Engineered fusion proteins for efficient protein secretion and

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Engineered fusion proteins for efficient protein secretion and purification of a human growth factor from the green microalga Chlamydomonas reinhardtii Thomas Baier, Dana Kros, Rebecca Christine Feiner, Kyle Jonathan Lauersen, Kristian Mark Müller, and Olaf Kruse ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00226 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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ACS Synthetic Biology

Engineered fusion proteins for efficient protein secretion and purification of a human growth factor from the green microalga Chlamydomonas reinhardtii Author List: Thomas Baier1, Dana Kros1, Rebecca C. Feiner2, Kyle J. Lauersen1, Kristian M. Müller2 and Olaf Kruse1,* *Corresponding Author: Olaf Kruse, [email protected] Phone: +49 521 106-12258, Fax: +49 521 106-12290

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

Abstract Light-driven recombinant protein (RP) production in eukaryotic microalgae offers a sustainable alternative to other established cell-culture system. RP production via secretion into the culture medium enables simple product separation from the cells adding a layer of process value in addition to the algal biomass which can be separately harvested. For the model microalga Chlamydomonas reinhardtii, a broad range of molecular tools have been established to enable heterologous gene expression, however, low RP production levels and unreliable purification from secretion concepts have been reported. Domesticated C. reinhardtii strains used for genetic engineering are often cell-wall deficient. These strains nevertheless secrete cell-wall components such as insoluble (hydroxy)proline-rich glycoproteins into the culture media which hinder downstream purification processes. Here, we attempted to overcome limitations in secretion titers and improve protein purification by combining fusion partners that enhance RP secretion and enable alternative aqueous two-phase (ATPS) RP extraction from the culture medium. Protein fusions were strategically designed to contain a stably folded peptide which enhanced secretion capacities and gave insights into (some) regulatory mechanisms responsible for this process in the algal host. The elevated protein titers mediated by this fusion were then successfully applied in combination with a fungal hydrophobin tag, which enabled protein purification from the complex microalgal extracellular environment by ATPS, to yield functional recombinant human epidermal growth factor (hEGF) from the algal host. Keywords: Microalga. Chlamydomonas reinhardtii. Transgene expression. Recombinant protein secretion. Hydrophobin. Detergent-based aqueous two-phase extraction.


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Microalgae naturally contain a great diversity of biotechnologically interesting products such as lipids, pigments, and polysaccharides.1 Eukaryotic algal cells also hold potential as platform organisms for recombinant protein (RP) production owing to a range of favorable characteristics such as sub-cellular compartmentation, secretion,2 capacity for post-translational modifications,3 and sophisticated protein folding machineries.4 Compared to other eukaryotic RP platforms such as mammalian, insect, or plant cell culture, microalgal strains exhibit favorable properties such as inexpensive cultivation in simple mineral-salt solutions, rapid genetic manipulation, fast scaleup,5 lack of human pathogens, and reduced risk of culture contamination.3 However, generally poor transgene expression capacities and low RP titers have prevented extended production processes at commercial scales. The model green freshwater chlorophycean microalga Chlamydomonas reinhardtii currently has the most mature and well developed genetic tools for heterologous transgene expression of any eukaryotic alga. Engineering of both chloroplast and nuclear genomes has been demonstrated, expanding the range of possible RP expression applications with this host.6,7 RP producing C. reinhardtii can also be readily cultivated at a large scale, a 100 L outdoor greenhouse facility was used to produce 3.28 mg L-1 of the therapeutic bovine milk amyloid protein from C. reinhardtii expressing this protein from its chloroplast genome.8 Expression of RPs from the nuclear genome can allow targeting of RPs for secretion into the extracellular space, similar to strategies used in other RP cell culture production concepts.9–11 Secretion of a target RP into the culture medium results in a physical separation of the RP and the algal biomass, which can be independently harvested and may itself be of value for other applications. Successful RP secretion from the C. reinhardtii has been previously demonstrated using robust reporter proteins such as the Gaussia princeps luciferase (gLuc),12–14 mCherry15–17 and mVenus.18



To the best of our knowledge, only four biotechnologically relevant RPs have been secreted from C. reinhardtii: human erythropoietin (EPO),13 Trichoderma reesei xylanase I,19 human vascular endothelial growth factor VEGF,20 and the Lolium perenne ice binding protein (LpIBP).21 The accumulated protein titers differ from 100 µg L-1 EPO in the culture media13 of up to 10 mg L-1 LpIBP after 96 h when it was fused to the gLuc reporter at the C-terminus.21 It was also recently found that the modification of a secreted mVenus reporter protein to contain repetitive serineproline repeat sequences on its C-terminus triggered the addition of O-linked glycan residues and led to an increase of mVenus accumulation from 1.3 mg L-1 to 15 mg L-1 after 168 h of cultivation.18 Intensive domestication of C. reinhardtii has occurred throughout its history as a model organism by successive modification of the originally isolated wild-type alga.22 Chemical and UV induced mutagenesis has led to variant strains with reduced cell walls which are more amenable to extraction as well as agitation-based transformation techniques compared to their cell-wall containing progenitors.23 The C. reinhardtii wild-type cell wall is composed of seven principal (hydroxy)proline rich chaotrope soluble protein layers24 which form an insoluble glycoprotein framework.25,26 Many different cell wall deficient C. reinhardtii strains exist and the cw-15 variant as well as its derivatives27 are widely used as recipients for genetic transformation. These mutants accumulate the precursor proteins of the wall in normal amounts, however, fail to assemble them into intact layers.28 Unassembled cell wall proteins are secreted into the extracellular matrix29 and form protein aggregates which negatively affect downstream processes (e.g. filtration, chromatography). Although, affinity chromatography of RPs has been demonstrated from C. reinhardtii culture media, characteristically low yields have been observed due to inherent issues of low RP secretion amounts from the host and contaminating cell wall proteins which physically hinder affinity chromatography. Here, we investigate the potential use ACS Paragon Plus Environment

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of a C-terminal fusion protein21 to boost secreted RP titers, we couple this enhanced secretion to an alternative extraction method based on an amphiphilic hydrophobin protein tag and use a scalable, detergent-based aqueous two-phase protein extraction system (ATPS) to enable purification of target RPs from contaminating cell-wall proteins found in the extracellular space.30 The combined system enabled recombinant production via secretion of the human epidermal growth factor (hEGF) from C. reinhardtii which, for the first time, could be successfully purified from contaminating extracellular proteins in a native and functional form.



