at room

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temperature. After additional centrifugation (16,000 ×g for 3 min), the supernatants (200 µL)

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were collected and the samples supplemented with 20 µg/mL of 1-phenyloctadecane as an

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internal standard were analyzed using GC-MS on a GC-MSD Agilent Technologies (Santa

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Clara, CA, USA) system equipped with an Ultra-2 capillary column (31 m × 0.2 mm, film

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thickness 0.11 mm; Agilent Technologies) under the following conditions: carrier gas, He

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(0.8 mL/min); oven temperature: 120–290°C (increase rate 6°C/min); run time 24.33 min.

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The electron ionization mass spectrometer was operated under the following conditions: scan

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mode; electron energy, 70 eV. The electron ionization mode with ion-selected monitoring (69

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m/z, 93 m/z) range was 35–50 m/z.

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Squalene production in a scalable photobioreactor. For the large-scale culture, a

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polypropylene-based cast polypropylene film scalable photobioreactor17 (10 cm in diameter

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and 1.2 m in height; house-manufactured products; maximum 6 L) was used for the

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production of squalene. The photobioreactor was disinfected by ultraviolet exposure for 24 h

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until use. The exponentially growing cyanobacterial cells were inoculated into 4 L of the BG-

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11 medium to start the culture at an OD730 of 0.5 at 20°C. The vessel in the form of the

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photoreactor was set under the condition of continuous light (100 or 200 µmol photons·m-2·s-

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1

) measured by a photometer (LI-250A, LI-COR Bioscience, USA) and 25°C. CO2 (5% v/v)



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was pumped into the cell medium at a flow rate of 160 mL/min. For induction of the

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engineered strain, 0.5 mM IPTG was added to the culture medium at an OD730 of 1.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at

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DOI:

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AUTHOR INFORMATION

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Corresponding author

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*Phone: +82-31-290-7808. E-mail: [email protected].

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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This work was supported by Korea CCS R&D Center (KCRC) (2014M1A8A1049277) and

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the National Research Foundation of Korea (2017R1A2B2002566) funded by the Korean

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Government (2016, University-Institute Cooperation program). This work was also partially

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supported by a Golden Seed Project (213008-05-1-WT911) grant funded by the Ministry of

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Agriculture, Ministry of Oceans and Fisheries.


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REFERENCES (1) Thimmappa, R., Geisler, K., Louveau, T., O'Maille, P., and Osbourn, A. (2014) Triterpene biosynthesis in plants, Annu. Rev. Plant Biol. 65, 225-257. (2) Spanova, M., and Daum, G. (2011) Squalene - biochemistry, molecular biology, process biotechnology, and applications, Eur. J. Lipid Sci. Tech. 113, 1299-1320. (3) Reddy, L. H., and Couvreur, P. (2009) Squalene: A natural triterpene for use in disease management and therapy, Adv. Drug Deliv. Rev. 61, 1412-1426. (4) Fisher, K., Schofer, S. J., and Kanne, D. B. Squalane and isosqualane compositions and methods for preparing the same. U.S. Patent 8,586,814 B2, November 19, 2013. (5) Woo, H. M. (2017) Solar-to-chemical and solar-to-fuel production from CO2 by metabolically engineered microorganisms, Curr. Opin. Biotechnol. 45, 1-7. (6) Melis, A. (2009) Solar energy conversion efficiencies in photosynthesis: Minimizing the chlorophyll antennae to maximize efficiency, Plant Sci 177, 272-280. (7) Rodriguez, S., Denby, C. M., Van Vu, T., Baidoo, E. E., Wang, G., and Keasling, J. D. (2016) ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae, Microb. Cell Fact. 15, 48. (8) Kim, E. M., Eom, J. H., Um, Y., Kim, Y., and Woo, H. M. (2015) Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli, J. Agric. Food Chem. 63, 46064612. (9) Tai, M., and Stephanopoulos, G. (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production, Metab. Eng. 15, 1-9. (10) Formighieri, C., and Melis, A. (2015) A phycocyanin.phellandrene synthase fusion enhances recombinant protein expression and beta-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria), Metab. Eng. 32, 116-124. (11) Formighieri, C., and Melis, A. (2016) Sustainable heterologous production of terpene hydrocarbons in cyanobacteria, Photosynth. Res. 130, 123-135. (12) Gao, X., Gao, F., Liu, D., Zhang, H., Nie, X. Q., and Yang, C. (2016) Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO2, Energy Environ. Sci. 9, 1400-1411. (13) Choi, S. Y., Lee, H. J., Choi, J., Kim, J., Sim, S. J., Um, Y., Kim, Y., Lee, T. S., Keasling, J. D., and Woo, H. M. (2016) Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria, Biotechnol. Biofuels 9, 202. (14) Kim, W. J., Lee, S.-M., Um, Y., Sim, S. J., and Woo, H. M. (2017) Development of SyneBrick vectors as a synthetic biology platform for gene expression in Synechococcus elongatus PCC 7942, Front. Plant Sci. 8, 293. (15) Anthony, J. R., Anthony, L. C., Nowroozi, F., Kwon, G., Newman, J. D., and Keasling, J. D. (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene, Metab. Eng. 11, 13-19. (16) Woo, H. M., Murray, G. W., Batth, T. S., Prasad, N., Adams, P. D., Keasling, J. D., Petzold, C. J., and Lee, T. S. (2013) Application of targeted proteomics and biological parts assembly in E. coli to optimize the biosynthesis of an anti-malarial drug precursor, amorpha4,11-diene, Chem. Eng. Sci. 103, 21-28. (17) Yoo, J. J., Choi, S. P., Kim, B. W., and Sim, S. J. (2011) Optimal design of scalable photo-bioreactor for phototropic culturing of Haematococcus pluvialis, Bioprocess Biosyst. Eng. 35, 309-315. 13 ACS Paragon Plus Environment


