Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide

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Yeast-based synthetic biology platform for antimicrobial peptide production Jicong Cao, Cesar de la Fuente-Nunez, Rui Wen Ou, Marcelo DT Torres, Santosh G. Pande, Anthony J Sinskey, and Timothy K. Lu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00396 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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ACS Synthetic Biology

Yeast-based synthetic biology platform for antimicrobial peptide production Jicong Cao1,2,3*, Cesar de la Fuente-Nunez1,2,3*, Rui Wen Ou4, Marcelo Der Torossian Torres1,2,6, Santosh G. Pande5, Anthony J. Sinskey4 and Timothy K. Lu1,2,3,§ 1

Synthetic Biology Group, MIT Synthetic Biology Center, Department of Biological

Engineering, and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2

Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA

02139, USA 3

The Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA.

4

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

5

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA

02139, USA 6

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP

09210580, Brazil *

These authors contributed equally to the work.

§

Correspondence: [email protected].

Abstract Antibiotic resistance represents one of the most challenging global health threats in our society. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics for the treatment of drug-resistant infections. However, they are limited by their high manufacturing cost. Engineering living organisms represents a promising approach to produce such molecules in an inexpensive manner. Here, we genetically modified the yeast Pichia pastoris to produce the prototypical AMP apidaecin Ia using a fusion protein approach that leverages the beneficial properties (e.g., stability) of human serum albumin. The peptide was successfully isolated from the fusion protein construct, purified, and demonstrated to have bioactivity against Escherichia

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coli. To demonstrate this approach as a manufacturing solution to AMPs, we scaled-up production in bioreactors to generate high AMP yields. We envision that this system could lead to improved AMP biomanufacturing platforms.

Keywords:

synthetic

biology platform,

peptide production,

Pichia

pastoris,

yeast,

antimicrobials.

Introduction Antibiotic resistance represents one of the great global health challenges of our time and thus there is a growing need for new strategies to combat antibiotic resistance. A range of alternative therapies to conventional antibiotics have been developed over the years, including phage therapy, anti-virulence molecules, antibodies, lytic enzymes, lysins from phages, and antimicrobial peptides (AMPs) 1. AMPs are produced naturally by many organisms and act as a defense mechanism against invading pathogens. These molecules have evolved through billions of years of evolution as part of our innate immune system, and provide broad-spectrum protection against a range of microorganisms, including bacteria, fungi, viruses, and parasites. AMPs are small in size (typically between 12 and 50 amino acid residues in length 2), have an amphipathic and cationic structure, with a positive net charge between +2 and +9 due to excess in Lys (K) and Arg (R) residues (although there are exceptions such as Arg-rich AMPs with >+9 net charge 3), and have ~50% of their primary structure composed of hydrophobic residues that enable interactions with membranes and translocation into cells.

Unlike other larger proteins, AMPs lack a complex 3D structure, so they can be chemically synthesized in small amounts for lab-scale studies. However, chemical synthesis is very costly, ranging from $100-1000 for 1 mg of AMP. Thus, it is not suited for synthesis of large AMPs and is not scalable for large-scale production for clinical studies and commercial use. Recombinant expression, the most commonly used method to produce therapeutic proteins, is used in many labs for large-scale production of AMPs. Compared with chemical synthesis, recombinant expression is scalable, cheap, and can produce larger AMPs containing >30 amino acids. Moreover, the technologies for process development and scale up are mature and the existing facilities used for therapeutic protein production can be used for AMP production.


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E. coli is the most widely used host for the production of AMPs, but the main challenge associated with AMP expression is their natural lethality towards host bacteria 4. Additionally, the vast majority of AMPs have a positive net charge and are therefore susceptible to degradation by endogenous proteases. Thus, several fusion protein strategies have been developed to overcome these challenges