Results and Discussion Selection of an improved secretion signal It has been reported that C. reinhardtii contains more than 2000 endogenous protein secretion signal peptides and that the recombinant protein secretion efficiency is affected by which is used.16 Here, the previously reported N-terminal secretion signals of the C. reinhardtii proteins arylsulfatases 1 and 2 (ARS1, ARS2),13 carbonic anhydrase 1 (cCA),14 and iron assimilatory protein (FEA2)31 were compared quantitatively for their capacity to target a codon optimized G. princeps luciferase (gLuc) into the extracellular space. The four constructs were compared for their secretion efficiency by quantifying relative bioluminescence of the gLuc reporter in the culture medium 48 h hours after inoculation. All four constructs were found to target the reporter protein into the secretory pathway, however with different efficiencies (Figure 1). The cCA sequence was reported to allow efficient protein secretion of the pOptimized gLuc reporter construct.21,32 In this study, it allowed protein accumulation to comparable titers as the ARS1 and ARS2 sequences. The FEA2 secretion signal outperformed the previously used sequence and gave stronger bioluminescence signals, ~2.2 fold higher than the cCA signal. Recently, the secretion signal of the FEA1 protein was also successfully used to drive secretion15 showing the conserved effectivity of the secretion signal of iron assimilatory proteins in C. reinhardtii.31,33 Under iron deficient conditions, C. reinhardtii has a high affinity for iron absorption, partly enabled by the fast secretion of CrFEA1 and its homologs which are responsible for the transport of reduced Fe2+.31,34 Effective targeting to the extracellular space mediated by the FEA signal sequences is likely an additional factor for this rapid response to iron deficiency.


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LpIBP fusions and efficient recombinant protein secretion Although translocation of proteins across the endoplasmic reticulum (ER) membrane is the initial step of protein secretion, the path from the ER to the extracellular space involves many sorting steps, which continually select membrane and lumenal proteins for packaging and transport.35 Correct protein folding in the ER as well as accurate post-translational modification regulate final targeting to the extracellular space36 and affect the protein secretion efficiency. The C-terminus of target proteins play a unique regulatory role in ER transport as they have the capacity to trigger ER retention and prevent cell exit.37 In a previous report, the C-terminus of the mVenus reporter protein was modified by adding serine-proline-(SP20)-repeat sequences harboring O-linked glycan binding motifs which led to increased mVenus secretion levels from C. reinhardtii.18 Among the existing reports of C. reinhardtii as an RP secretion host, the LpIBP represents the most successful example of active target protein secretion with accumulation levels of 10 mg L-1 and a confirmed biological activity in concentrated media.21 Secretion of the LpIBP was conducted as a C-terminal fusion to the gLuc reporter14 and it was not further investigated whether the particular fusion orientation affects protein secretion or protein accumulation. To elucidate the role of LpIBP in relation to secretion efficiencies, the nucleotide sequence was amplified in this work to create both N- and C-terminal fusions to two different reporter proteins (gLuc, Figure 2A and mVenus, Supplementary Figure S1). Addition of the LpIPB in both positions caused slight reductions in the secretion levels of the mVenus fusion proteins, likely due to the increased protein size. The N-terminal LpIBP_gLuc construct exhibited similar secretion levels when compared to the gLuc-alone control. In contrast, when the LpIBP was placed as a C-terminal fusion (gLuc_LpIBP) the bioluminescence signal was markedly increased by ~3.5 fold. It is likely that this increase in protein secretion is due to a



favorable protein fold of the gLuc mediated by the LpIBP in this position, which leads to efficient transport through the secretory pathway and consequent protein export. The LpIBP tertiary structure is a tight ß-roll, which is highly stable and re-folds after denaturation38,39 and it is possible that protein stability encourages the passage of gLuc_LpIBP through the secretory pathway. In addition to factors such as amino acid composition and protein tertiary structure, post translational modifications are potential signals that may affect secretion levels. Addition of glycan residues can positively affect protein stability in the extracellular space and increase the accumulation titer.40 The LpIBP contains six potential N-glycosylation motifs and was found to be at least partially glycosylated as determined by a lower running behavior in SDS-PAGE and immunodetection when targeted to the cytoplasm than when secreted.14 To elucidate whether protein structure or glycosylation affect secretion of the gLuc_LpIBP fusion, we performed a truncation study of the LpIBP and compared the native glycosylated protein with a synthetic variant, which is predicted to be non-glycosylated (Figure 2B and C). Glycosylation, protein structure, and efficient export The six potential N-glycosylation motifs of the LpIBP were removed by amino acid substitutions and the modified sequence was fused to the C-terminus of the gLuc reporter as indicated in Figure 2B. Both, the native LpIBP and its non-glycosylated variant led to increased secretion levels of the gLuc reporter compared to the reporter alone, ~3.5 and ~3.3 fold higher respectively. The resulting bioluminescence signals of the non-glycosylated version were more consistent, whereas the original glycosylated sequence exhibited a variety of outliers in the analyzed mutant population. N-glycosylation may contribute to protein stability in the extracellular space,40