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(18) Shaw, A. J., Lam, F. H., Hamilton, M., Consiglio, A., MacEwen, K., Brevnova, E. E., Greenhagen, E., LaTouf, W. G., South, C. R., van Dijken, H., and Stephanopoulos, G. (2016) Metabolic engineering of microbial competitive advantage for industrial fermentation processes, Science 353, 583-586. (19) Zhang, D., Jennings, S. M., Robinson, G. W., and Poulter, C. D. (1993) Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme, Arch. Biochem. Biophys. 304, 133-143. (20) Arai, R., Ueda, H., Kitayama, A., Kamiya, N., and Nagamune, T. (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein, Protein Eng. 14, 529-532. (21) Golden, S. S., Brusslan, J., and Haselkorn, R. (1987) Genetic engineering of the cyanobacterial chromosome, Methods Enzymol. 153, 215-231. (22) Lee, H. J., Choi, J., Lee, S.-M., Um, Y., Sim, S. J., Kim, Y., and Woo, H. M. (2017) Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria, J. Agric. Food Chem. 65, 1087-1092. (23) Chwa, J. W., Kim, W. J., Sim, S. J., Um, Y., and Woo, H. M. (2016) Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition, Plant Biotechnol. J. 14, 1768-1776. (24) Zhang, C., Chen, X., Stephanopoulos, G., and Too, H. P. (2016) Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli, Biotechnol. Bioeng. 113, 1755-1763.


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

Relevant characteristics

Strains E. coli DH5α24 S. elongatus PCC 7942 SeHL3313 SeSC33S13 SeSC34S SeSC35S SeSC36S SeSC37S SeSC38S SeSC39S SeSC33SII SeSC34SII SeSC35SII

F-(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r– m+) recA1 endA1 relA1 deoR96 S. elongatus PCC 7942 S. elongatus NSI::Bb1s-dxs-idi-ispA S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·LF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-ispA·LF2·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·LF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::k-PcpcB1-cpcB1·LF2·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS NSIII::Bb1c-SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII::Bb1k-SQS NSIII::c-PcpcB1cpcB1·SF·SQS S. elongatus NSI::Bb1s-dxs-idi-ispA NSII:: k- PcpcB1-cpcB1·SF·SQS NSIII::cPcpcB1-cpcB1·SF·SQS

Plasmidsa pSe3Bb1c-eyfp14 pSe1Bb1s-dxr-idi-ispA13 pSe2Bb1k-SQS13 pSe3Bb1c-SQS pSe2Bb1k-ispA·SF·SQS pSe2Bb1k-ispA·LF·SQS pSe2Bb1k-ispA·LF2·SQS pSe2k-cpcB1·SF·SQS pSe2k-cpcB1·LF·SQS pSe2k-cpcB1·LF2·SQS pSe3c-cpcB1·SF·SQS

pUC, Cmr, LacI, Ptrc, BglBrick sites, NSIII targeting SyneBrick vector pUC, Spcr, LacI, Ptrc, dxr(se.co), idi(se.co), ispA(se.co), NSI target sites pUC, Kmr, LacI, Ptrc, SQS(se.co), NSII target sites pUC, Cmr, LacI, Ptrc, SQS(se.co), NSIII target sites pUC, Kmr, LacI, Ptrc, ispA·SF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, LacI, Ptrc, ispA-LF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, LacI, Ptrc, ispA·LF2·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·SF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·LF·SQS(se.co), fusion protein, NSII target sites pUC, Kmr, PcpcB1, cpcB1·LF2·SQS(se.co), fusion protein, NSII target sites pUC, Cmr, PcpcB1, cpcB1·SF·SQS(se.co), fusion protein, NSIII target sites

a

Kmr, kanamycin resistance; Spcr, spectinomycin resistance; Cmr , chloramphenicol resistance; the dxs

gene (E. coli), encoding 1-deoxy-D-xylulose-5-phosphate synthase; the idi gene (E. coli), encoding 1deoxy-D-xylulose-5-phosphate reductase; the ispA gene (E. coli), encoding farnesyl diphosphate synthase (IspA); SQS (S. cerevisiae), encoding truncated squalene synthase; (se.co), codon-optimized for S. elongatus PCC 7942.13,19 Peptide sequences for the linkers: SF, short flexible linker (4 aa), GGGS12; LF, long flexible linker (7 aa), GSGGGGS12; LF2, long flexible linker 2 (25 aa), LS(GGGGS)4AAA20. Note that all strains and plasmids were constructed in this work unless otherwise cited.



