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Anaerobic production of poly(3-hydroxybutyrate) (PHB) and its precursor 3-hydroxybutyrate (3-HB) from synthesis gas by autotrophic clostridia Sebastian Flüchter, Stéphanie Follonier, Bettina SchielBengelsdorf, Frank R. Bengelsdorf, Manfred Zinn, and Peter Dürre Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00342 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Anaerobic production of Poly(3hydroxybutyrate) (PHB) and its Precursor 3Hydroxybutyrate (3-HB) from Synthesis Gas by Autotrophic Clostridia Sebastian Flüchter,1 Stéphanie Follonier,2,3 Bettina Schiel-Bengelsdorf,1 Frank R. Bengelsdorf,1 Manfred Zinn,2 and Peter Dürre,1* 1Institut

für Mikrobiologie und Biotechnologie, Universität Ulm, Albert-Einstein-Allee

11, 89081 Ulm, Germany 2University

of Applied Sciences and Arts Western Switzerland (HES-SO Valais),

Institute of Life Technologies, Route du Rawyl 64, 1950 Sion, Switzerland 3Current

Address: Lonza AG, Clinical Development-Microbial (USP), Lonzastrasse,

3930 Visp, Switzerland

KEYWORDS: Clostridium, 3-hydroxybutyrate, PHB, poly(3-hydroxybutyrate), polyhydroxyalkanoates, syngas

ACS Paragon Plus Environment

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AUTHOR INFORMATION Corresponding Author Prof. Dr. Peter Dürre Institut für Mikrobiologie und Biotechnologie Universität Ulm Albert-Einstein-Allee 11 D-89081 Ulm Germany e-Mail: [email protected] Tel. +49-731-5022710 Fax +49-731-5022719


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Abstract Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant C. coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and C. ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29 % PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12 % PHB per CDW.


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Introduction Although plastic materials have become indispensable in our modern life and their production increased steadily and became one of the fastest growing global industries over the last decades1, world is now facing a global crisis of plastic pollution. A massive switch to biobased and biodegradable plastics (preferably from substrates not competing with human nutrition) could be a solution. However, such materials are still too costly in production to be considered for large scale fabrication, as commercial production is based on carbohydrates (e.g. glucose + propionate, fatty acids, sucrose, sugar cane) as substrate.2 Poly(3-hydroxyalkanoates) (PHAs) such as poly(3hydroxybutyrate) (PHB) are suitable biobased and biodegradable candidates to replace petroleum-based non-degradable plastics2. The monomer of PHB, 3-hydroxybutyrate (3-HB) can also serve as chiral building block for food supplements, pharmaceuticals, cosmetics, and other fine chemicals.3 Little is known about natural PHB synthesis in Clostridium species. In 1973, Emeruwa and Hawirko published a report on PHB production in C. botulinum and in 1997, Girbal et al. discovered this metabolic feature in C. acetireducens.4,5 On the other hand, autotrophic clostridia such as C. ljungdahlii and C. autoethanogenum are able to grow on waste gases containing CO, H2, and CO2 (such mixtures are usually called syn(thesis)gas), which thus provide a very cheap substrate. This group of bacteria


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employs the Wood-Ljungdahl pathway for producing acetate, ethanol, and 2,3butandiol from gases and is collectively called "acetogens".6 C. autoethanogenum is already been used at industrial scale. A plant, producing app. 46,000 t of ethanol per year, was built close to a steel mill in Caofeidian (China) and uses the respective waste gases as substrate.7 It is a joint venture of LanzaTech and the Shougang Group. The biomass, which is accumulating during the process, is used as animal feed. If such acetogenic clostridia could be metabolically engineered to become PHB producers, the synthesis of this biobased and biodegradable polymer could become economically competitive to naphta-based plastics, due to the cheap substrate costs. This scheme is depicted in Figure 1. Here, we elucidate natural PHB production in clostridia, describe metabolic engineering of the acetogens C. coskatii and C. ljungdahlii to become producers of 3-HB or PHB, respectively, and provide an analysis of the syngas-derived PHB (A preliminary account of some of the data presented in this report was given at the 16th International Symposium on Biopolymers, Beijing, China, October 21-24, 2018).


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Figure 1: Schematic overview of potential autotrophic 3-HB and PHB synthesis pathways in C. ljungdahlii and C. coskatii, their substrate utilization, and product formation. Naturally occurring products

ethanol

and

acetate

are

shown

in

black,

recombinant

products

poly(3-

hydroxybutyrate) and 3-hydroxybutyrate are shown in purple. If CO is used as carbon source, CO2 will be an end product as well. Abbreviations: [H], redox equivalent (one electron + one proton); PTS, phosphotransferase system; RNF, proton-translocating ferredoxin:NAD+ oxidoreductase complex; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NAD+, nicotinamide adenine dinucleotide oxidized; NADH, nicotinamide adenine dinucleotide reduced; Fd, oxidized ferredoxin; Fd2-, reduced ferredoxin; THF, tetrahydrofolate; H2, hydrogen; CO, carbon monoxide; CO2, carbon dioxide.

Material and methods Bacterial strains and growth conditions.


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Escherichia coli DH5α was used for general cloning procedures. E. coli was cultivated in 5-mL test tubes containing LB medium 8 (Luria-Bertani) with tryptone 10 g L-1, NaCl 10 g L-1, yeast extract 5 g L-1), and the respective antibiotic (thiamphenicol 20 µg mL-1, chloramphenicol 30 µg mL-1, and ampicillin 100 µg mL-1). Incubation was performed in an orbital shaker (3020, GFL, Burgwedel, Germany) at 175 rpm and 37 °C. LB agar plates were prepared with addition of 15 g L-1 agar. Clostridium coskatii PTA-10522 and C. ljungdahlii DSM 13583 cells were cultivated at 37 °C under strictly anaerobic batch conditions in infusion bottles (Thermo scientific) containing modified Tanner medium (Tanner

mod.

medium).9

Tanner

mod.

medium

contained

per

1 L:

2-(N-

morpholino)ethanesulfonic acid (MES) 10.0 g, yeast extract 0.5 g for autotrophic growth or 2.0 g for heterotrophic growth, mineral solution 10 mL, trace element solution 10 mL, vitamin solution 10 mL, resazurin 0.5 mg, cysteine-HCl x H2O 0.5 g. Mineral solution contained per 500 mL: NaCl 40.0 g, NH4Cl 50.0 g, KCl 5.0 g, KH2PO4 5.0 g, MgSO4 x 7 H2O 10.0 g, CaCl2 x 2 H2O 2.0 g. Vitamin solution contained per 1 L: nitrilotriacetic acid 2.0 g, MnSO4 x H2O 1.0 g, Fe(NH4)(SO4)2 x 6 H2O 0.8 g, CoCl2 0.2 g, ZnSO4 x 7 H2O 0.2 g, CuCl2 0.02 g, NiCl2 x 6 H2O 0.02 g, Na2MoO4 x 2 H2O 0.02 g, Na2SeO3 x 5 H2O 0.02 g, Na2WO4 x 2 H2O 0.02 g. For preparing trace element solution, nitrilotriacetic acid was dissolved first and pH was adjusted to 6.5 using 2 M KOH. Then, the other components were added and the final pH was adjusted to 7.0 using 2 M KOH. Vitamin solution contained per 1 L: pyridoxine-HCl 10.0 mg, thiamine-HCl x 2 H2O 5.0 mg, riboflavine