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however, was not responsible for the increased rates of secretion observed here for the gLuc_LpIBP reporter fusion. The LpIBP is composed of eight ß-helical coils with two flat surfaces, one of which is responsible for its ice binding activity (Figure 2C).41 To elucidate the impact of protein structure on secretion, a truncation study according to the LpIBP tertiary structure was performed using the nonglycosylated variant amino acid sequence. Truncations of six (267 bp), four (183 bp) and two (96 bp) helical coils of the LpIBP were generated and compared to the full length non-glycosylated sequence (8 coils, 354 bp) as gLuc-LpIBP(n-coils) reporter fusions. All truncations exhibited higher bioluminescence levels compared to the levels of the gLuc control, however, without correlation to the coil number. A truncated four-coil LpIBP sequence is predicted to maintain its ß-roll tertiary structure (iTasser,42 Supplemental Figure S2) and exhibited the highest secretion level. Other truncations (two coils or six coils respectively) had reduced secretion levels compared to the full-length LpIBP. The modular setup of the pOptimized vector contains a native linker sequence composed of a FactorXa site and restriction enzyme recognition sites (amino acid sequence: IEGR-DI) downstream of the gLuc sequence. Eliminating this linker sequence by directly fusing the LpIBP to the gLuc C-terminus resulted in comparable secretion levels as the levels of the gLuc control and loss of the boosting effect mediated by the LpIBP likely due to improper protein folding (Supplementary Figure S2). We quantified the fraction of target RPs in the media and the remaining protein in the biomass by Western blotting and immunodetection (Figure 2D). Whereas for the gLuc construct noticeable amounts of the protein remained intracellular, we found that the addition of LpIBP efficiently mediated export of the reporter from the cell, as minimal signals were detected in the biomass. Elevated secretion levels were also observed when a second reporter protein (mVenus) was fused to the N-terminus of the gLuc_LpIBP (mVenus_gLuc_LpIBP, Supplementary Figure S3). We conclude from these results ACS Paragon Plus Environment


that the gLuc_LpIBP serves as an improved and very efficient transport vehicle to more effectively secrete N-terminally fused target RPs. Fusion protein design and cultivation for secreted RP production The improved secretion of the gLuc_LpIBP 4-coil reporter combined with the FEA2 secretion signal used here (hereafter, gLuc4c) not only improves screening efforts, but can be used for efficient protein targeting into the extracellular space. In this study, the human epidermal growth factor (hEGF) was used as a RP target to test protein secretion efficiency with this construct. Human EGF is a small, mitotic growth stimulating polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices as well as cell cultures at low concentrations.43 It has been previously reported that authentic structural conformation and optimal biological activity can be detected when three disulfide bonds are present in the protein.44,45 The capacity for disulfide bridge formation and accurate eukaryotic protein folding makes C. reinhardtii a suitable protein production host for this and other complex RP targets.46 To this end, a codon-optimized gene sequence coding for the hEGF protein was cloned in order to generate a fusion of this protein to the N-terminus of the gLuc4c (Figure 3). For subsequent downstream processes, we added a hydrophobin (HFBI) protein tag into the transport vehicle between the gLuc and LpIBP (gLuc_HFBI_4c), which should enable aqueous two-phase protein separation from contaminating extracellular proteins. The increased secretion mediated by the Cterminal LpIBP was maintained with this larger construct (Figure 3A), although the overall protein secretion was lower, likely due to the complex HFBI protein structure. The FEA2 secretion signal, hEGF and HFBI sequences were designed to contain copies of the rbcS2 intron 1 since this was shown to contribute to high expression rates.47 When cultivated under mixotrophic conditions, growth of the expressing mutant was not affected when compared to the


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UVM4 parental control (Figure 3B). Final cell densities and cell dry weight were 1.75±0.06 x107 cells mL-1 and 0.82±0.04 g L-1 for the hEGF expressing mutant and 1.8±0.06 x107 cells mL-1 and 0.74±0.04 g L-1 for the parental UVM4 control. Protein samples were taken in 24 h intervals and analyzed by Western blot and immunodetection. The target protein was detected in the culture media after 48 h past inoculation with accumulated RP titers of 75 - 100 µg L-1 (Figure 3C), which corresponds to 0.2-0.25 % of the total secreted protein amount (40 mg L-1, Figure 3D). Protein accumulation during the stationary phase was detected, however, the quality of protein separation was reduced due to the presence of insoluble cell wall proteins, and multiple bands were observed indicating protein degradation. Therefore, we determined that culture harvest in early stationary phase is most suitable to high-quality RP production. Protein extraction from C. reinhardtii culture mediated by ATPS Secreted RPs from C. reinhardtii have been shown to have desired target activities even in concentrated media samples.21 However, low protein titers and media composition do not always allow the direct application of C. reinhardtii culture supernatants or concentrated protein mixtures. It has been shown that affinity chromatography with lyophilized culture supernatant is highly inefficient, due to the presence of cell wall proteins that hinder downstream processes by clogging columns and filter membranes.13 Here, we demonstrate a simple and rapid alternative liquid/liquid protein extraction technique as a part of an efficient RP purification strategy based on aqueous two-phase separation in micellar solutions after addition of a non-ionic surfactant.48,49 At temperatures above a detergent cloudpoint, liquid-detergent solutions separate into a surfactant-depleted phase (aqueous phase) and a surfactant-rich phase. These two distinct phases cause a separation wherein hydrophilic proteins remain in the aqueous phase and hydrophobic (e.g. membrane) proteins are extracted into the ACS Paragon Plus Environment


surfactant-rich phase.48 Here, we found that phase separation of complete C. reinhardtii culture as well as clarified culture medium can be induced by the addition of the non-ionic detergent Triton™ X-114 at low concentrations and incubation at 30 °C for 30 min (Figure 4A). The volume of the detergent-rich phase (lower phase) and resulting extraction efficiency correlates with the detergent concentration: 4 % (v/v) Triton™ X-114 results in ~50 % of the initial volume as lower phase. The majority of the proteins found in the extracellular space of C. reinhardtii cultures are chaotropic soluble (hydroxy)proline rich glycoproteins which form insoluble aggregates when concentrated. Here, when a Triton™ X-114-based ATPS was applied to C. reinhardtii culture, the majority of the secretome was found to partition into the aqueous phase (Figure 4B) where hydrophilic proteins are reported to accumulate.48 The partitioning behavior can be leveraged to isolate desired RPs of interest when they are fused to amphiphilic HFBI protein tags.50 The T. reesei HFBI is a small, highly surface active protein that is used in several biotechnological applications, such as surface modification, immobilization, and protein purification.51,52 Due to its affinity for the detergent (lower) phase,51,53 target RP_HFBI fusions can be concentrated and separated from non-tagged proteins, which accumulate in the aqueous (upper) phase. Following separation, recovery of the target RP_HFBI is conducted on the detergent rich phase using isobutanol to yield active target RPs.30 This technique was shown be scalable to larger volumes54 and is currently used to generate active RPs from insect and plant cell culture.30,50,55

