BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Expression of enzymes for 3′-phosphoadenosine-5′-phosphosulfate(PAPS) biosynthesis and their preparation for PAPSsynthesis and regeneration

Payel Datta1,2 & Li Fu 1,2 & Wenqin He1,2 & M. A. G. Koffas1,2,3 & J. S. Dordick1,2,3,4 & R. J. Linhardt1,2,3,4

Received: 24 April 2020 /Revised: 22 May 2020 /Accepted: 31 May 2020# Springer-Verlag GmbH Germany, part of Springer Nature 2020

AbstractThe synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the actionsulfotransferases using the cofactor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Three enzymes are essential for the in vitrosynthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5′-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS).The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biologytool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different locationenable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1),ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400–1380 mg/Lwith accumulation of 34–66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, wereused for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupledwith a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing theenzymeASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153mg/L enzymewithaccumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach tooptimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be appliedtowards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated anautoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV).

Key points• ePathBrick used to optimize expression levels of enzymes.• Protocols have been used for the production of recombinant enzymes.• High cell density fed-batch fermentations with high yields of soluble enzymes.• Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

Keywords Heparin . 3′-phosphoadenosine-5′-phosphosulfate (PAPS) . PAPS-ASTIV recycling system . Fed-batchfermentation . High cell density autoinduction . ePathBrick platform

Introduction

Sulfation of biomolecules is ubiquitous in eukaryotic cellsand plays a pivotal role in various biological processesinvolving homeostasis and hormone metabolism; inactiva-tion and drug metabolism; activation and inactivation ofmutagens and xenobiotics; and post-translational modifica-tion of proteins, carbohydrates, and polysaccharides(Günal et al. 2019; Hale et al. 2020; Italia et al. 2020;Kamiyama et al. 2003; Klaassen and Boles 1997;Koprivova and Kopriva 2016; Lin 2004; Piecewicz and

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00253-020-10709-6) contains supplementarymaterial, which is available to authorized users.

* M. A. G. Koffaskoffam@rpi.edu

* J. S. Dordickdordick@rpi.edu

* R. J. Linhardtlinhar@rpi.edu

Extended author information available on the last page of the article

https://doi.org/10.1007/s00253-020-10709-6

http://crossmark.crossref.org/dialog/?doi=10.1007/s00253-020-10709-6&domain=pdfhttp://orcid.org/0000-0003-2219-5833https://doi.org/10.1007/s00253-020-10709-6mailto:koffam@rpi.edumailto:dordick@rpi.edumailto:linhar@rpi.edu

Sengupta 2011; Xiong et al. 2014). The sulfation of bio-molecules is catalyzed by tissue-specific sulfotransferasesfound in the Golgi and cytosol of eukaryotes (Coughtrie2016; Goettsch et al. 2006; Kamiyama et al. 2003). Thecofactor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS)is nearly universally used as a sulfate donor in the sulfationof a target biomolecule (Badri et al. 2019; Burkart et al.2000; Zhou et al. 2011). Therefore, the synthesis and re-generation of PAPS are critical for the action ofsulfotransferases and their use in biosynthesis. PAPS issynthesized in the cytosol, through the sequential actionof adenosine-triphosphate (ATP) sulfurylase (ATPS, ATPsu l f a t e adeny ly l t r ans fe ra se ) and adenos ine -5 ′ -phosphosulfate (APS) kinase (APSK, ATP adenylylsulfate3′-phosphotransferase) (Burkart et al. 2000; Zhou et al.2011). ATPS catalyzes the sulfation of ATP with inorganicsulfate to yield (APS) and inorganic pyrophosphate (PPi)(Fig. 1). The APS is then converted to PAPS by APSK inthe presence of ATP (Burkart et al. 2000; Zhou et al.2011). The resulting PAPS is then either utilized by cyto-solic sulfotransferases or translocated into the Golgi(Schwarz et al. 1984) for use by Golgi-associatedsulfotransferases (Coughtrie 2016; Goettsch et al. 2006;Kamiyama et al. 2003).