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Figure 1. Engineering of S. elongatus PCC 7942 to improve the photosynthetic squalene production from CO2 by protein engineering. (A) The metabolic pathway for squalene production from CO2 was engineered by overexpressing the dxs, idi, and ipsA genes from E.

coli (OverMEP module at neutral site I [NSI]) and expressing the SQS gene from S. cerevisiae (SQS module at neutral site II [NSII]).13 The engineered strain was named SeSC33S (a parental strain for this work). For protein engineering, the SQS modules were engineered with two different fusion partners (IspA or CpcB1) and three different linker domains (SF, LF, LF2) and introduced to the strain SeHL33 lacking the SQS gene at NSII. Six strains (SeSc34S, SeSc35S, SeSc36S, SeSc37S, SeSc38S, and SeSc39S) were constructed and confirmed by colony PCR using a pair of primers (1F/1R and 2F/2R; Table S1). The corresponding DNA fragments (NSI and NSII) are shown in gel images with white arrows. The DNA sequences were also verified to be correct. (B) Growth (OD730) and squalene production (mg/L/OD730) from the engineered strains are shown. No squalene is formed in the wild-type.13 Strain SeSc33S was used as a control. Solid symbols represent strains with the IspA-SQS fusion protein (SeSC34S, SeSC35S, SeSC36S), and open symbols represent strains with the CpcB1-SQS fusion protein (SeSC37S, SeSC38S, SeSC39S). The genotypes of the recombinant strains are described in Table 1. All data are the mean ± standard deviation from triplicate cultures. Abbreviations: dxs, 1-deoxy-D-xylulose-5phosphate synthase gene of E. coli; idi, isopentenyl diphosphate isomerase gene of E. coli;

ispA, farnesyl diphosphate synthase (IspA) gene of E. coli; SQS (Sc), squalene synthase gene of S. cerevisiae; G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-Derythritol; CDP-MEP, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate; MEcPP, 2Cmethyl-D-erythritol-2,4-cyclodiphosphate;

HMBPP,

(E)-4-hydroxy-3-methylbut-2-enyl-

diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, 16 ACS Paragon Plus Environment

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farnesyl diphosphate; PcpcB1, promoter region of the upstream 500 bp of the cpcB1 operon from S. elongatus PCC 7942; cpcB1, encoding the phycocyanin β-subunit protein from S.

elongatus PCC 7942; SF, short flexible linker; LF, long flexible linker; LF2, long flexible linker 2; WT, wild type.

Figure 2. Overexpression of duplicated squalene synthase gene copies in S. elongatus PCC 7942 to enhance squalene contents. (A) Scheme of combinatorial overexpression with the fused squalene synthase protein (a CpcB1-[SF]-SQS fusion protein). Four strains (SeSc33SII, SeSc34SII, SeSc37S, and SeSc35SII) were constructed and confirmed by colony PCR using a pair of primers (2F/2R and 3F/3R; Table S1). The corresponding DNA fragments (NSI and NSII) are shown in gel images with white arrows. The genotypes of the recombinant strains are described in Table 1. WT and NC stand for the wild type and negative control, respectively. (B) Growth (OD730) and squalene production (mg/L/OD730) from engineered strains are shown. Squalene contents in the strain with overexpression of one copy of the gene (grey bars) or overexpression of two copies of the gene (black bars) are shown. All data are the mean ± standard deviation from triplicate cultures.

Figure 3. Scalable production of squalene using a photobioreactor. (A) Schematic diagram and photographic images of the scalable production system with engineered cyanobacteria to produce squalene from 5% (v/v) CO2 and 95% (v/v) air. The 6-L V-shaped cylindrical bioreactor was optimized and used for scalable production.17 (B and C) The growth (OD730) and photosynthetic squalene production (mg/L/OD730) of the wild type as a control (black squares) and engineered S. elongatus PCC 7942 strains SeSC33S (red circles; grey bars) and SeSC35SII (blue triangles; black bars) under a light intensity of 100 or 200 µmol photon·m2

·s-1. All data are the mean ± standard deviation from duplicate cultures. 17 ACS Paragon Plus Environment


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Figure 1


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Figure 2



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Figure 3


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Synechococcus elongatus PCC 7 - ACS Publications

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