Aqueous two-phase separation was tested using clarified culture media from C. reinhardtii strains engineered to secrete either the gLuc protein (vector I, Figure 3A) or gLuc_HFBI (vector IV, Figure 3A). Expressing transformants were cultivated for 48 h, the culture media was clarified by centrifugation and phase separation was induced at 30 °C for 30 min after addition of 4 % (v/v)


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Triton™ X-114. Protein analysis of the two resulting phases indicated that the majority of secreted proteins, including the secreted gLuc protein, remained in the aqueous phase (Figure 4B). The fusion of gLuc with the amphiphilic HBFI protein resulted in its affinity to the detergent phase and partitioning of gLuc_HFBI into the lower phase (Figure 4B). As the partitioning to each phase is passive, a small fraction of the gLuc_HFBI remained in the upper phase along with hydrophilic proteins. In addition to the temperature and detergent concentration, protein size and hydrophobicity of fusion partners are likely factors that affect partitioning behavior of the HFBI tagged fusion proteins.50,56 We tested the effectiveness and biocompatibility of this purification method by expressing the 6 kDa large hEGF protein sequence fused the N-terminus of the gLuc_HFBI_LpIBP4c shuttle protein (predicted molecular mass: 43kDa) as an example of a valuable protein with potential biopharmaceutical and analytical applications. Both full culture and supernatants of mixotrophically cultivated C. reinhardtii cultures were subjected to ATPS (Figure 4C). Culture medium harvested after 48 h was clarified by centrifugation prior to target protein extraction. The resulting lower-phase protein fraction from clarified medium was subjected to an additional polishing step by a StrepII-Tag® affinity chromatography (Supplementary Figure S4) to remove residual detergent and allow biological activity testing in a wound healing assay using human cell culture. A total of 15 µg recombinant hEGF fusion protein was isolated from 1 L initial culture volume (starting concentration: 56 µg L-1, Supplementary Figure S4). Elutions exhibited high purity with concentrations up to 5.3 µg mL-1. When applied to human cell culture in a wound healing assay, the purified hEGF exhibited comparable biological activity at 1 nM concentration as a commercial hEGF (Gibco™, Thermo Scientific) (Figure 5). A wound created by scratching the confluent A-431-cell monolayer was



closed by hEGF induced cell migration with a speed of 10.83 ± 0.26 µm h-1 for the purified hEGF (commercial hEGF control: 13.14 ± 0.23 µm h-1). A-431 cells subjected to an equal volume of buffer solution and no hEGF did not exhibit wound healing activity in the observed time frame and appeared to have reduced fitness. When 10 nM hEGF was applied, morphological changes were observed as reported for higher hEGF concentrations in general,57,58 suggesting that high concentrations of hEGF induced terminal differentiation and detachment of A-431 cells. In this work, the hEGF was expressed as a complex fusion protein from the algal cells in order to facilitate its secretion and enable the ATPS purification from contaminating (hydroxy)proline rich cell-wall glycoproteins. Although this fusion contained other large peptide moieties, such as the luciferase reporter and HFBI, it was able to show the native biological activity of the hEGF in cell culture, indicating that this strategy is effective for functional RP extraction from algal cultures. The success of the HFBI-based ATPS extraction may be improved by new, recently identified hydrophobin protein variants which exhibit increased affinity for the detergent phase and may increase the relative extraction yields than those observed here.53 In this work, a new and strategically designed fusion protein complex was developed to enable efficient protein secretion and purification from a microalgal system. This fusion construct was designed for robust secretion mediated by protein tertiary structure considerations and enabled rapid detection of expression strains at the agar plate level by bioluminescence screening. It was determined that placing the LpIBP on the C-terminus of the gLuc reporter mediated efficient export of the protein complex from the algal cell and increased overall RP yields. In combination with the HFBI amphiphilic protein tag, we were then able to effectively extract desired RP targets from contaminating cell wall proteins in the culture medium by employing ATPS. The protein product was found to maintain its native biological activity when applied to human cell culture. These results represent a practical strategy for the use of photosynthetic microalgae for ACS Paragon Plus Environment

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sustainable production of hEGF and will likely enable light-driven production of other high-value RPs as well.

Methods DNA assembly, construct design, and cloning Nucleotide sequences coding for the predicted N-terminal secretion signals of C. reinhardtii arylsulfatases ARS1 (27 amino acids, NCBI accession number: XM_001692070.1), ARS2 (22 aa, NCBI: XM_001691920.1), carbonic anhydrase 1 (20 aa, cCA, NCBI: XM_001692239.1) and iron assimilatory protein 2 (24 aa, FEA2, NCBI: XM_001693913.1) were de novo assembled using complementary oligonucleotides containing terminal recognition sites for respective restriction enzymes (Supplementary Table S1). Cloning and expression was conducted in the pOptimized

vectors

pOpt_gLuc_Paro

and

pOpt_mVenus_Paro

(NCBI:

KM061059,

KM061060.1).32 The 354 bp codon optimized Lolium perenne ice binding protein (LpIBP) sequence was PCR amplified (Q5® High-Fidelity DNA Polymerase, M0491, NEB) from a previously constructed plasmid using respective primers (Supplementary Table S1).21 The amplified product was inserted in respective reporter expression vectors to create N- and C-terminal fusion proteins in frame with secreted Gaussia princeps luciferase (gLuc) and the mVenus reporters in the pOptimized vector backbones.32 Six potential N-glycosylation motifs (N-{P}-[ST]-{P}) were identified in the LpIBP using the Prosite database,59 removed in silico from the original LpIBP sequence by nucleotide modifications to cause amino acid substitutions (N11V, N34D, N41D, N72V, N79T, N98D) and a potential non-glycosylated sequence was chemically synthesized