Understanding the biochemistry and activity of enzymesinvolved in PAPS synthesis is critical for myriad applications,including the cost-efficient chemoenzymatic synthesis of sulfat-ed polysaccharides like HS and heparin (Badri et al. 2018;

Zhang et al. 2009). HS is important in signaling in developmen-tal biology, physiology, and pathophysiology (Lindahl et al.1998; Soares da Costa et al. 2017; Zhang et al. 2009).Heparin is important pharmacologically as an anticoagulantdrug (Linhardt 2003). The biosynthesis of HS/heparin biosyn-thesis takes place in the Golgi of mammalian mast cells andinvolves the chemical N-deacetylation, N-sulfation ofheparosan to afford N-sulfoheparosan, followed byepimerization of the glucuronic acid residues to iduronic acid,catalyzed by C5-epimerase, and a series of HS-sulfotransferase-catalyzed reactions at the 2, 6, and 3-OH positions in the pres-ence of PAPS (Liu and Linhardt 2014; Zhang et al. 2009). Thisapproach has been used in vitro in the chemoenzymatic synthe-sis of HS/heparin (Fu et al. 2017). An efficient one-potchemoenzymatic synthesis of PAPS has been reported that in-volves APSK, ATPS, and PPA (Fig. 1a) (Burkart et al. 2000;Zhou et al. 2011). PAPS can then be used in sulfation reactionsand subsequently recycled using aryl sulfotransferase IV(ASTIV) (Fig. 1b) (Fu et al. 2017; Paul et al. 2012).Applications of the ASTIV-PAPS recycling system have beenused successfully towards production of biosynthetic heparin,and for enhancing the anticoagulant activity of bovine intestinalheparin (Fu et al. 2017).

An effective chemoenzymatic process requires the suf-ficient production of the three enzymes involved inPAPS synthesis as well as ASTIV involved in PAPSrecycling. To this end, we have focused on enzyme pro-duction using high cell density fed-batch fermentations

Fig. 1 Enzymes in PAPSsynthesis and regenerationsystem. a PAPS is synthesizedthrough the addition of inorganicsulfate to ATP and the addition ofphosphate to APS (Burkart et al.2000; Zhou et al. 2011). b Oncegenerated, PAPS serves as adonor for sulfotransferases andcan be regenerated in vitro usingp-nitrophenol sulfate (PNPS) as asacrificial sulfate donor

(Tables 1 and 2). In addition, we demonstrate that theseenzymes can be used in the chemoenzymatic synthesis ofdefined HS polysaccharides.

Materials and methods

Materials, bacterial strains, and plasmid constructs

Chemicals for media formulation, enzyme purification, en-zyme quantification, and PAPS production were purchasedfrom Sigma-Aldrich (St. Louis, MO). Antibiotics and IPTG

were purchased from Gold Biotechnology (St. Louis, MO).The bacterial strains used in the study are listed in Table 1.

High cell density non-autoinduction fed-batch fer-mentation of PAPS synthesis enzyme

Enzyme fermentations were performed in 5-L BioFlo 320Eppendorf or 1-L Applikon bioreactors. The fermentationsconsisted of a batch phase, followed by a fed-batch phase.The batch media and feed compositions for each strain areshown in Table 2. The batch phase was initiated with theinoculation of seed culture in batch media (starting OD600 =

Table 1 Strain, gene sequenceaccession numbers, and plasmidinformation (see Table S1 forprimer information and cloningstrategy for the genes ATPS,APSK and PPA)

Strain Description Source

DH5α General cloning host Invitrogen

BL21Star™ (DE3) F-ompT hsdSB (rB-, mB

-) galdcmrne131 (DE3) NovagenRosetta-gami B (DE3) F– ompT hsdSB (rB

– mB–) gal dcm lacY1 ahpC

(DE3) gor522::Tn10 trxBpRARE (CamR, KanR, TetR)

Novagen

Gene Organism Accession numbers

APSK Penicillium chrysogenum U39393.1ATPS Kluyveromyces lactis CP021243.1PPA Kluyveromyces lactis CP047127.1