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5.0 mg, D-Ca-pantothenate 5.0 mg, lipoic acid 5.0 mg, p-aminobenzoic acid 5.0 mg, nicotinic acid 5.0 mg, vitamin B12 5.0 mg, biotin 2.0 mg, folic acid 2.0 mg. For heterotrophic and autotrophic growth, the infusion bottles differed in size. Heterotrophic and autotrophic growth experiments for 3-HB production were performed with a working volume of 50 mL in 125-mL bottles and 500-mL bottles, respectively, in order to increase the volume of the gas atmosphere in the latter case. For PHB production, the bottle volumes were 500 mL and 1000 mL, respectively, with a working volume of 200 mL or 500 mL. Heterotrophic growth experiments were conducted under N2 + CO2 (80 % + 20 %) atmosphere without shaking and 40 mM of fructose was added under aseptic conditions from a 1.1 M stock solution. Autotrophic growth was performed after 3 adaptation steps on gaseous substrate consisting of CO + H2 + CO2 + N2 (40 mol% + 40 mol% + 10 mol% + 10 mol%) at a pressure of 100 kPa without shaking. The pressure was regularly monitored and the gas phase exchanged, when a drop to app. 20 % of the starting value was observed (about once a week). Growth experiments with C. coskatii and C. ljungdahlii were performed in triplicate.

Construction of plasmids encoding 3-HB and PHB synthetic pathways All genes used for subcloning and the final plasmids constructed are listed in Table S1. All primers used for amplification of DNA fragments are listed in Table S2. Plasmid p83_PHB_C.nec for PHB synthesis based on Cupriavidus necator genes.


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For the construction of this plasmid, genomic DNA from C. necator DSM 428 was used. Genes required for PHB synthesis (phaCAB) were amplified using primers C.nec_phaCAB_F1_XhoI and C.nec_phaCAB_R1_NheI. The genes included PHA synthase (phaC; H16_A1437), a β-ketothiolase (phaA; H16_A1438), and an acetoacetylCoA reductase (phaB; H16_A1439). The amplified fragment had a size of 3,875 bp and was subcloned into 'pJet1.2/blunt Cloning Vector' leading to pJet1.2_phaCAB_Cnec (intermediate plasmid 1). This plasmid was digested using restriction enzymes XhoI and NheI. Subsequently, the phaCABC.nec gene cluster was cloned into pMTL8315110, which was digested with restriction ezymes XhoI and NheI as well (intermediate plasmid 2). Ppta-ack promoter was amplified from genomic DNA of C. ljungdahlii using primers C.lju_pta-ack_F1_MluI and C.lju_pta-ack_R1_XhoI resulting in a 159-bp fragment. The promoter was subcloned into 'pJet1.2/blunt Cloning Vector' resulting in pJet1.2_Ppta.ack_C.lju_MluI/XhoI (intermediate plasmid 3). A double digestion of intermediate plasmid 3 (2,960 bp + 153 bp + 20 bp) and intermediate plasmid 2 (pMTL83151_phaCABCnec) (8,130 bp + 16 bp) using restriction enzymes MluI, XhoI, followed by a purification and a ligation step, led to the final construct p83_PHB_C.nec. This plasmid was transformed into heat shock-sensitive E. coli DH5α cells. After transformation, positive clones where screened via colony PCR. One positive clone was inoculated, the plasmid was isolated and sent to GATC Biotech (GATC Biotech,


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Konstanz, Germany) for sequencing. The sequencing result showed that the plasmid was free of mutations. Plasmid p83_PHB_B.thai for PHB synthesis based on Burkholderia thailandensis genes. For construction of this plasmid, genes required for PHB synthesis (phaCABB.thai) were amplified from genomic DNA of B. thailandensis DSM 13276 using primers B.thai_phaCAB_F1_Eco147I and B.thai_phaCAB_R1_NheI. The amplified genes had a size of 3,962 bp and were subcloned into 'pJet1.2/blunt Cloning Vector' resulting in pJet1.2_phaCABB.thai (intermediate plasmid 4). By using the inserted restriction sites of the cloned phaCABB.thai fragment, a digestion was performed using restriction enzymes NheI and Eco147I. The digestion product phaCABB.thai was cloned into Clostridium-E. coli shuttle plasmid pMTL83151 resulting in pMTL83151_phaCABB.thai (intermediate plasmid 5). Ppta-ack promoter was amplified from genomic DNA of C. ljungdahlii using primers C.lju_pta-ack_F1_KpnI and C.lju_pta-ack_R1_Eco147I. The amplification of the 159-bp fragment was followed by a ligation into 'pJet1.2/blunt Cloning Vector' resulting in pJet1.2_pta.ack_C.ljung_KpnI/Eco147I (intermediate plasmid 6). The constructed plasmids pMTL83151_phaCABB.thai and pJet1.2_pta.ack_C.ljung_KpnI/Eco147I were digested using KpnI and Eco147I restriction enzymes, followed by a ligation of both fragments leading to the final plasmid p83_PHB_B.thai. This plasmid was transformed into heat shock-sensitive E. coli DH5α cells. After transformation, 3 clones were obtained. Plasmid DNA of these clones was isolated and one plasmid was found


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positive

after

a

PCR

using

primers

C.lju_pta-ack_F1_KpnI

and

C.lju_pta-

ack_R1_Eco147I. The plasmid was sent to GATC Biotech (GATC Biotech, Konstanz, Germany) for sequencing. The result of the sequencing reaction showed that the plasmid was free of mutations. Plasmid p83_tcb for 3-hydroxybutyrate synthesis based on clostridial genes. For the construction of the 3-hydroxybutyrate formation plasmid p83_tcb, the thiolase A gene (thlA; CA_C2873), the acetoacetyl-CoA:acetate/butyrate CoA transferase genes (ctfA/ctfB; CA_P0163/ CA_P0164), and the 3-hydroxybutyrate dehydrogenase gene (bdhA; CDIF630_02933) were used. The genes thlA and ctfA/B originated from C. acetobutylicum ATCC 824 and bdhA from Clostrioides difficile DSM 27543. Thiolase A and acetoacetyl-CoA: acetate/butyrate CoA transferase genes were previously cloned, together with the acetoacetate decarboxylase gene (adc; CA_P0165) under the control of thiolase promoter PthlA of C. acetobutylicum, resulting in pMTL83151_act.11 In order to replace the gene adc by bdhA (3-hydroxybutyrate dehydrogenase), the plasmid pMTL83151_act was digested using KpnI and NotI restriction enzymes to cut out adc. The bdhA gene was amplified using Kapa HIFI polymerase (Kapa Biosystem, SigmaAldrich Chemie GmbH, Munich, Germany) from genomic DNA of C. difficile ATCC 630 using primer Pep_bdhA_rev along with Pep_thlA_fw and then subcloned into pJet1.2/blunt cloning vector (Thermo Fisher Scientific) resulting in pJet1.2_t2cb (intermediate plasmid 7). The bdhA gene was subsequently amplified from plasmid