(Genscript). Truncations were created by PCR amplification according to the predicted tertiary ßroll structure of the LpIBP protein41 (6 coils: 267 bp, 4 coils: 183 bp, 2 coils: 96 bp) and fused to the C-terminus of the gLuc reporter. The 53 amino acid sequence of the human epidermal growth factor (hEGF, NCBI: NP_001171601.1) and the 97 aa sequence of Trichoderma reesei Hydrophobin II (HFBI, Hydrophobin type I, NCBI: XP_006964181.1) were back-translated to the highest frequency C. reinhardtii codon bias (Kazusa database) and de novo assembled using complementary oligonucleotides (Supplementary Table S1). The FEA2 secretion signal, hEGF, and HFBI sequences were PCR amplified and re-assembled by overlapping extension PCR as previously described47 to contain a copy of the C. reinhardtii rbcS2 intron 1 (NCBI: X04472.1). All cloning was performed using FastDigest restriction enzymes (Thermo Scientific) and the Rapid Dephos & Ligation Kit (Roche) using heat shock transformation of chemically competent Escherichia coli DH5a cells. Selection was performed overnight on 300 mg L-1 Ampicillin containing LB-agar plates and colonies were checked by colony PCR. Plasmid sequences were confirmed by Sanger sequencing (Sequencing Core Facility, CeBiTec). The full gene sequence for the FEA2_hEGF_gLuc_HFBI_LpIBP including introns has been stored as ACS registry part ID ACS_000727 and is shown in Supplementary Figure S5. C. reinhardtii cultivation, transformation and mutant screening Cultivation of C. reinhardtii strain UVM460 was conducted under mixotrophic conditions with Tris acetate phosphate (TAP)61 medium on agar plates or liquid medium in shake flasks with 150200 µE m-2 s-1 light. Cell densities were determined with a Z2™ COULTER COUNTER®


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Analyzer (Beckman Coulter Inc.) and cell dry weight (CDW) by centrifugation (3,000 x g for 3 min) and drying of the biomass pellet at 105 °C overnight. Nuclear transformation was carried out by glass beads agitation as previously described using 10 µg linearized plasmid DNA.62 Transformants were selected and maintained on TAP agar plates containing 10 mg L-1 paromomycin. Initial screening for expression of the gene of interest cassette was performed for a transformant population (>300 isolated transformants) at agar-plate level after 3 d of subcultivation. For bioluminescence analysis, plates were incubated with 10 µM coelenterazine (pjk GmbH, 102171) in luciferase assay buffer (100 mM K2HPO4 pH 7.6, 500 mM NaCl and 1 mM EDTA) as a substrate and relative bioluminescence signals were analyzed using the NightShade LB 985 (Berthold Technologies) plant imaging CCD camera system with an infrared CutOff BG40 filter (640 nm). For each construct, 20 expressing transformants were selected, cultivated in microtiter plates until stationary phase was reached and quantitative expression was determined normalized to the respective cell densities. Cultures were transferred to 2 mL reaction tubes, the culture media was clarified by centrifugation (3,000 x g for 3 min), and 50 µL of the supernatant was mixed with bioluminescence assay buffer followed by direct injection of 10 µM coelenterazine immediately before measurement. Luciferase activity was detected in technical triplicates using the FLUOstar OPTIMA (BMG LABTECH) with 10 s as acquisition time. Protein extraction and SDS-PAGE Samples of the clarified culture media were incubated with trichloroacetic acid (TCA, final concentration 10 % (v/v)) for 30 min at 4 °C followed by centrifugation for 30 min at 14,000 x g and 4 °C. Resulting protein pellets were washed with ice-cold acetone, centrifuged, and finally resuspended in protein sample buffer (60 mM Tris pH 6.8, 2 % (w/v) SDS, 10 % (v/v) glycerol, ACS Paragon Plus Environment


0,01 % (w/v) bromophenol blue). Intracellular protein samples were taken by resuspending cell pellets in protein sample buffer according to their respective cell density. Proteins were separated by Tris-glycine-SDS-PAGE.63 Gels were stained using Colloidal Coomassie Brilliant Blue G25064 or subjected to Western blotting on nitrocellulose membranes (Amersham, GE Healthcare) prior to immunodetection using either a HRP-linked mouse-anti-StrepII® monoclonal antibody (1:5000, in TBS including 5 % (w/v) BSA and milk powder (blocking buffer), Iba Life Science, 2-1509-001) or a rabbit-anti-gLuc antibody (1:5000 in blocking buffer, NEB, E8023S) followed by a HRP-linked goat-anti-rabbit IgG (1:10000 in blocking buffer, Agrisera AB, AS09602). Visualization was performed using the Pierce™ ECL Western blotting substrate (Thermo Fisher Scientific) and the Fusion Fx7 CCD- camera (peQLab GmbH, VWR). Target protein concentration was determined by immunodetection and signals were compared to a recombinantly expressed and purified mVenus protein standard (StrepII®-Tag affinity chromatography, Iba Life Science) quantified by Lowry DC-Protein Assay (Bio-Rad, CA, USA) using 0.1-1.5 mg mL-1 BSA as a reference. Detergent-based aqueous two-phase system (ATPS) Expressing mutants were cultivated for 48 h and culture medium was harvested from the algal biomass by centrifugation for 3 min at 3,000 x g. Clarified medium was pre-warmed to 30 °C and mixed with the non-ionic detergent Triton™ X-114 (final concentration: 4 % (v/v), Sigma Aldrich). The emulsion was transferred to a pre-warmed conical separation funnel and incubated at 30 °C until complete phase separation was observed (~30-60 min). The detergent-rich, lowerphase fraction was isolated and regenerated with successive wash steps of isobutanol (Carl Roth GmbH) using ~3-5 times the volume of the lower phase. The buffer was changed for the resulting protein samples to PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH ACS Paragon Plus Environment