Plasmid Description Reference

pETM6 T7 promoter, ColE1 ori. AmpR (Xu et al. 2012)

pETM6_ATP

pETM6_ATPSH

pETM6 carrying gene atps

pETM6 carrying gene atps fused with Histag in N-terminus

This study

This study

pETM6_APSK

pETM6_APSKH

pETM6 carrying gene apsk

pETM6 carrying gene apsk fused with His tag in N-terminus

This study

This study

pETM6_PPA

pETM6_PPA

pETM6 carrying gene ppa

pETM6 carrying gene ppa fused with His tag in N-terminus

This study

This study

pETM6_PSKP pETM6 carrying genes in the order of atps,apsk ppa in pseudo-operon configuration

This study

pETM6_PSPK pETM6 carrying genes in the order of atps,ppa and apsk in pseudo-operon configuration

This study

pETM6_PKSP pETM6 carrying genes in the order of apsk,atps, and ppa in pseudo-operon configuration

This study

pETM6_PKPS pETM6 carrying genes in the order of apsk,ppa and atps in pseudo-operon configuration

This study

pETM6_PPSK pETM6 carrying genes in the orderof ppa, atps and apsk in pseudo-operonconfiguration

This study

pETM6_PPKS pETM6 carrying genes in the order of ppa,apsk and atps in pseudo-operon configuration

This study

pCDFDuet-1_PPA pCDFDuet-1 carrying gene ppa This study

pETDuet-1_ATPS_APSK

pETDuet-1 carrying genes ATPS and APSK This study

pET28a(+)_ASTIV pET28a(+) carrying gene ASTIV fromR. norvegicus

Gift from Pr. Jian Lu,UNC

pMalc2x_6OST3 pMalc2x carrying the catalytic domainof mouse 6OST-3 (Pro121–Pro450)

(Chen et al. 2005)

0.25 ± 0.1). The fermentation and bioreactor conditions areprovided in supporting information Table S2. The fed-batchphase was initiated when OD600 reached 3.5 ± 2.0. The cellswere fed at an initial feed rate of 2.5 mL/h/L and the feedingrate was adjusted based on fluctuations in pH and dissolvedoxygen (DO) levels. The cells were induced at 22 °C at anOD600 of 6.5 ± 2.0. Overnight induction at 22 °C was per-formed with 0.5 mM IPTG. Post-induction, the cells wereharvested by centrifugation at 5000×g for 20 min at 4 °C.The cell paste was weighed and stored at − 80 °C.

High cell density autoinduction fed-batch fermenta-tion of ASTIV enzyme

Enzyme fermentations were performed in 5-L BioFlo 320Eppendorf or 1-L Applikon bioreactors. The fermentationsconsisted of a batch phase, followed by a fed-batch phase.The batch media and feed compositions for each strain areshown in Table 2. The batch phase was initiated with theinoculation of seed culture in batch media (starting OD600 =0.25 ± 0.1). The fermentation and bioreactor conditions areprovided in supporting information Table S2. The fed-batchphase was initiated when OD600 reached 3.5 ± 2.0 and tem-perature was reduced to 22 °C. The cells were fed at an initialrate of 2.5 mL/h/L and the feeding rate was adjusted, based onfluctuations in pH and dissolved oxygen (DO) levels. Thelactose in the feed served as the inducer and overnight induc-tion at 22 °C was performed. Post-induction, the cells wereharvested by centrifugation at 5000×g for 20 min at 4 °C. Thecell paste was weighed and stored at − 80 °C.

Quantification of enzyme yield using SDS-PAGEanalysis

Enzyme concentrations were determined using semi-quantitative SDS-PAGE analysis with bovine serum albumin

(BSA) used as a standard. Briefly, the cell pellet (5 g wetweight) was dispersed in 25 mL of purification buffer(25 mM Tris-HCl (Bio-Rad, USA), 500 mM NaCl (Sigma,USA), pH 7.5) containing 8000 kU/LDNAse I (Sigma, USA),and protease inhibitors (SIGMAFAST™ Protease InhibitorCocktail Tablets, EDTA-Free, Sigma, USA). The cells werelysed using sonication, followed by centrifugation at13,500×g for 40 min at 4 °C. The cell lysate (soluble enzymefraction) was carefully transferred to a clean tube, and the cellpellet (insoluble fraction) was dissolved in 2.5 mL lysis buffer.Both the undiluted and the diluted fractions were evaluatedusing SDS-PAGE performed using a MiniProtean Tetra sys-tem (Bio-Rad, Hercules, CA). Denatured enzymes (10 μL of a1:1 (v:v) boiled enzyme solution and sample buffer) and pro-tein ladder (Precision Plus Protein™ Kaleidoscope™Prestained Protein Standards, Bio-Rad) were resolved usinga 4–15% precast gel (Mini protean TGX gels, Bio-Rad) at 220V. Gels were washed with deionized water, stained withGelCode™ Blue Safe Protein Stain (ThermoFisher, City,State), and destained with deionized water.