5–7

, since fused proteins can mask AMP activity, therefore reducing

their toxicity towards the host and protecting the AMPs from potential proteolytic cleavage. However, these methods lead to low protein expression levels of about 10-30 mg L-1 (fusion proteins) and 1-5 mg L-1 (peptides) 6,8–11. A promising alternative to using bacterial cells as hosts for heterologous expression of AMPs is the use of yeast to produce these agents, as these organisms have the advantage of being resistant to AMP-mediated killing. Moreover, specific yeast species, such as Pichia pastoris, can efficiently secrete peptides to the medium to increase protein titers and reduce purification burden 12. Here, we used P. pastoris as a heterologous AMP expression host because it has the following advantages over traditional prokaryotic and mammalian expression systems: 1) it limits secretion of host cell proteins but is able to secrete large amounts of heterologous recombinant proteins, which significantly reduces the burden of downstream purification; 2) the growth rate of P. pastoris is faster than that of mammalian hosts such as Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney 293 (HEK 293) cells, and it can easily grow in inexpensive media; 3) it is free of endotoxins and viruses; and 4) in addition to the endogenous alcohol oxidase 1 (AOX1) inducible promoter, various new carbon source-dependent and independent inducible promoters have been developed for protein expression in P. pastoris 13,14. Recently, our lab developed a recombinase-based gene integration method for rapid and efficient insertion of large DNA fragments into the genome of P. pastoris, an approach that was used to successfully produce a number of different proteins 14–16.

Proline-rich antimicrobial peptides (PrAMPs) are a large family of AMPs. PrAMPs are generally cationic and are often present in conserved patterns intercalated with arginine residues 17

. They are expressed as part of the immune response to bacterial invasion

18

and act by killing

microbes via membrane targeting, stimulating the immune response, penetrating into cells and carrying payloads through the biomembrane into cells

19,20

, and affecting protein synthesis

21

.

Apidaecins were the first reported class of PrAMPs; Casteels et al. discovered these agents upon



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infection of adult bees with bacteria and subsequent monitoring of the immune response

22

.

Apidaecins are small, proline-rich peptides of 18 amino acids produced by the honeybee (Apis mellifera). Apidaecin Ia (GNNRPVYIPQPRPPHPRI) and Ib (GNNRPVYIPQPRPPHPRL) are able to inhibit the viability of a number of Gram-negative bacterial cells, such as Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas syringae 23,24. Unlike most other AMPs that kill bacteria by disrupting their cell membrane, apidaecins instead operate by targeting intracellular ribosomes to inhibit translation 21.

In this work, we developed an HSA-based AMP expression system in Pichia pastoris for the production of AMPs. Our method enables rational production of AMPs, thus conferring an advantage over previously described approaches, which rely on random mutagenesis of Pichia strains to secrete peptides

25

. We used apidaecin as a model AMP, and used HSA to enhance

protein secretion and provide increased stability. We demonstrate that the apidaecin peptide generated using this HSA-apidaecin fusion protein approach was biologically active against E. coli and that the titer of the fusion protein could achieve >700 mg L-1. We envision that this Pichia-based method can be used for large-scale production of AMPs for clinical and industrial applications.

Results and Discussion Construction of the HSA-AMP fusion protein expressing P. pastoris strain In prior work, we developed an efficient DNA integration method to insert genes of interest in the P. pastoris genome using a serine recombinase

14

. Specifically, we constructed a

P. pastoris strain with a landing pad with BxbI attP sites integrated in the genome at the Trp2 locus. When the plasmid of interest containing the corresponding BxbI attB site is cotransformed with a plasmid expressing BxbI recombinase, the BxbI recombinase inserts the entire plasmid into the BxbI attP site within the yeast cell genome 14,16 (Fig. S1A). To efficiently secrete the peptide of interest from P. pastoris, we constructed a fusion protein consisting of the alpha mating factor secretion signal, human serum albumin (HSA), and the AMP apidaecin (Fig 1A). HSA is known to facilitate protein secretion in P. pastoris, and a variety of HSA fusion protein have been produced in high titers (10-30 g L-1) 26. To purify and release AMPs, we also


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inserted a polyhistidine tag (His-tag) containing six histidines, and a TEV protease cleavage site between HSA and apidaecin (Fig. 1B). The TEV protease recognizes the amino acid sequence ENLYFQ/X and cleaves between glutamine (Q) and X, where X is preferably Q or serine (S), but can be any of the other naturally occurring 17 amino acids with the exception of proline (P). The first amino acid within the sequence of apidaecin is glycine (G), which should result in high cleavage efficiency. The coding sequence of the fusion protein was inserted after a methanolinducible promoter, AOX1 promoter, which can be repressed by glycerol and activated by methanol. The resulting plasmid, named pJC235, also contained a zeocin-resistance gene for drug selection and a BxbI attP site for recombinase-based gene integration.