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DNA using primer J5_bdhA_fw and J5_bdhA_rev, of which 5’ ends were compatible with the digested fragment of pMTL83151_act. Both fragments were linked using circular polymerase extension cloning (CPEC)12 resulting in the final plasmid p83_tcb. Subsequently, plasmid DNA was transformed and amplified in E. coli DH5α cells. Then, plasmid DNA was isolated and the correctness of assembled genes verified by Sanger sequencing provided by GATC Biotech (Constance, Germany). Plasmid DNA from E. coli DH5α cells was isolated using 'Zyppy™ Plasmid Mini Prep' (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. Plasmid p83_PHB_Scaceti for PHB synthesis based on clostridial genes. For the construction of this plasmid, genes encoding thiolase A (thlA; CSCA_2635), 3hydroxybutyryl-CoA

dehydrogenase

(hbd;

CSCA_2636),

and

crotonase

(crt;

CSCA_2637) from C. scatologenes ATCC 25775 were cloned together with (R)-enoyl-CoA hydratase (phaJ; CLAOCE_21160) and PHA synthase (phaEC; CLAOCE_21150/21140) from C. acetireducens DSM 10703 under the control of Ppta-ack (C. ljungdahlii DSM 13583) into pMTL83151 Clostridium-E. coli shuttle plasmid. The clustered genes thlA, hbd, and crt were initially cloned into pMTL83151, together with Ppta-ack promoter (intermediate plasmid 8). Plasmid p83_PHB_Scaceti was constructed by using CPEC cloning. The clustered genes phaJEC were amplified from genomic DNA of C. acetireducens using primers C.aceti_phaC_fw and C.aceti_phaJ_rev to construct pJet1.2_C.aceti_PHB_locus (intermediate plasmid 9). This plasmid served as template to amplify the phaJEC cluster


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by using overlapping primers J5_phaJEC_fw and J5_phaJEC_rev. Additionally, the DNA fragment containing the pMTL83151 backbone together with the Ppta-ack promoter and the genes thlA, hbd, and crt from intermediate plasmid 8 were amplified using primer J5_scat7_fw and J5_scat7_rev. The CEPC merging of both fragments resulted in the final plasmid p83_PHB_Scaceti. Subsequently, plasmid DNA was amplified in E. coli cells, isolated, and verified by Sanger sequencing. CPEC primer design was performed using J5 device editor.13 Standard oligonucleotides were designed using CLC Main Workbench (Version 7.6.4.; Qiagen Bioinformatics). 2.2.3 Transformation of plasmids C. ljungdahlii has become a model strain for syngas fermentation and is phylogenetically closely related to C. coskatii, sharing 98.3 % genome similarity.14,15 C. coskatii is only described in patent literature and was lacking a functional transformation protocol. C. coskatii and C. ljungdahlii were transformed by applying the protocol published by Leang et al.16 Briefly, all oxygen-sensitive steps were carried out in an anaerobic chamber with a N2 + H2 (95 % + 5 %) atmosphere. In order to generate competent clostridial cells, an overnight culture grown at 37 °C in 100 mL Tanner mod. medium supplemented with 40 mM fructose and 40 mM DL-threonine was harvested at an optical density (OD600) of app. 0.4 by centrifugation (9,418 g, 10 min, 4 °C). Cells were washed twice with 50 mL anoxic SMP buffer (270 mM sucrose, 1 mM MgCl2, 7 mM sodium phosphate, pH 6) and suspended in 0.6 mL of the same buffer. Afterwards, 120


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µL anoxic anti-freezing buffer (60 % (v/v) DMSO and 40 % (v/v) SMP buffer, pH 6) were added to the competent cells. These cells were used directly or stored in cryo tubes at 80 °C for future use. The transformation was carried out using 25 µL competent C. ljungdahlii or C. coskatii cells mixed with 3-5 µg of plasmid DNA. The mixture was transferred to a pre-cooled 0.1-cm gap electroporation cuvette (Biozym Scientific GmbH, Vienna, Austria). Electroporation of clostridial cells was achieved using a 'GenePulser® II with Pulse Controller Plus' (Bio-Rad, Hercules, CA, USA) adjusted to 0.625 kV, resistance of 600 Ω, and a capacitance of 25 µF. Cells were recovered in a Hungate tube filled with 5 mL Tanner mod. medium without antibiotic. The OD600 nm was controlled after transformation and as soon as the value doubled, thiamphenicol was added to a final concentration of 20 µg mL-1. In general, growth occurred 4-5 days after addition of antibiotic. A further transfer into fresh medium was performed before genomic DNA was isolated to verify 16S rDNA and the cloned plasmid by sequencing. Genomic DNA from clostridial cells (C. ljungdahlii, C. coskatii, and C. acetireducens) was isolated using 'MasterPureTM Gram+ DNA Purification Kit' (Biozym Scientific GmbH, Vienna, Austria).

Transmission electron microscopy (TEM) and fluorescence microscopy In order to obtain TEM pictures, C. coskatii and C. ljungdahlii samples were taken from heterotrophically grown (1-2 mL) and autotrophically grown cultures (3-5 mL) and


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centrifuged (604 g, room temperature for 5 min). Subsequently, the supernatant was discarded and cells were washed with fresh medium. Suspended cells were centrifuged again (5 min, 604 g, room temperature). The cells were suspended in a 1:1 mixture of Tanner mod. medium and fixation solution containing 5 % (v/v) glutaraldehyde, 58.4 mM sucrose, 0.2 M phosphate buffer (PBS, pH 7.3). Afterwards, cells were washed 5 times with 0.01 M PBS buffer and postfixed in 2% (v/v) aqueous osmium tetroxide. Cells were dehydrated in graded series of 1-propanol, block stained in 1% (w/v) of uranyl acetate and embedded in Epon (Sigma-Aldrich Chemie GmbH, Munich, Germany). Ultra-thin sections (80 nm) were contrasted with 0.3 % (w/v) lead citrate for 1 min and imaged in a Jeol 1400 TEM device (Jeol GmbH, Freising, Germany) at an acceleration voltage of 120kV. Samples for fluorescence microscopy were obtained from heterotrophically grown cells using a protocol of Galià et al.17 Respective images were taken using a Zeiss AxioObserver.Z1 Microscope (Zeiss, Oberkochen, Germany) equipped with an AxioCam MRm camera (Zeiss, Oberkochen, Germany) and a Plan-Apochromat 63x/1.40 Oil Ph3 objective (Zeiss, Oberkochen, Germany). All images were taken and adjusted with the same settings. An excitation/emission wavelength of 559/636 nm, together with an exposure time of 50 ms for Nile red was chosen. Pictures in bright field were set with a contrast of black/white 0/4,096 and for fluorescence 2,000/4,096, along with gamma values of 1.00.