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7.4) using Vivaspin® 20 columns (MWCO: 10 kDa, Sartorius AG). As an optional polishing step the resulting protein fraction was purified by affinity chromatography using the StrepII-Tactin® system according to manufacturers’ protocols (Iba Life Science). Protein samples were taken from the initial culture media and the upper phase for reference by TCA precipitation as described before. Wound Healing Assay The human squamous carcinoma cell line A-431 (DSMZ, accession no.: 91) was used to determine the biological activity of hEGF proteins in a wound healing assay.65 A-431 cells (2x105 cells/ well) were seeded in 24-well plates (TC-treated, Sarstedt) and cultivated in RPMI1640 culture media (D8758, Sigma Aldrich; supplemented with 10 % fetal calf serum (FCS) and 1 % P/S (P4333, Sigma Aldrich)) at 37 °C and 5 % CO2 for 24 h until a confluent monolayer was formed. Cells were serum-starved overnight in RPMI-1640 without FCS. Two scratches per well were made using a sterile pipet tip. After a washing procedure using PBS, cells were treated with either the purified or a commercial hEGF (PHG0311, Gibco™, Thermo Scientific), both at concentrations of 1 nM and 10 nM in RPMI-1640 with 1 % P/S. Control cells received an equal volume of buffer (1000 µL RPMI-1640 with 25 mM Tris, 37.5 mM NaCl, 0.25 mM EDTA und 0.625 mM Desthiobiotin, pH 8). Cells were maintained in cultivation conditions and cell migration was observed using a Leica DMI6000 microscope at selected time points ranging from 0 h to 8 h of incubation. The area of cell-free surfaces was determined using the MRI Wound Healing Tool plug-in for the ImageJ software.66 A linear regression of the surface area versus time was used to determine the cell migration rate. Figure Captions:



Figure 1: Comparative analysis of the four endogenous C. reinhardtii secretion signals. Arylsulfatases ARS1 (27 amino acids, NCBI accession no.: XM_001692070.1) and ARS2 (22 aa, XM_001691920.1), carbonic anhydrase 1 (20 aa, cCA, XM_001692239.1) and iron assimilatory protein 2 (24 aa, FEA2, XM_001693913.1) were used to target the gLuc reporter encoded in the vector pOpt_gLuc_Paro (NCBI: KM061059.1) into the extracellular space. Boxplots represent the bioluminescence signals per 106 cells of 20 analyzed mutants (from >300 initially screened transformants) after 48 h mixotrophic cultivation. The vertical depth represents the secretion intensity of the analyzed population per construct. Minimal and maximal values are indicated by black lines while quartiles and mean value are indicated by a blue box. H R i – the HSP70/RBCS2i1 promoter, 3’ UTR – 3’ untranslated region of the rbcS2 gene. gLuc – G. princeps luciferase. i2 – rbcS2 intron 2

Figure 2: Investigation of the Lolium perenne ice binding protein as a fusion partner for the gLuc reporter. A) Vector pOpt_gLuc_Paro (NCBI: KM061059.1) with either N- or C-terminal fusion of the LpIBP and secretion mediated by the FEA2 secretion signal. Boxplots represent the bioluminescence signals per 106 cells normalized to the respective cell densities of 20 expressing mutants (from >300 initially screened transformants). B) Modification of the LpIBP by replacing the six predicted N-glycosylation motifs with indicated amino acid changes. Both versions of the LpIBP were fused C-terminally to the gLuc reporter and bioluminescence was quantified. C) Tertiary structure of the LpIBP (PDB: 3ULT)41 and amplified truncations according to the number of coils. Truncation bioluminescence signals were compared to the gLuc parental control and the full-length sequence. D) Immunodetection using protein samples from cell pellet and supernatant after centrifugation of culture. Equal amounts of protein of three representative mutants were loaded normalized to cell densities. Intracellular protein samples represent 2x106 cells and secreted proteins represent proteins from 1 mL of the culture after TCA precipitation. H R i – the HSP70/RBCS2i1 promoter, 3’ UTR – 3’ untranslated region of the rbcS2 gene. gLuc – G. princeps luciferase. i2 – rbcS2 intron 2. CBB – Coomassie Brilliant Blue, α-gLuc antibody.

Figure 3: Secretion constructs of the hEGF protein including the HFBI and LpIBP4c tag. A) Fusion of the hEGF with an improved gLuc reporter sequence and the FEA2 secretion signal (vector I) and an additional 4-coil truncated LpIBP at the C-terminus (vector II). Additionally, the HFBI sequence was cloned at the C-terminus of the gLuc reporter (vector III and IV). Boxplots represent secretion levels by quantifying the bioluminescence signal as described above. B) Mixotrophic cultivation in stirred flasks of a representative mutant expressing the hEGF secretion and purification construct (vector IV). Cell densities and cell dry weight were determined daily in triplicates and compared to the UVM4 parental control. C) Protein samples were taken and concentrated by TCA-precipitation. Equal volumes per day representing 1 mL of culture media were separated on a 12 % SDS-PAGE gel. Proteins were visualized by Coomassie staining or immunodetection using an α-gLuc antibody. D) Production titers of the ACS Paragon Plus Environment

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hEGF_gLuc_HFBI_LpIBP fusion protein. Secreted target protein amounts were detected using 1 mL of precipitated media and compared to a recombinant protein standard purified from E. coli using the HRP-linked α-StrepII®-Tag antibody. H R i – the HSP70/RBCS2i1 promoter, 3’ UTR – 3’ untranslated region of the rbcS2 gene. gLuc – G. princeps luciferase. i – rbcS2 intron 1. i2 – rbcS2 intron 2