We co-transformed pJC235 and another plasmid expressing BxbI recombinase (pPP43) into P. pastoris with the landing pad, and selected zeocin-resistant clones with YPD zeocin plates. The correct insertion of the gene was verified using colony PCR (Fig. S1B). To test protein expression levels, we cultured the strain in BMGY medium for 48 h and induced protein expression in BMMY medium for an additional 96 hours at 30 °C. The supernatants were then collected every 24 h and protein expression levels were measured using SDS-PAGE and Commassie blue. SDS-PAGE analysis showed that the fusion protein HSA-apidaecin was about 70 kDa, consistent with the expected molecular weight of 70.68 kDa. We also observed that protein expression lasted for at least 96 h and the protein expression level was similar at the induction temperature of 25 °C. The culture supernatant after 96 h of induction at 30 °C in shake flasks was used for downstream processing (Fig. 2).

Expression and purification of apidaecin AMP in shake flasks To show that the yeast-generated apidaecin AMP was biologically active, we purified the peptide and tested its bacterial killing activity against Escherichia coli. The fusion protein present in the cell supernatant was purified using immobilized metal ion affinity chromatography (IMAC) and SDS-PAGE showed that most of the host proteins had been removed. We then removed the HSA portion of the fusion protein by adding TEV protease to the purified fusion proteins. The HSA-AMP fusion protein was about 70.7 kDa, whereas the AMP alone was about 2.1 kDa. Using a 10 KDa cut-off centrifuge filtration column, we retained HSA and other large unremoved host proteins in the column, and collected the small AMP in the flow-through. SDS-



PAGE analysis verified the efficient separation of the two portions, and the successful isolation of apidaecin in the flow-through medium. Apidaecin characterization was performed by RPHPLC/MS (Figs. S2 and S3).

Antibacterial assays To assess the antimicrobial activity of the homemade apidaecin peptide, we performed killing assays against the E. coli strain BL21. Bacteria were grown overnight at 37 °C and 250 rpm in BM2 with glucose medium and a subculture was prepared and incubated in the same conditions until it reached OD600 of 0.5. Then, bacteria were incubated with the peptides as described in Fig. 3-A. The peptides were diluted 10-fold and plated onto agar petri dishes prior to incubation for 22 h at 37 °C. The antibacterial efficacy of the homemade peptide was then compared to that of chemically synthesized apidaecin controls at increasing concentrations (50, 20 and 5 µg mL-1). The peptide killed approximately 80% of the bacteria (Fig 3-B), therefore we can roughly estimate that its concentration is within the range of 20 to 50 µg mL-1.

The same conditions were used in the growth curve assays, which evaluated the ability of the HSA-apidaecin fusion protein to block the growth of E. coli strain BL21 (Fig. 3-C). Chemically synthesized peptides (50 and 25 µg mL-1) were used as active controls, and at both concentrations, they completely inhibited bacterial growth after 24 h. Conversely, the HSAapidaecin fusion protein did not interfere with bacterial growth, which was expected since the apidaecin portion is probably not able to interact with the membrane and thus penetrate into the cell to act on its ribosomal target

21

. These results are consistent with those presented in a

previous study describing the lack of antibacterial activity of an E. coli producing a carrier AMP fusion protein 6.

Scale-up production of biologically active AMP apidaecin in bioreactors To demonstrate the scalability of the system and further increase protein production yield, we used a methanol-limited culture condition to induce the expression of the fusion protein. The AMP production process in bioreactor involves three stages: inoculum seed preparation, biomass accumulation, and protein production. One colony of P. pastoris cells was inoculated into BMGY medium and grown overnight at 30 °C and 250 rpm. The overnight


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grown culture was inoculated at an OD600 of 0.05 in 500 mL BMGY medium in a 2-L bioreactor and grown at 30 °C with agitation (600-1,200 rpm). When the carbon source (glycerol) was consumed entirely, which was indicated by a dissolved oxygen (DO) spike, the glycerol fedbatch phase was initiated to build sufficient biomass prior to AMP production. After 4 h, the glycerol feed was terminated and methanol, the carbon source and protein production inducer, were added when the DO spike was observed. The detailed protocol and operating parameters are shown in Table 1.