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Isolation of PHB 26 x 500 mL of recombinant C. coskatii [p83_PHB_Scaceti] cells were grown under heterotrophic growth conditions with fructose as carbon source in Tanner mod. medium for about 120 h. Afterwards, cells were centrifuged (10 min, 11,325 g, 4 °C) using a Sorvall Lynx 4000 Superspeed Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) equipped with rotor FiberlightTM F12-6 x 500 LEX Fixed Angle Rotor (Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed once with 0.8 % (w/v) NaCl and centrifuged again (10 min, 11,325 g, 4 °C). The supernatant was discarded, cells were pooled together in a small amount of 0.8 % NaCl solution, stored in a freezer at -20 °C for 12 h and subsequently lyophilized for 72 h at -54 °C using freeze dryer ALPHA 1-4 LDplus (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). For PHB extraction, freeze-dried biomass (6.74 g) was mixed with 120 mL dichloromethane and stirred at room temperature for 2 h. The suspension was filtered two times and rinsed with dichloromethane. The solution was concentrated using a rotary evaporator (40 °C, 350 mbar). Afterwards, potential PHB was suspended in 10 mL dichloromethane and 75 mL of ice-cold methanol was added dropwise. The solution was filtered (Filter papers grade 388, Sartorius AG, Göttingen, Germany) and dried under a fume hood.18 PHB (244.8 mg) was further used for NMR, DSC, and GPC analysis.


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High Performance Liquid Chromatography (HPLC) analysis Fructose consumption and product formation was monitored using 500 µL of cell suspension withdrawn using a syringe. Samples were subsequently centrifuged (17,950 g, 30 min, 4 °C) and the supernatant was stored at -20 °C until HPLC analysis. Fructose consumption and production of acetate, ethanol, 3-HB, and 2,3-butanediol were measured using an Agilent 1260 Infinity Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a refractive index detector operated at 35 °C. 20 µL of culture supernatant were injected into the system while 5 mM of H2SO4 was used as mobile phase at a flow rate of 0.7 mL min-1. Separation was achieved using a CS-Chromatographie organic acid column with a length of 150 mm (CS-Chromatographie Service GmbH, Langerwehe, Germany) kept at 40 °C.

Gas Chromatography (GC) analysis for PHB quantification PHB was quantified after methanolysis with sulfuric acid, methanol, and chloroform.19 Monomeric methylesters were analyzed using a gas chromatography Perkin Elmer Clarus 680 GC system (Perkin Elmer, Waltham, MA, USA) equipped with an Elite-


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FFAP capillary column (Perkin Elmer, Waltham, MA, USA) having 30 m length, 0.32 mm ID, and 0.25 µm df. 2 µL of sample volume from the organic phase were injected into the GC system with a split ratio of 20. As carrier gases, 45 mL min-1 H2 and 450 mL min-1 synthetic air was used. A temperature program was used for efficient separation of hydroxyalkanoic acid methyl esters (120°C for 5 min; temperature ramp of 10 °C min1

until 220 °C; 220°C for 5 min), which were detected using a flame ionization detector

(FID). PHB from natural origin (Sigma-Aldrich Chemie GmbH, Munich, Germany) served as external quantification standard.

Nuclear magnetic resonance (NMR) NMR spectra were measured with a Bruker UltraShield 400 MHz NMR spectrometer (Bruker Biospin AG, Fällanden, Switzerland) at 295 K using a 5 mm BBI probe. The polymer was dissolved in CDCl3 before the analyses. Chemical shifts are given in ppm relative to the remaining signals of chloroform as internal reference (1H NMR: 7.26 ppm;

13C

NMR: 77.0 ppm). 1H NMR spectrum was recorded at 400.13 MHz with the

following parameters: 8.00 µs 90° pulse length, 6’983 Hz spectral width, 55 865 data points, 16 scans, and relaxation delay 10 s.

13C

NMR spectra were recorded at 100.61

MHz with 1H WALTZ 16 decoupling. Other parameters were chosen as follows: 14.50 µs 90° pulse length, 25’063 Hz spectral width, 32768 data points, 568 scans, 1.0 s relaxation delay, and field 1’800 Hz decoupling.


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Differential scanning calorimetry (DSC) The polymer was analyzed by DSC (Mettler Toledo DSC 823e, Mettler Toledo GmbH, Giessen, Germany) to determine the glass transition temperature (Tg), melting temperature (Tm), and degradation temperature (Td). The sample (7.48 mg) was first heated under helium atmosphere from 25 °C to 210 °C at a rate of 20 °C min-1 in order to suppress memory effect, then cooled down to -100 °C at a rate of -50 °C min-1, kept 5 minutes at -100 °C, and finally heated to 210 °C at a rate of 10 °C min-1.

Gel permeation chromatography (GPC) The average molecular weight (Mw) and the polydispersity index (PDI) of the polymer were measured by GPC using a Waters 6000 HPLC system (Waters Corporation, Millford, MA, USA) with a 1260 Agilent refractive index detector (Agilent Technologies, Santa Clara, CA, USA). About 5 mg of polymer were dissolved in 1 mL chloroform and 10 µL of this solution were injected in the system. Separation was achieved at 40 °C in a GPC column (PLgel 5 µm MiniMIX-C 250 × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA) coupled to a precolumn (PLgel 5 µm MiniMIX-C Guard 50 × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA) using chloroform as mobile phase with a flow rate of 0.3 mL min-1. Calibration was done with 10 polystyrene bead standards having a Mw


19


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ranging from 580 to 3’039’000 (GPC/SEC Calibration Kit PL2010–0100, Agilent Technologies, Santa Clara, CA, USA).

Results Construction of a 3-HB synthesis pathway As a straightforward approach, the genes phaCAB of the natural PHB producer strains Cupriavidus necator (3,875-bp fragment) and Burkholderia thailandensis (3,962-bp fragment) were subcloned under control of the promoter Ppta-ack from Clostridium ljungdahlii into the pMTL83151 Clostridium-E. coli shuttle plasmid (Supplementary Fig. 1a,b) and subsequently transformed into C. ljungdahlii. However, no recombinant PHBproducing strains of C. ljungdahlii could be obtained. This result could be due to three obvious reasons: i) different codon usage, ii) possible toxic effects of 3-HB monomer formation, and iii) the production of the recombinant proteins is toxic for the cells. The codon usage of C. necator and B. thailandensis differs significantly from C. ljungdahlii, thus possibly hindering efficient translation and enzyme expression. Codon optimization could help to overcome this obstacle. Therefore, codon-optimized phaCAB genes of C. necator based on the codon preference for C. ljungdahlii and C. coskatii were designed. However, these codon-optimized genes could not be correctly subcloned into E. coli, since mutations occurred in irregular intervals during the cloning procedure. Therefore, this approach was abandoned. In order to exclude a toxic effect of 3-HB, a


20

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Biomacromolecules

respective production plasmid was constructed. For the heterologous production of 3HB, a novel recombinant pathway was assembled consisting of the genes thlA (CA_C2873), ctfA/B (CA_P0163 and CA_P), and bdhA (CDIF630_02933) encoding thiolase A, CoA transferase A/B (from C. acetobutylicum), and a 3-hydroxybutyrate dehydrogenase (from Clostridioides difficile), respectively (Fig. 2a). The bdhA gene from C. difficile ATCC 630 showed high similarities to the gene encoding the well characterized enzyme BdhA (Rsph17025_1507) of Rhodobacter sphaeroides (Fig. 2b). Thus, it was likely that the enzyme reduces acetoacetate stereospecifically to (R)hydroxybutyrate by oxidizing NADH. According to the KEGG database the gene encoding BdhA in C. difficile is unique in clostridial species. Expression of the subcloned genes was controlled by the constitutive promoter PthlA (from C. acetobutylicum), and the final 8,343 bp plasmid was designated p83_tcb (Fig. 2c).