Figure 4: Detergent-based aqueous two-phase system as a liquid/liquid extraction method. A) Scheme of phase separation after addition of the nonionic detergent to culture media with resulting aqueous-phase (upper phase) and detergent-rich phase (lower phase). Separation can be induced in whole culture volumes at low detergent concentrations at incubation temperatures above the respective cloud point (25 °C for Triton™ X-114). B) Two-phase separation of clarified culture media after addition of 4 % (v/v) Triton™ X-114. Partitioning behavior of proteins is shown with secreted proteins in the culture medium of a gLuc protein alone or a gLuc_HFBI fusion protein. ‘medium’ indicates a protein sample taken from clarified medium before ATPS. ‘Upper p.’ and ‘lower p.’ indicate protein samples from the aqueous or detergent rich phases, respectively. Secreted proteins were visualized after SDS-PAGE by either Coomassie staining or immunodetection using a rabbit α-gLuc antibody. Arrows indicate the amount of the target protein in the lower phase. C) Scale-up of extraction was performed using 1 L of clarified culture media. The secreted fusion protein hEGF_gLuc_HFBI_LpIBP was extracted and recovered as described before. Protein samples were analyzed by Coomassie stain or immunodetection using α-StrepII®-Tag-HRP-linked antibody. Protein titers were compared to a recombinant protein standard.

Figure 5: Wound healing assay using human A-431 cells. Cells were seeded in 24-well plates and cultivated in RPMI (supplemented with 10% FCS, 1% P/S) culture media at 37 °C and 5 % CO2 for 24 h before serum starvation. Wounds were created, unbound cells removed with PBS and cells incubated with 0 nM, 1 nM and 10 nM concentrations of algal-produced or a commercial hEGF (Gibco™, Thermo Scientific) protein. Images were taken using a Leica DMI6000 and cell-free areas and cell migration were determined using the ImageJ software.66 Scale bars represent 250 µm.



Supporting Information: Supplementary Table S1: List of primers used in this study Supplementary Figure S1: Fusion of the LpIBP at the N- and C-terminus of the mVenus reporter protein in the pOpt_mVenus_Paro (NCBI: KM061060.1) vector Supplementary Figure S2: Fusion of the full length LpIBP sequence and a 4 coil truncation at the C-terminus of the gLuc protein. Supplementary Figure S3: Application of the gLuc_LpIBP4c fusion as a transport vehicle for improved protein export. Supplementary Figure S4: Quantification of the purified hEGF used for wound healing assay. Supplementary Figure S5: Complete vector sequence of the final vector VI: FEA2_hEGF_gLuc_HFBI_LpIBP4coil used for expression and purification

Supporting Information Figure Captions: Supplementary Table S1: List of primers used in this study. Supplementary Figure S1: Fusion of the LpIBP at the N- and C-terminus of the mVenus reporter protein in the pOpt_mVenus_Paro (NCBI: KM061060.1) vector. Targeting to the extracellular space was mediated by the FEA2 secretion signal. The secretion titer was quantified by YFP fluorescence and compared to the mVenus parental construct. Boxplots represent fluorescence in correlation to the cell density of 20 representative mutants per construct cultivated for 48 h mixotrophically in microtiter plates. H R i – the HSP70/RBCS2i1 promoter, 3’ UTR – 3’ untranslated region of the rbcS2 gene. gLuc – G. princeps luciferase. i2 – rbcS2 intron 2 Supplementary Figure S2: Fusion of the full length LpIBP sequence and a 4 coil truncation at the C-terminus of the gLuc protein. Tertiary structure was predicted using the iTasser software. Both fusion partners exhibit predicted ß-roll coil structures. Bioluminescence signals were taken from mutants expressing the fusion construct of the gLuc_4c LpIBP without a linker sequence where tertiary structure formation is inhibited and the increase in secretion levels is no longer observed. Supplementary Figure S3: Application of the gLuc_LpIBP4c fusion as a transport vehicle for improved protein export. The second reporter, mVenus, was fused to the N-terminus of the gLuc_LpIBP4 as an example of target proteins. Whereas the direct fusion of the LpIBP to the ACS Paragon Plus Environment

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mVenus reporter did not change secretion levels (Supplementary Figure S1), the fusion of mVenus_gLuc_LpIBP4c exhibits a stronger bioluminescence compared to the mVenus_gLuc reporter construct. H R i – the HSP70/RBCS2i1 promoter, 3’ UTR – 3’ untranslated region of the rbcS2 gene. gLuc – G. princeps luciferase. i2 – rbcS2 intron 2 Supplementary Figure S4: Quantification of the purified hEGF used for wound healing assay. One Liter of C. reinhardtii culture media was clarified by centrifugation and subjected to an ATPS as described. The recovered protein from the lower phase was additionally purified by affinity chromatography to remove residual detergent. Samples were taken from all intermediate steps and the elution fractions 2-5. Proteins were separated in 12 % SDS-PAGE gels and visualized by western blotting followed by immunostaining (HRP-linked α- StrepII antibody, Iba Life Science) as well as Colloidal Coomassie staining method. Supplementary Figure S5: Complete vector sequence of the final vector VI: FEA2_hEGF_gLuc_HFBI_LpIBP4coil used for expression and purification. Features are indicated by the respective color. The sequence information of the expression vector construct VI: pOpt_FEA2_hEGF_gLuc_HFBI_LpIBP was deposited to the ACS Synthetic Biology registry with part ID ACS_000727. Abbreviations: TAP – Tris acetate phosphate medium FEA2 – Chlamydomonas reinhardtii Fe-assimilatory protein 2 secretion signal cCA – Chlamydomonas reinhardtii carbonic anhydrase (CAH1) secretion signal gLuc – Gaussia princeps Luciferase LpIBP – Lolium perenne Ice Binding Protein HFBI – Trichoderma reesei Hydrophin I ATPS – detergent-based aqueous two-phase system Acknowledgements The authors would like to acknowledge the Industrial Biotechnology Graduate Cluster (CLIBGC) (to TB), the European Union’s Horizon 2020 grant agreement No. 640720 Photofuel (to OK). The authors would like to express thanks to Prof. Dr. Ralph Bock for the strain UVM4.