We observed that cells grew quickly in the glycerol fed-batch phase while the growth rate decreased in the methanol fed-batch phase. There was nearly no fusion protein in the glycerol fed-batch phase, whereas it began to accumulate in the methanol fed-batch phase (Fig. 4). SDSPAGE analysis showed that the fusion protein titer was >700 mg L-1 after 84 h of induction with methanol. This HSA-AMP fusion protein titer represents a substantial improvement over previously published methods using E. coli as a heterologous host to generate other AMP fusion proteins, which resulted in yields of 5-30 mg L-1 5,8–11. In this work, we developed a platform in P. pastoris for the production of AMPs using a HSA fusion-based system. The HSA-apidaecin fusion protein was efficiently secreted to the culture supernatant, achieving a titer of >700 mg L-1. The active apidaecin peptide was released from the fusion protein after purification with TEV protease. We validated that the AMP conserved its antimicrobial activity. Compared with producing AMPs using E. coli, our P. pastoris-based method has the potential reduce downstream processing cost by eliminating the cell lysis and cell debris removal steps. In addition, traditional strain optimization processes are laborious, time consuming, and often unpredictable. Our work reports a novel rational design approach based on an HSA-fusion protein system. Carrier-protein-based AMP expression platforms have been previously described in E. coli

5

and cell-free systems

27

, but not in yeast.

Collectively, these results demonstrate that our yeast-based system can be used as strategy for the scalable and cost-effective production of AMPs.



Materials and Methods Construct design and P. pastoris strain construction DNA sequences were synthesized as gBlocks (IDT Technologies, IA) and cloned into the protein expression plasmid using Gibson assembly. Two flexible linkers, GGGS and SSG, were placed between the HSA sequence and His-tag sequence to ensure that the His-tag was fully exposed for efficient IMAC purification. The TEV protease recognition site ENLYFG preceded the SSG linker and was followed by the apidaecin sequence. The transformed E. coli colonies were selected on LB agar plates with 25 µg mL-1 zeocin (ThermoFisher, MA). Five µg protein expression plasmid (pJC235) and 5 µg the BxbI expression plasmid (pPP43) were cotransformed into the P. pastoris strain with landing pad as described, resulting in the HSAapidaecin expressing strain JC235. The transformed P. pastoris colonies were selected on Yeast extract-Peptone-Dextrose (YPD) agar plates with 75 µg mL-1 zeocin and the integrations were verified by colony PCR using Robust 2G Polymerase (Kapa Biosystems, MA). The sequences of the plasmids used in this study can be found in the supplementary and all constructs will be deposited on Addgene. YPD agar recipe: 1% yeast extract, 2% peptone, 2% dextrose (glucose), and 2% agar.

Protein expression and production in shake flasks and SDS PAGE analysis One colony was inoculated in 2 mL Buffered Glycerol-Complex Medium (BMGY) media at 250 rpm at 30 °C. Next day, the overnight culture was inoculated at OD of 0.05 in 50 mL BMGY media at 250 rpm at 30 °C. Two days later, the cells were pelleted down through centrifuge and cultured in 50 mL Buffered Methanol-Complex Medium (BMMY) media with 1% L81 antifoam at 250 rpm at 30 °C. Every 24 hours, 1% methanol was added into the culture and 1 mL culture was taken out for SDS analysis. The culture supernatants were mixed with 4X loading buffer (ThermoFisher, MA) and 10% β-mercaptoethanol (Sigma-Aldrich, MO), and heated at 90 °C for 10 min. The mixtures were loaded into NuPAGE 4-12% Bis-Tris Protein Gels (ThermoFisher, MA) and run at 200V for 40 min. The gels were strained with InstantBlue (Sigma-Aldrich, MO). Below are the media recipes used for these experiments:


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BMGY media recipe: 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH = 6.0), 1.34% yeast nitrogen base (YNB), 4×10-5% biotin, and 2% glycerol. BMMY media recipe: 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH = 6.0), 1.34% YNB, 4×10-5% biotin, and 1% methanol.