a

b

c

Figure 2. a) Metabolic pathways of acetogens leading to natural and recombinant products. Natural pathways of C. ljungdahlii and C. coskatii are shown in black and synthetic ones in purple. Gene product Aor shown in grey is present in C. ljungdahlii, but not in C. coskatii; b) tBLASTx comparison of bdhA genes (encoding 3-hydroxybutyrate dehydrogenase from

c


21


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Clostridioides difficile and Rhodobacter sphaeroides) showing 39.5 % similarity; c) plasmid p83_tcb for the synthesis of 3-hydroxybutyrate.

3-HB production in clostridial acetogens The synthetic 3-HB production pathway encoded on plasmid p83_tcb was used to construct the recombinant strains C. coskatii [p83_tcb] and C. ljungdahlii [p83_tcb]. C. coskatii [p83_tcb] produced heterotrophically 21.7 ± 0.3 mM of 3-HB, 64.9 ± 0.1 mM of acetate and consumed 33.5 ± 1 mM fructose, while reaching an optical density (OD600) of 3.6 ± 0.2 within a 71 h growth experiment. The control experiments using the parental strains C. coskatii (wildtype) and C. coskatii [pMTL83151] produced 80.3 ± 0.1 mM and 81.1 ± 0.5 mM acetate as sole end product and yielded an OD600 of 3.4 ± 0.2 and 3.4 ± 0.1, respectively (Fig. 3a). Autotrophically grown C. coskatii [p83_tcb] cells produced 0.98 ± 0.12 mM of 3-HB, 34.5 ± 2.6 mM of acetate, 1.4 ± 0.01 mM of ethanol and reached a max. OD600 of 0.40 ± 0.05 and after 32 days. C. coskatii (wildtype) and C. coskatii [pMTL83151] did not produce 3-HB, but reached slightly higher OD600 values of 0.63 ± 0.03 and 0.52 ± 0.08 (Fig. 3b). Autotrophically and heterotrophically produced 3-HB was a stable metabolic end product in C. coskatii [p83_tcb] and its detection provided evidence for both the functionality of the new synthetic 3-HB pathway expressing BdhA and a nontoxicity of this compound at least in the concentrations detected. The recombinant C. ljungdahlii [p83_tcb] strain did not produce any detectable amounts of 3-HB (< 1mM)


22

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Biomacromolecules

neither under heterotrophic nor under autotrophic growth conditions, but showed in both cases an increased ethanol production compared to experiments performed with C. ljungdahlii (wildtype) and C. ljungdahlii [pMTL83151] (Supplementary Fig. 2a, b).

Figure 3. Production of 3-hydroxybutyrate (3-HB) using C. coskatii [p83_tcb]. Growth experiments were performed in triplicates in comparison to strains C. coskatii (wildtype) and C. coskatii [pMTL83151] as controls (represented by filled squares, circles and triangles, respectively). a) 3-HB production under heterotrophic conditions using fructose as carbon source; b) 3-HB production under autotrophic conditions with syngas atmosphere. OD600 displayed in black, fructose in red, acetate in blue, ethanol in pink, 3-hydroxybutyrate in purple, and pH in brown. Detailed numbers are presented in Supplementary Table 2.

Construction of a clostridial PHB synthesis pathway PHB production has been reported for two clostridia, C. botulinum and C. acetireducens. 4,5

As codon-optimization of the proteobacterial pathway genes had been unsuccessful,

respective clostridial genes should be used for metabolic engineering. First, the C.


23


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botulinum genome was checked for PHB synthase genes. Surprisingly, no such genes could be detected. Also, BLAST comparisons of known PHB synthase genes with the C. botulinum genome did not lead to positive results. The original publication from Emeruwa and Hawirko4 based the conclusion of PHB synthesis by C. botulinum solely on granule formation detected by electron microscopy. However, such structures can also be resulting from glycan polymers, often reported for clostridia (e.g. granulose in case of C. saccharobutylicum).20 Genes required for glycan synthesis could indeed be detected in C. botulinum, in accordance with earlier reports of production of such compounds in C. botulinum21,22 Thus, C. acetireducens was checked next for the presence of PHB synthesis genes. In this organism, PHB production has been experimentally verified.5 As no genome sequence was available, the genome of C. acetireducens was sequenced.23 Indeed, PHB synthesis genes could be identified, which were used for recombinant pathway construction (Fig. 4a). The three genes phaJ (CLAOCE_21160) and phaEC (CLAOCE_21150 and CLAOCE_21140) are clustered in C. acetireducens and encode a (R)-enoyl-CoA hydratase as well as a type III PHA synthase. The genes phaJ and phaEC from C. acetireducens share high sequence similarity compared to genes encoding enzymes involved in PHB formation such as phaJ from Rhodospirillum rubrum or Haloferax mediterranei (Rru_A2964, HFX_1483; Fig. 4b) and phaEC from Synechocystis sp. PCC 6803 (Fig. 4c). Furthermore, Clostridium ludense (T514DRAFT_02527 and T514DRAFT_02526)

and

Clostridium

tetanomorphum

(Ga0042847_11162

and


24

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Biomacromolecules

Ga0042847_11163) also encode homologs of both phaJ and phaEC from C. acetireducens, while C. homopropionicum only carries homologs of phaEC (Ga0098769_127143 and Ga0098769_127142). All three clostridial species have not been investigated regarding PHB production. The other three genes of the PHB synthesis pathway originated from Clostridium scatologenes, namely thlA (CSCA_2635), hbd (CSCA_2636), and crt (CSCA_2637) that encode thiolase A, 3-hydroxybutyryl-CoA dehydrogenase, and crotonase, respectively. The decision to choose these genes from C. scatologenes instead of C. acetobutylicum was based on clustering in C. scatologenes and potentially problematic enzymatic features of ThlA of C. acetobutylicum (regulated by redox potential). All six assembled genes were set under the control of the constitutive Ppta-ack promoter (from C. ljungdahlii), resulting in plasmid p83_PHB_Scaceti (Fig. 4d).