PAPS production using APSK, ATPS, and PPA

PAPS synthesis was performed using a modified protocol,based on a previously described method (Burkart et al.2000; Zhou et al. 2011). Briefly, following fermentation, cellswere harvested by centrifugation at 5000×g for 20min at 4 °C.The supernatant was discarded, and the cell paste was storedin − 80 °C. On the day of the experiment, PPA expressingcells and ATPS+APSK expressing cells were thawed in lysisbuffer (20 mM tris(hydroxymethyl) amino (Tris)–HCl,500 mM NaCl, pH 8.0) and lysed using a microfluidizer at 4°C. After centrifugation at 12,000×g for 20 min at 4 °C, thelysate was filtered using a 0.45-μm filter. The filtered celllysate was utilized for PAPS synthesis. Briefly, the cell lysate(0.3 mg/mL PPA and 0.6 mg/ml APSK+ATPS) was added to

Table 2 Batch media and feed composition

Strain Description Composition

Recombinant E. coli BL21 Star™ (DE3)expressing ASTIV (kanamycin, 50 μg/ml)ASTIV

Autoinduction batch media Ammonium chloride (31 mM), Glycerol (7 g/L),KH2PO4 (25 mM), K2HPO4 (25 mM), lactose (2 g/L),MgSO4.7H20 (1.8 g/L), sodium chloride (5 g/L),tryptone (10 g/L), yeast extract (12 g/L)

Autoinduction feed 40% Glycerol, supplemented with 67 g/L NH4Cland 100 g/L lactose

Recombinant E. coli BL21 Star™ (DE3)expressing PPA (streptomycin 50 μg/ml)andecombinant E. coli BL21 Star™ (DE3)expressing ATPS and APSK (carbenicillin, 50 μg/ml)

Non-autoinduction batch media Glycerol (7 g/L), MgSO4.7H20 (1.8 g/L),sodium chloride (5 g/L), tryptone (10 g/L),yeast extract (12 g/L)

Non-autoinduction feed 50% glycerol, supplemented with 67 g/L NH4Cland 100 g/L yeast extract

a reaction mixture that contained variable concentrations ofATP sodium (0 mM to 22.5 mM), 100 mM Na2SO4, 10 mMMgCl2, 10 mM LiCl2, 50 mM Tris-HCl (pH 8.0), and 0.02%(w/v) sodium azide. The reaction was performed for 16 h at 30°C. Following the reaction, the reaction mixture was centri-fuged at 12,000×g for 20 min. The supernatant was removed,clarified, and subjected to ion exchange chromatography,wherein PAPS was eluted with 5 mM sodium phosphate, pH8.0, and 500 mM NaCl. PAPS was desalted using size exclu-sion chromatography (Sephadex G-10 resin) using high-performance liquid chromatography (HPLC) grade water asthe mobile phase. The desalted PAPS was lyophilized andconcentrated in HPLC grade water, aliquoted, and stored at− 80 °C. The final product was assessed for yield, PAPSpurity, and concentration.