Peptide purification Thirty mL culture supernatants were loaded into GE Healthcare HisTrap HP columns (GE Healthcare, NY), and were washed with 10 mL wash buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). The purified fusion proteins were eluted with 2.5 mL elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4). Eluate were then passed through a PD-10 Desalting Column (GE Healthcare, NY) to change the buffer to Phosphate Buffered Saline (PBS) (ThermoFisher, MA). Purified HSA-Apidaecin fusion protein was then subjected to TEV protease (ThermoFisher, MA) digestion at 16°C for 16 hours. The HSA part and the Apidaecin were then separated using a Amicon Ultra 0.5-mL Centrifugal Filters with 10 KDa cut-off membrane (Sigma-Aldrich, MO).

Bacterial killing assays Escherichia coli strain BL21 was grown in LB medium overnight at 37 °C and stirring on (250 rpm). Subcultures of the cells were prepared in LB medium (1:100 dilution) and grown until OD600 reached 0.5. After that, the solutions were diluted (1:10,000) and added to 96-well plates. A dilution series of the chemically synthesized peptides, ranging from 50 to 5 µg mL-1, and the purified peptide were incubated with the bacteria (E. coli BL21) at 37 °C for 11 h. 10fold dilution series of each well were made in water, platted in LB agar plates and incubated at 37 °C for 24 h. The colonies (CFU/mL) were counted visually and experiments were made in triplicates.

P. pastoris fermentation 1. Cell growth stage:



One colony of the HSA-apidaecin expressing strain was inoculated in 5 mL BMGY medium and grown at 30°C, 250 rpm for inoculum production. The overnight grown culture was subsequently inoculated at an OD600 of 0.05 in 500 mL BMGY medium (2% glycerol) supplemented with 0.08% antifoam 204 in a 2-L Bioengineering AG bioreactor (Bioengineering RALF Plus Trio, Switzerland). During batch phase, the operating parameters were set as follows: temperature was set at 30°C, air was supplied at 30 L h-1 constant flow rate, dissolved oxygen (DO) was set at 30% of air saturation by cascading DO onto pure oxygen addition up to 7.5 L h-1 and agitation from 600 to 1200 rpm, the gas flow was 1-1.25 vessel volume per min (vvm), pH was maintained at 6.0 with 5 mol L-1 ammonium hydroxide and 5 mol L-1 phosphoric acid addition as needed. When DO fell below 20%, the glycerol or methanol feed was stopped, and adjustments were made to aeration following the Invitrogen Pichia Fermentation Process Guidelines (https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_prot.pdf).

When the DO spike appeared, which indicated the total consumption of the carbon source, glycerol, the operating parameters were set as follows: dissolved oxygen (DO) was set at 30% of air saturation by cascading DO onto air (30-32.5 L h-1), pure oxygen (0-25 L h-1) and agitation (600-900 rpm) pH and temperature were maintained at 6.0 and 30°C, respectively. The glycerol fed-batch, to build additional biomass prior to HSA-apidaecin production, was initiated by adding 50% glycerol supplemented with 1.2% PTM1 at constant 10 mL h-1 feed rate for 4 consecutive hours.

2. Protein production stage: Starvation phase was initiated to ensure that the glycerol is completely utilized in the media. When the DO spike is observed, the operating parameters were set as follow: dissolved oxygen (DO) was set at 30% of air saturation by cascading DO onto air (30-45 L h-1), pure oxygen (0-11.3 L h-1) and agitation (600-975 rpm). The induction phase was initiated by supplementing 2.5X BMMY medium (contains 5% methanol) at constant 20 mL h-1 feed rate for 10 consecutive hours to provide carbon source and nutrients for protein production. After the rich medium feed, 100% methanol supplemented with 1.2% PTM1 was added continuously at a constant feed rate of 1 mL h-1. One mL of culture was sampled every 12 hours to analyze protein production by SDS-PAGE.