25


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c

a

b

d phaJ – R hodospirillum rubrum ATCC 11170

phaJ – Clostridium acetireducens DSM 10703

phaJ – H aloferax m editerranei ATCC 335 00 100 %

250 bp

29 %

Figure 4. Genes and enzymes of a novel PHB pathway. a) synthetic PHB pathway expressed in C. coskatii b) tBLASTx comparison of phaJ genes (encoding (R)-enoyl-CoA hydratase from Clostridium acetireducens, Haloferax mediterranei, and Rhodospirillum rubrum showing 48 or 52 % similarity; c) plasmid p83_PHB_Scaceti for the synthesis of PHB); d) tBLASTx comparison of phaJEC genes (encoding (R)-enoyl-CoA hydratase and PHA synthase from C. acetireducens, C. homopropionicum, Synechocystis sp., C. ludense, and C. tetanomorphum). The visualization was done using the program Easyfig.24 Abbreviations: Pta, phosphotransacetylase; Ack, acetate kinase; AdhE, acetaldehyde/alcohol dehydrogenase; ThlA, thiolase A (C. acetobutylicum); CtfA/B, coenzyme A transferase subunits A and B (C. acetobutylicum); ThlA, thiolase A (C. scatologenes); Hbd, 3-hydroxybutyryl-CoA dehydrogenase (C. scatologenes); Crt, crotonase (C. scatologenes); PhaJ, (R)-enoyl-CoA hydratase (C. acetireducens); PhaEC, PHA synthase (C. acetireducens); [H], redox equivalent (one electron + one proton).

PHB production in clostridial acetogens Plasmid p83_PHB_Scaceti was transformed into C. coskatii and C. ljungdahlii, resulting in

construction

of

strains

C.

coskatii

[p83_PHB_Scaceti]

and

C. ljungdahlii


26

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Biomacromolecules

[p83_PHB_Scaceti] and successful PHB production. C. coskatii [p83_PHB_Scaceti] produced 3.4 ± 0.3 % of PHB per cell dry weight (CDW) and 56.5 ± 0.9 mM of acetate after 120 h in a heterotrophic growth experiment. The strain consumed 25.4 mM of fructose and reached an OD600 of 2.4 ± 0.1. The parental strain C. coskatii (wildtype) and C. coskatii [pMTL83151] produced no PHB but higher amounts of acetate (69.1 ± 0.3 and 76.5 ± 0.5 mM, respectively) as shown in Figure 5a. Under autotrophic conditions C. coskatii [p83_PHB_Scaceti] produced 1.2 ± 0.1 % PHB/CDW. Additionally, the strain produced acetate (53.2 ± 2.5 mM) as major and ethanol (2.6 ± 0.2 mM) as minor end product and reached a OD600 of 0.43 ± 0.02 using syngas as substrate (Fig. 5b). C. ljungdahlii [p83_PHB_Scaceti] produced heterotrophically 1 ± 0.04 % PHB/CDW while consuming 41.9 ± 1.3 mM of fructose. Autotrophically, this strain produced 1.2 ± 0.1 % PHB/CDW, which is in the range of PHB production of C. coskatii [p83_PHB_Scaceti] (Supplementary Fig. 2c, d). These results confirm the functionality of the new synthetic PHB production pathway expressed in clostridial acetogens, either using fructose or synthesis gas as carbon substrate.


27


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a

b

Figure 5. Production of PHB using C. coskatii [p83_PHB_Scaceti] compared to the control strains C. coskatii (wildtype) and C. coskatii [pMTL83151] (represented by filled squares, circles and triangles, respectively). a) PHB production under heterotrophic conditions using fructose as substrate; b) PHB production under autotrophic growth conditions with syngas atmosphere. OD600 displayed in black, fructose in red, acetate in blue, ethanol in pink, and pH in brown. Bars represent quantified PHB/CDW ratios of C. coskatii [p83_PHB_Scaceti]. Detailed numbers are presented in Supplementary Table 2.

Verification and characterization of PHB produced by recombinant acetogens In order to visualize the electron transparent intracellular granules of PHB in the electron dense cytosol, respective recombinant cells were analyzed by transmission electron microscopy (TEM). The number and size of PHB granules provide an indication on the quality of the produced polymer and its distribution in the cell population. Experiments conducted under heterotrophic condition revealed that C. coskatii [p83_PHB_Scaceti] produced increasing amounts of PHB over the course of time (Fig. 6a). After 23 h, few cells contained PHB granules with a rather small diameter of about 0.18 µm. After 31 h of incubation, the average diameter of PHB granules increased to approximately 0.26 - 0.41 µm and cells harbored 3 - 4 PHB granules. After


28

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Biomacromolecules

47 h, some cells contained 5 - 6 granules with a diameter of approximately 0.45 - 0.49 µm. The control strain C. coskatii [pMTL83151] did not show any electron transparent granule formation over the course of time (Fig. 6b). TEM micrographs obtained from heterotrophically grown C. ljungdahlii [p83_PHB_Scaceti] cells showed PHB granules with 2-fold larger diameter (0.8 µm), compared to C. coskatii [p83_PHB_Scaceti] (Supplementary Fig. 3a). TEM micrographs obtained from C. coskatii [p83_PHB_Scaceti] cells grown under autotrophic conditions demonstrated the presence of PHB granules (Fig. 6c, 6d), even below the quantification limit of < 1 % PHB/CDW (Fig. 5b). The formation of PHB became apparent after 14 days of growth, as indicated by small granules (approx. 36 nm). The granules increased in diameter and reached approximately 0.29 µm after 26 days and 1 µm after 46 days. At that time, respective cells were deformed into a clubshaped structure by the intracellular PHB granules. In C. ljungdahlii [p83_PHB_Scaceti] cells, PHB granules were observed with diameters reaching approximately 2.2 µm (Supplementary Fig. 3b). Thus, it can be concluded that the strains C. coskatii [p83_PHB_Scaceti] and C. ljungdahlii [p83_PHB_Scaceti] grown on syngas were able to produce PHB and store it intracellularly.


29


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Figure 6: Transmission electron microscopy micrographs of recombinant C. coskatii strains over course of time while using fructose or syngas as carbon and energy source. a) C. coskatii [p83_PHB_Scaceti] grown on fructose with electron transparent intracellular granules of PHB; b) C. coskatii [pMTL83151] grown on fructose showing no granules of PHB (served as negative control); c) C. coskatii [p83_PHB_Scaceti] grown on syngas showing granules of PHB from day 14 onwards; d) C. coskatii [pMTL83151] showing no granules of PHB (served as negative control). Scale bars represent 3 µm.

In order to characterize the chemical properties of PHB, C. coskatii [p83_PHB_Scaceti] cells were grown on 40 mM fructose and harvested at an OD600 of 1.91 after 120 h. The PHB production was confirmed by Nile red staining of cells (Fig. 7 a, b and Supplementary Fig. 4) Subsequently, PHB was extracted using dichloromethane and


30

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Biomacromolecules

purified by precipitation in cold methanol. A total amount of 244.8 mg of PHB was recovered from 6.74 g of freeze-dried biomass, which corresponds to 3.6 % PHB/CDW. Nuclear magnetic resonance (NMR) exhibited an atomic shift pattern of 1H and

13C

typical for PHB, providing evidence that the polymer purified from C. coskatii [p83_PHB_Scaceti] was indeed a PHB homopolymer (Fig. 7c, d). The thermal properties measured by differential scanning calorimetry (DSC) also supported a PHB homopolymer structure (Fig. 7e): glass transition temperature (Tg) of 4.1 °C, crystallization temperature (Tc) of 54.4 °C, melting temperature (Tm) of 174.3 °C, crystallization enthalpy (ΔHc) of 30.3 J g-1, and a melting enthalpy (ΔHm) of 79.4 J g-1. Gel permeation chromatography (GPC) revealed a calculated molecular weight (Mw) of 207,464 g mol-1 with a polydispersity index (PDI) of 3.3 (Fig. 7f).