Analysis of PAPS by HPLC-MS

Liquid chromatography mass spectrometry (LC-MS) anal-yses were performed on an Agilent 1200 LC/MSD instru-ment (Agilent Technologies, Inc. Wilmington, DE). Theinstrument was equipped with a 6300 ion-trap and a binarypump and a Poroshell 120 EC-C18 column (2.0 × 100 mm,2.7 μm, Agilent, USA) was used for the experiments.Eluent A contained water/acetonitrile (85:15, v/v) and elu-ent B contained water/acetonitrile (35:65, v/v). Both elu-ents were supplemented with 12 mM tributylamine (TrBA)and 38 mM NH4OAC. For separation of ATP and PAPS, alinear gradient from 0 to 18 min (0–80% of solution B) wasused at 80 μL/min. For continuous detection by MS, thecolumn effluent entered the electrospray ionization MSsource. The electrospray interface was set in negative ion-ization mode with a skimmer potential of − 40.0 V, a cap-illary exit of − 40.0 V, and a source temperature of 350 °C,to obtain the maximum abundance of the ions in a full-scanspectrum (100–900 Da). Nitrogen (8 L/min, 40 psi) wasused as a drying and nebulizing gas.

Purification and immobilization of ASTIV enzymes

The cell pellet (5 g wet weight) of His6-tagged-ASTIV wasdispersed in 25 mL of purification buffer (25 mM Tris-HCl(Bio-Rad, USA), 500 mM NaCl (Sigma, USA), and 30 mMimidazole, pH 7.5) containing 8000 kU/L DNAse I (Sigma,USA), and protease inhibitors (SIGMAFAST™ ProteaseInhibitor Cocktail Tablets, EDTA-Free, Sigma, USA). Thecells were lysed using sonication, followed by centrifugationat 13,500×g for 40 min at 4 °C. The enzymes from the solublefraction were purified using 5 mL of Ni-NTA Sepharose (GEHealthcare, Sweden) according to manufacturer’s instruc-tions. The purity and MW of the enzyme was assessed usingSDS-PAGE gel analysis.

ASTIV activity assay

The ASTIV activity assay was performed as previously de-scribed (Paul et al. 2012) with some modification using aShimadzu UV-1650PC. Briefly, the enzyme was purifiedusing Ni-NTA Sepharose and quantified using nanodrop spec-troscopy at 280 nm. The activity was determined by incubat-ing 0.1 mg of purified ASTIV with 5 mM PNPS, 0.05 mM 3′adenosine-5′ phosphate (PAP), in phosphate-buffered saline(pH 7.0) (15 min, 37 °C, absorbance at 400 nm). The specificactivity was calculated using an extinction coefficient of 1.05× 10−2 M−1 cm−1.

Production, purification, and immobilization of MBP-tagged-6OST-3 enzyme

Recombinant E. coli Rosetta-gami B (DE3) strain (Novagen,Cambridge, MA) expressing the plasmid pMalc2x_6OST3(the catalytic domain of mouse 6OST-3, Pro121–Pro450)was used for production of MBP-tagged-6OST-3 (Chenet al. 2005). The non-autoinduction fermentation protocolfor PAPS biosynthetic enzymes was used for production ofthe MBP-tagged-6OST3 enzymes. The cell pellet (5 g wetweight) of MBP-tagged-6OST-3 was dispersed in 25 mL ofpurification buffer (25 mM Tris-HCl (Bio-Rad, USA),500 mM NaCl (Sigma), pH 7.5) containing 8000 kU/LDNAse I (Sigma), and protease inhibitors (SIGMAFAST™Protease Inhibitor Cocktail Tablets, EDTA-Free). The cellswere lysed using sonication, followed by centrifugation at13,500×g for 40 min at 4 °C. The enzymes from the solublefraction were purified using 5 mL of amylose resin (NEB,USA) according to manufacturer’s instructions. The purityand MW of the enzyme was assessed using SDS-PAGE gelanalysis.

6-O-sulfation of N-sulfo heparosan using ASTIV andPAPS recycling system

NSH was prepared using a method previously described(Wang et al. 2011).N-sulfoheparosan (NSH) (2mg) was treat-ed with 6OST-3, using a modified protocol, based on a previ-ously described method (Xiong et al. 2013). The sulfationreaction was coupled with the PAPS recycling system thatconsisted of PNPS, PAPS, and ASTIV. The detailed reactionconditions were as follows: 0.1 mg/mL NSH, 0.5 mg/mL6OST-3, and 10 mM and 250 μM of PNPS and PAPS, re-spectively. Reactions were incubated at 37 °C for 40 h in50 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH7.2). After the reaction was complete, the mixture containingN-sulfo, 6-O-sulfo heparosan (NS6S) was boiled for 10 minfollowed with 5000×g centrifugation (10 min) to spin downprecipitated free enzymes. The supernatant was collected,

desalted, and freeze-dried for further analysis, including disac-charide analysis and NMR.