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Acknowledgements This work was supported by the Ramon Areces Foundation (to C.F.-N.), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2016/24413-0) (to M.D.T.T), Broad Institute of MIT and Harvard (Shark Tank Award) (to J.C.) and DTRA (DTRA HDTRA1-15-1-0050) (to T.K.L.).

References (1) de la Fuente-Nunez, C., Torres, M. D., Mojica, F. J., and Lu, T. K. (2017) Next-generation precision antimicrobials: towards personalized treatment of infectious diseases. Curr. Opin. Microbiol. 37, 95–102. (2) Hwang, P. M., and Vogel, H. J. (1998) Structure-function relationships of antimicrobial peptides. Biochem. Cell Biol. 76, 235–46. (3) Venkatesh, T. C. and Y. P. (2016) Occurrence, Functions and Biological Significance of Arginine-Rich Proteins. Curr. Protein Pept. Sci. (4) Bommarius, B., Jenssen, H., Elliott, M., Kindrachuk, J., Pasupuleti, M., Gieren, H., Jaeger, K.-E., Hancock, R. E. W., and Kalman, D. (2010) Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 31, 1957–1965. (5) Li, Y. (2011) Recombinant production of antimicrobial peptides in Escherichia coli: A review. Protein Expr. Purif. 80, 260–267. (6) Ishida, H., Nguyen, L. T., Gopal, R., Aizawa, T., and Vogel, H. J. (2016) Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System. J. Am. Chem. Soc. 138, 11318–11326. (7) Wright, O., Yoshimi, T., and Tunnacliffe, A. (2012) Recombinant production of cathelicidinderived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag. N. Biotechnol. 29, 352–358. (8) Yang, Y. H., Zheng, G. G., Li, G., Zhang, X. J., Cao, Z. Y., Rao, Q., and Wu, K. F. (2004) Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli. Protein Expr. Purif. 37, 229–235. (9) Moon, J.-Y., Henzler-Wildman, K. A., and Ramamoorthy, A. (2006) Expression and



purification of a recombinant LL-37 from Escherichia coli. Biochim. Biophys. Acta 1758, 1351– 8. (10) Li, Y., Li, X., and Wang, G. (2006) Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expr. Purif. 47, 498–505. (11) Li, Y., Li, X., Li, H., Lockridge, O., and Wang, G. (2007) A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation. Protein Expr. Purif. 54, 157–165. (12) Ahmad, M., Hirz, M., Pichler, H., and Schwab, H. (2014) Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 98, 5301–5317. (13) Prielhofer, R., Maurer, M., Klein, J., Wenger, J., Kiziak, C., and Gasser, B. (2013) Induction without methanol : novel regulated promoters enable high-level expression in Pichia pastoris 1– 10. (14) Perez-Pinera, P., Han, N., Cleto, S., Cao, J., Purcell, O., Shah, K. A., Lee, K., Ram, R., and Lu, T. K. (2016) Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. Nat. Commun. 7, 12211. (15) Landry, M. P., Ando, H., Chen, A. Y., Cao, J., Kottadiel, V. I., Chio, L., Yang, D., Dong, J., Lu, T. K., and Strano, M. S. (2017) Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays. Nat. Nanotechnol. 12, 368–377. (16) Purcell, O., Opdensteinen, P., Chen, W., Lowenhaupt, K., Brown, A., Hermann, M., Cao, J., Tenhaef, N., Kallweit, E., Kastilan, R., Sinskey, A. J., Perez-Pinera, P., Buyel, J. F., and Lu, T. K. (2017) Production of Functional Anti-Ebola Antibodies in Pichia pastoris. ACS Synth. Biol. acssynbio.7b00234. (17) Graf, M., Mardirossian, M., Nguyen, F., Seefeldt, A. C., Guichard, G., Scocchi, M., Innis, C. A., and Wilson, D. N. (2017) Proline-rich antimicrobial peptides targeting protein synthesis. Nat. Prod. Rep. 34, 702–711. (18) Zasloff, M. (2002) ANTIMICROBIAL PEPTIDES IN HEALTH AND DISEASE 347, 1199–1200. (19) Wang, F., Wang, Y., Zhang, X., Zhang, W., Guo, S., and Jin, F. (2014) Recent progress of