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Author Information 1

Bielefeld

University,

Faculty

of

Biology,

Center

for

Biotechnology

(CeBiTec),

Universitätsstrasse 27, 33615 Bielefeld, Germany. Phone: +49 521 106-12258, Fax: +49 521 106-12290 2

Bielefeld University, Faculty of Technology, Cellular and Molecular Biotechnology,

Universitätsstrasse 25, 33615 Bielefeld, Germany. Phone: +49 521 106-106-6323, Fax: +49 521 106-156318 Author ORCID iDs: Kyle J. Lauersen: 0000-0002-5538-7201 Olaf Kruse: 0000-0001-9874-382X Kristian M. Müller: 0000-0002-7914-0625 Rebecca C. Feiner: 0000-0002-8784-0875

Author Contribution TB, KL, RF and DK designed and performed the experiments. TB and RF analyzed the results and TB, KL, KM and OK wrote and edited the manuscript. All authors have read and approved the final version of this manuscript. Conflict of Interest The authors declare that they have no conflict of interest.


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20


Page 30 of 35

10

1 2 3 4

0 medium

ACS Paragon Plus Environment U

medium

L

U

L

H

R

ARS1

ARS2

1 2 3 4 5 6 7 8

gLuc i2

i

cCA

FEA2

3‘ UTR

2E+04 ACS Synthetic Biology Bioluminescence / 106 cells

Page 31 of 35

1E+04

8E+03

4E+03

ACS Paragon Plus Environment 0E+00

ARS1

ARS2

cCA

FEA2

gLuc

H

R

FEA2

LpIBP

i2

3‘ UTR

ACS Synthetic Biology 1E+05 Bioluminescence / 106 cells

A

Page 32 of 35

8E+04

CBB

α-gLuc

Bioluminescence / 106 cells


1 5E+04 2N-term 3 LpIBP 3E+04 4C-term 5 0E+00 6 gLuc N-term 7 B 8 C 9 LpIBP LpIBP 10 3‘ PDB: 3ULT i2 R i H UTR 11 12 13 14 N11D N34D N41D N72V N79T N98D 15 16 1E+05 17 18 8E+04 19 20 6E+04 6E+04 21 22 4E+04 4E+04 23 24 2E+04 2E+04 25 0E+00 26 0E+00 gLuc without with gLuc 2 coils 4 coils 27 N-glycosylation N-glycosylation 28 29 gLuc_LpIBP gLuc D 30 intracellular secreted intracellular secreted 1 2 3 1 2 3 1 2 3 1 2 3 31 [kDa] [kDa] 40-40 4032 33 35-35 3534 25-25 2535 36 5555-55 37 4040-40 38 3535-35 ACS Paragon Plus Environment 39 2525-25 40 41

C-term

6 coils

-40 -35 -25

-55 -40 -35 -25

full length

A

FEA2

hEGF

gLuc

i

i

i2

Page 33 of 35 ACS Synthetic Biology

I

H

R i

3‘ UTR

LpIBP

II

HFBI i


1 2 III3 4 5 IV 6 7 8 92E+04 10 2E+04 11 12 1E+04 13 14 5E+03 15 16 0E+00 17 18 B 19 20 20 21 15 22 23 10 24 25 5 26 27 0 28 29 30 C31 [kDa] 32 24 h 7033 4034 35 257036 37 4038 2539 40 D 41 42 43 44 45 46 47 48

I

II

III

IV

Cell dry weight g L-1

1

0 0h

24 h

48 h

cell density EGF CDW EGF

72 h

96 h

cell density UVM4 CDW UVM4

EGF

UVM4

48 h 72 h 96 h

CBB

α-gLuc

48 h 72 h 96 h 24 h

180

standard [ng] 130 100 75

α-Strep

supernatant UVM4 EGF


CBB

quantification after 48 h

Cell density x106

2

A Two-phase system

B

Synthetic Biology PartitioningACS behavior with HFBI-Tag

Page 34 of 35

CBB

α-Strep

CBB

α-Strep

1 addition of detergent: gLuc gLuc + HFBI 2 4 % (v/v) TritionX-114 upper 3 i phase 4 5 lower medium upper p. lower p. medium upper p. lower p. 6 phase 30 °C 7 8 9 10 11 12 FEA2 hEGF gLuc HFBI LpIBP C13Protein extraction of hEGF 14 15 addition of phase detergent protein standard 16 culture detergent separation removal [kDa] medium upper p. lower p. 150 ng 100 ng 75 ng 5517 4018 3519 2520 5521 4022 3523 2524 5525 4026 clarified medium 3527 2528 1305529 ACS Paragon Plus Environment 4030 3531 25-

Page 35 of 35 14

ACS Synthetic BiologyHuman A-431

buffer control

hEGF (C. reinhardtii) 1 nM

Cell migration in µm/h

hEGF (control) 1 nM

0h 3h 6h

1 12 2 10 3 4 8 5 6 6 4 7 8 2 9 0 10 nM trol nM nM nM 11 buffer con hardtii) 1 ardtii) 10 control) 1 ontrol) 10 h n ( (c rei rein GF GF (C. 12 hE (C. hE GF GF hE hE 13 buffer control y= -340.46x + 567219 14 9E+05 hEGF (C.reinhardtii)1 nM y= -25137.59x + 416058 15 8E+05 hEGF (control) 1 nM y= -27256,75x + 528639 hEGF (C.reinhardtii)10 nM y= 173,69x + 580129 16 7E+05 hEGF (control) 10 nM y=-91,41x + 524139 17 6E+05 18 19 5E+05 20 4E+05 21 3E+05 22 2E+05 23 ACS Paragon Plus Environment 24 1E+05 0 1 2 3 4 5 6 7 8 9 25 time in h area in µm2

cell line

Engineered fusion proteins for efficient protein secretion and

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