31


a

C. coskatii [p83_PHB_Scaceti]

b

C. coskatii [pMTL83151]

bright-field

red

c

d

e

f

Page 32 of 60

merge

Figure 7: Micrographs of C. coskatii [p83_PHB_Scaceti] stained with lipophilic fluorescence dye Nile red and chemical properties of produced PHB. a) Fluorescence microscopical images of C. coskatii [p83_PHB_Scaceti] cells; b) respective images of C. coskatii [pMTL83151] serving as negative control. Scale bars represent 10 µm; c) 1H NMR spectra of isolated PHB; d)

13C

NMR spectra of isolated PHB; e) DSC to analyze

the glass transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm); f) GPC to determine the average molecular weight and the polydispersity index (PDI).

4. Discussion


32

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Biomacromolecules

There are few publications describing microbial strains producing 3-HB. In literature, production of 3-HB mainly focused on in vivo/ex vivo depolymerization of synthesized PHB/PHA25,26 or by generating metabolic pathways directly leading to 3-HB. Metabolic pathways leading to 3-HB have been established for example in recombinant strains of Escherichia coli27,28, Synechocystis sp. PCC 680329, or Clostridium ljungdahlii30 by using genes encoding for thiolase A, acetacetyl-CoA reductase, 3-hydroxybutyryl-CoA dehydrogenase, and thioesterase II (PhaA or Thl, PhaB or Hbd, and TesB) or phosphotransbutyrylase with butyrate kinase (Ptb and Buk), respectively. Here, a novel synthetic 3-HB production pathway was engineered by using genes encoding for thiolase A, CoA-transferase A/B, and (R)-3-hydroxybutyrate dehydrogenase. This new pathway enabled C. coskatii [p83_tcb] to produce (R)-3-HB under heterotrophic (21.7 ± 0.3 mM) and autotrophic (0.98 ± 0.12 mM) conditions, clearly providing a proof of concept. The assumption that the (R)-3-HB enantiomer is produced, is solely based on the described properties of the enzyme (R)-3-hydroxybutyrate dehydrogenase. Recently, Woolston et al.30 achieved titers in a comparable range using an engineered C. ljungdahlii strain (growing either heterotrophically on fructose or autotrophically on CO2 + H2) that redirects the carbon flux by a CRISPR interference approach. Even so, it has to be pointed out that the pathway (using PhaA, Hbd, TesB) implemented by Woolston et al.30 yields (S)-3-HB. In contrast, only the (R)-configuration of 3-HB possesses a chiral center that can be incorporated into the polymer.


33


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The recombinant Synechocystis strain produces autotrophically up to 4.8 mM (R)3-HB during photosynthetic cultivation29, demonstrating a higher production titer compared to the mentioned C. coskatii and C. ljungdahlii strains. A phototrophic cultivation approach in large scale is challenging due to an unfavorable surface to volume ratio limiting the CO2 consumption driven by a light-dependent energy metabolism. Light-independent 3-HB production by syngas fermentation thus seems feasible. The major bottleneck in the newly presented synthetic pathway is the conversion of acetacetyl-CoA to acetoacetate by a CoA transferase, which depends on high concentrations of acetate. The enzyme used originates from C. acetobutylicum and is known to have a high Km value for acetate of 1200 mM.31 By using a CoA transferase with a lower Km value for acetate, production efficiencies and product titer could possibly be improved. A lower Km might trigger 3-HB production in the respective recombinant C. ljungdahlii strain as well, which did not produce any detectable amounts of 3-HB. In contrast to C. coskatii, C. ljungdahlii possesses aor genes encoding an aldehyde:ferredoxin oxidoreductase.15 Aor catalyzes the reaction of acetate to acetaldehyde, which is further converted by an alcohol dehydrogenase to ethanol. It is likely that higher intracellular acetate concentrations fostered 3-HB production in C. coskatii [p83_tcb], while C. ljungdahlii [p83_tcb] could not reach the respective acetate threshold due to the conversion of acetate to ethanol via Aor.32 Interestingly, C.


34

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Biomacromolecules

ljungdahlii [p83_tcb] showed increased ethanol titers in comparison to the control strains. ScanProsite33 analysis of the BdhA sequence revealed a common feature of short-chain alcohol dehydrogenases (140-168 aa with an active site at 153 aa) that suggests that BdhA can use acetaldehyde as alternative substrate and form ethanol. Substrate specificity of BdhA from Rhodobacter sphaeroides was shown to be not restricted to acetoacetate; however, the conversion of acetaldehyde to ethanol was not tested.34 Generally, aor lacking strains such as C. coskatii might be more suitable for recombinant product formation (e.g. acetone, isopropanol, 3-hydroxybutyrate) that depend on high concentrations of acetate when using ctfA/B. Although BdhA of Clostridioides difficile was not enzymatically characterized before, it was preferred over BdhA of R. sphaeroides because of a higher similarity in codon usage to acetogenic clostridia (68 % GC bdhAR.sph vs. 34 % GC bdhAC.dif compared to 31 % GC in the genomic DNA of C. ljungdahlii and C. coskatii), which is known to effect translation efficiencies significantly.35,36 For PHB production, initially two different PHB synthesis pathways have been considered. The first involved the genes phaA, phaB, and phaC of Cupriavidus necator and Burkholderia thailandensis and was used to engineer PHB production in heterologous hosts several times before. However, the native subcloned genes turned out not to be functional in respective recombinant C. ljungdahlii strains and the codon optimized genes could not even be subcloned in E. coli. It must be noted that a recent publication


35


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reported codon optimization of C. necator phaCAB genes and successful expression of PHB formation in C. autoethanogenum.37 Due to our failure in obtaining codon-optimized genes, we designed a novel synthetic PHB pathway consisting of genes from bacteria, phylogenetically more closely related to C. ljungdahlii and C. coskatii. C. acetireducens is a non-motile, Gram-positive, anaerobic, rod-shaped bacterium and was first isolated and described by Örlygsson et al.38 The strain oxidizes various amino acids (e.g. alanine, leucine, isoleucine, valine, serine, threonine) and uses acetate as an electron acceptor. Already, this initial description provided the first hint that C. acetireducens is able to produce PHB by showing electron transparent granules in the cytosol of the strain. Then, Girbal et al.5 reported that C. acetireducens accumulates PHB when grown on leucine and acetate. The pathway for PHB formation in C. acetireducens is still unclear, but genome analysis indicated an enoyl-CoA hydratase-mediated PHB synthesis pathway. C. acetireducens genome sequence analysis revealed the presence of a phaJEC gene cluster. The amino acid sequence of PhaJ (CLAOCE_21160) annotated as (R)enoyl-CoA hydratase, exhibits 48% similarity to the PhaJ protein of Haloferax mediterranei R-4 ATCC 33500.39 In H. mediterranei, PhaJ acts as (R)-enoyl-CoA hydratase and converts crotonyl-CoA to (R)-3-hydroxybutyryl-CoA, whereas the PHB metabolism is proposed to be fueled either by glucose or via ß-oxidation of alkanoic acids with crotonyl-CoA as central intermediate.40 The presence of phaJ in C. acetireducens, C. ludense, and C. tetanomorphum (which are all butyrate producers) and its absence in C.