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cell-penetrating peptides as new carriers for intracellular cargo delivery. J. Control. Release 174, 126–136. (20) Li, W., Tailhades, J., O’Brien-Simpson, N. M., Separovic, F., Otvos, L., Hossain, M. A., and Wade, J. D. (2014) Proline-rich antimicrobial peptides: Potential therapeutics against antibiotic-resistant bacteria. Amino Acids 46, 2287–2294. (21) Krizsan, A., Prahl, C., Goldbach, T., Knappe, D., and Hoffmann, R. (2015) Short ProlineRich Antimicrobial Peptides Inhibit Either the Bacterial 70S Ribosome or the Assembly of its Large 50S Subunit. ChemBioChem 16, 2304–2308. (22) Casteels, P., Ampe, C., Jacobs, F., Vaeck, M., and Tempst, P. (1989) Apidaecins: antibacterial peptides from honeybees. EMBO J. 8, 2387–2391. (23) Casteels, P., Romagnolo, J., Castle, M., Casteels-Josson, K., Erdjument-Bromage, H., and Tempst, P. (1994) Biodiversity of apidaecin-type peptide antibiotics. Prospects of manipulating the antibacterial spectrum and combating acquired resistance. J. Biol. Chem. 269, 26107–26115. (24) Schmidt, R., Knappe, D., Wende, E., Ostorházi, E., and Hoffmann, R. (2017) In vivo Efficacy and Pharmacokinetics of Optimized Apidaecin Analogs. Front. Chem. 5, 1–13. (25) Chen, X., Li, J., Sun, H., Li, S., Chen, T., Liu, G., and Dyson, P. (2017) High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin. Sci. Rep. 7, 14543. (26) Record, V. T. U. T. UNLOCK PICHIA Pichia pastoris Protein Expression Excellence UNLOCK PICHIA Pichia pastoris Protein Expression Excellence. (27) Pardee, K., Slomovic, S., Nguyen, P. Q., Lee, J. W., Donghia, N., Burrill, D., Ferrante, T., McSorley, F. R., Furuta, Y., Vernet, A., Lewandowski, M., Boddy, C. N., Joshi, N. S., and Collins, J. J. (2016) Portable, On-Demand Biomolecular Manufacturing. Cell 167, 248–259.e12.



Tables Table 1. Operating parameters used for HSA-apidaecin fusion protein fermentation.


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Figures

Figure 1. Schematic representation of the pAOX1-HSA-apidaecin expression vector and detailed information about the HAS-apidaecin fusion protein. (A) Schematic representation of the pAOX1-HSA-apidaecin expression vector and (B) amino acid sequence of the HSAapidaecin fusion protein. The histidine tag containing six histidines (H) is colored in green. The TEV protease recognition site is highlighted in red. The apidaecin peptide sequence is highlighted in blue.



Figure 2. Workflow for apidaecin production. (A) Overview of the production scheme for the HSA-apidaecin fusion protein and the release of apidaecin from the fusion protein. (B) SDSPAGE analysis of HSA-apidaecin expression. (C) SDS-PAGE analysis of the His-tag-based purification of HSA-apidaecin. A Ni Sepharose column was used to purify the fusion protein containing the His-tag. 1 µg standard HSA (red text), and samples (black text) were loaded in each lane. (D) SDS-PAGE analysis of the release and purification of apidaecin. TEV protease cleaved the fusion protein to release apidaecin, which was later separated with molecular weight filtration. Samples were loaded in each lane.


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Figure 3. Antimicrobial activity of yeast-produced apidaecin peptide. (A) Scheme of experimental workflow followed to assess antimicrobial activity. (B) Bacterial counts (CFU/mL) of E. coli BL21 treated with control groups using chemically synthesized peptide (CSP) at decreasing concentrations, or treated with P. pastoris-produced homemade apidaecin (“Homemade Peptide”). Three independent biological replicates were performed. Statistical significance was determined by Student’s t test. **** corresponds to p

Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide

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