36

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Biomacromolecules

homopropionicum (which is not producing butyrate) indicates the use of the different isomers for different clostridial pathways. (S)-3-hydroxybutyryl-CoA is obviously required for butyrate formation, whereas (R)-3-hydroxybutyryl-CoA is the starting point for PHB synthesis. Thus, the phaJ gene represents an important metabolic branch point in clostridia, which must be tightly regulated. Autotrophic PHB production by e.g. C. necator is long been known, leading to yields of app. 40% PHB/CDW.41 However, until recently syngas could not be used as the only carbon source. Then, a recombinant C. necator strain expressing the cox gene cluster coxMSLDEFG of Oligotropha carboxidivorans OM5 was constructed, which expressed the respective aerobic carbon monoxide dehydrogenase.41 This strain showed an 20 % increased PHB production when cells were cultivated under 80% of air, 10% CO, and 10 % H2 compared to cells that do not express the cox gene cluster. This is a very promising approach as well, however, technically somewhat hampered by using the very explosive syngas-oxygen mixture. Currently, only recombinant C. necator and recombinant autotrophic acetogenic clostridia are able to grow on syngas and to form PHB concomitantly. PHB synthesis has also been demonstrated with Rhodospirillum rubrum using syngas in combination with a heterotrophic substrate under dark and anaerobic growth conditions.42,43 Detailed analysis showed that PHB synthesis was significantly enhanced when in addition to syngas acetate was provided in the medium.44 However, when there was no syngas present, acetate could not be


37


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metabolized by R. rubrum under dark and anaerobic culture conditions and consequently no cell growth was detectable.45 TEM

micrographs

of

C.

coskatii

[p83_PHB_Scaceti]

and

C.

ljungdahlii

[p83_PHB_Scaceti] clearly indicated that only some cells produce high amounts of PHB (thus providing a proof of concept), while others did not show any PHB formation. This heterogeneity might come from an uneven distribution of PHB granules during cell division, most likely due to the lack of DNA/PHB-binding phasins that are present in natural PHB producers, such as PhaM in C. necator.46 A further issue could have been the low gaseous supply under applied batch cultivation conditions. The gas transfer rates can be improved by cultivating respective strains in bioreactors, while applying continuous gas sparging with overpressure.47 Such an approach could significantly improve PHB production efficiencies, as shown for ethanol production with C. ljungdahlii C-01 that rose from 38.4 g L-1 d-1 at 1 bar to 360 g L-1 d-1 at 6 bar in a continuous fermentation process.48 The quality of the clostridial PHB was in the range of what can be achieved by C. necator and other known PHB producers. NMR and DSC analysis

clearly

confirmed

the

identity

of

PHB

isolated

from

C.

coskatii

[p83_PHB_Scaceti]. GPC measurements revealed a polymer size of about 207,464 g mol1

which is six times smaller than of PHB isolated from the natural producer C. necator

(approximately 1,200,000 g mol-1) using a comparable isolation method.49 The size of the polymer chain often depends on the organism and the expressed aliphatic structural


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proteins bound to the surface of the biopolymer (often referred to as phasins)50, but on the time point of analysis as well.51 Phasins are a very diverse group of proteins characterized in different PHB-producing organisms.52 However, no annotated phasinencoding genes could be detected in the genome sequence of the natural PHB producer C. acetireducens. Further work is needed to elucidate the regulation of the granule size in recombinant clostridial acetogens or in C. acetireducens itself as well as the possible benefits of expressing phasin genes heterologously along with the PHB synthesis gene cluster.

Conclusions Clostridia are somewhat underrepresented when elucidating bacterial PHB metabolism. However, some species do contain type III PHA synthases (phaEC). It is interesting to note that those species able to produce butyrate possess in addition an (R)-enoyl-CoA hydratase (phaJ), suggesting that (R)-3-hydroxybutyryl-CoA is the starting point for PHB synthesis, whereas the (S) isomer is used for butyrate formation. The successful transfer and expression of phaJ and phaEC in autotrophic gas-fermenting clostridia leading to PHB formation now opens the possibility to establish an economically viable route to biodegradable plastics from waste and greenhouse gases, as the gas


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fermentation technology has meanwhile matured and is already performed at industrial scale. A novel metabolic pathway leading to 3-hydroxybutyrate has also been engineered that will allow similarly better economic production of this platform chemical.

Supporting information Plasmids for synthesis of PHB using C. necator and B. thailandensis genes (Figure S1), heterotrophic and autotrophic growth experiments with wildtype and recombinant C. ljungdahlii strains for production of 3-HB or PHB (Figure S2), transmission electron microscopy pictures of recombinant C. ljungdahlii at different time points during growth on fructose or syngas (Figure S3), fluorescence microscopy of heterotrophically grown recombinant C. ljungdahlii stained with lipophilic fluorescence dye Nile red (Figure S4), genes subcloned and plasmids constructed (Table S1), primers used for plasmid construction (Table S2), genetic features of PHB and 3-HB synthesis gene clusters from C. necator and B. thailandensis as well as genes from C. scatologenes, C. acetireducens, C. acetobutylicum, and C. difficile (Table S3); maximal optical density, fructose consumption, and product formation data of wildtype and recombinant C. ljungdahlii and C. coskatii strains shown in Fig. 3 and Fig. S2 (Table S4).


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Acknowledgment This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 311815 (SYNPOL project). We thank Renate Kunz from Central Facility for Electron Microscopy of Ulm University as well as Antoine Fornage, Mònica Bassas-Galià, and Fabrice Micaux from HES-SO Valais-Wallis for technical assistance. Furthermore, we thank Daniel Heinrich and Matthias Raberg for answering many questions regarding PHB purification and analysis.

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For table of contents only


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Figure 1: Schematic overview of autotrophic 3-HB and PHB synthesis pathways in C. ljungdahlii and C. coskatii, their substrate utilization, and product formation. Naturally occurring products ethanol and acetate are shown in black, recombinant products poly(3-hydroxybutyrate) and 3-hydroxybutyrate are shown in purple. If CO is used as carbon source, CO2 will be an end product as well. Abbreviations: [H], redox equivalent (one electron + one proton); PTS, phosphotransferase system; RNF, proton-translocating ferredoxin:NAD+ oxidoreductase complex; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NAD+, nicotinamide adenine dinucleotide oxidized; NADH, nicotinamide adenine dinucleotide reduced; Fd, oxidized ferredoxin; Fd2-, reduced ferredoxin; THF, tetrahydrofolate; H2, hydrogen; CO, carbon monoxide; CO2, carbon dioxide. 264x182mm (300 x 300 DPI)


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b phaJ – Rhodospirillum rubrum ATCC 11170

phaJ – Clostridium acetireducens DSM 10703

phaJ – Haloferax mediterranei ATCC 33500 250 bp

100 % 29 %


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