Disaccharide compositional analysis

NSH and NS6S samples (200 μg each) were completelydigested using a mixture of heparinase I, II, and III (10 mUeach) in 200 μL 50 mM ammonium acetate buffer (pH 7.2) at37 °C for 12 h. The resulting disaccharides were recoveredusing 10 KDa molecular weight cut-off (MWCO) ultrafiltra-tion units (Millipore, MA) and washed with deionized water.The ultrafiltrates containing disaccharides were collected andfreeze-dried for disaccharide analysis.

Disaccharide analysis was carried out with high-pressure liquid chromatography (HPLC)–ultraviolet spec-trometry (UV). The analyses were performed with aShimadzu LC-20 AD pump, Shimadzu CBM-20A con-troller, Shimadzu SIL-20AHT auto-sampler, ShimadzuCTO-20 AC column oven, and a Shimadzu SPD-20AVUV detector (Shimadzu, Kyoto, Japan). A Spherisorb-SAX chromatography column (4.0 × 250 mm, 5.0 μm,Waters) was equilibrated with 1.8 mM monobasic sodiumphosphate (eluent A, pH 3.0) and followed with ingredi-ent elution after injection using 1.8 mM monobasic sodi-um phosphate with sodium perchlorate (eluent B, pH 3.0).Disaccharide standards were purchased from Iduron(Manchester, UK). The weight/weight (w/w) percentagefor each disaccharide was calculated. Peak areas wereused for quantification.

NMR spectroscopy

NSH and NS6S samples (each 1.5 mg) were analyzed with1D 1H nuclear magnetic resonance (NMR). The 1D 1H-NMR experiments were performed on a Bruker Advance II600 MHz spectrometer (Bruker Bio Spin, Billerica, MA)with Topsin 2.1.6 software (Bruker). Samples were eachdissolved in 0.5 mL D2O (99.996%, Sigma-Aldrich) andfreeze-dried repeatedly to remove the exchangeable pro-tons. The samples were dissolved in 0.4 mL D2O and trans-ferred to NMR microtubes (outside diameter, 5 mm, Norell(Norell, Landisville, NJ)). All NMR experiments were car-ried out with the conditions as previously reported. (Fuet al. 2014; Fu et al. 2016)

Data analysis

All data analysis was performed using Bio-rad Image Labsoftware, Microsoft Excel, and GraphPad (GraphPad Prism,San Diego, CA, USA).

Results

Expression of PAPS synthesis enzymes and PAPSePathbrick screening platform

ATPS, APSK, and PPA are used for the in vitro synthesis ofPAPS. The enzymes were cloned into the pETM6 vector,expressed as His-tagged enzymes in recombinant E. coli cells.All three enzymes, ATPS (57.3 KDa), APSK (24.6 KDa), andPPA (20.5 KDa) were expressed as His-tagged proteins inE. coli (Fig. 2).

PAPS expression analysis was evaluated usingePathBrick platform (He 2017; Xu et al. 2012). TheePathBricks is a versatile platform used for rapid designand optimization of metabolic pathways in E. coli; in thecurrent study, the ePathbrick platform has been success-fully used as a tool to study gene expression of PAPSsynthesis enzymes. The PAPS ePathbrick platform dem-onstrated that the ratio of PPA, APSK, and ATPS impactPAPS production. Increasing ATPS concentration nega-tively impacted PAPS production (Fig. 3). Two ap-proaches were evaluated: (1) three genes in a single plas-mid pETM6_PSKP used for expression and (2) threegenes placed in a pseudo-operon configuration in differentgene orders to optimize PAPS production (He 2017). Thisexpression system demonstrated the feasibility of crude

Fig. 2 Shake flask experiments and SDS-PAGE gel of the expression ofPAPS synthesis related genes. Lane 0, protein ladder; lane 1, solublefraction of ATPS; lane 2, purified ATPS (57.3 KDa); lane 3, solublefraction of APSK; lane 4, purified APSK (24.6 KDa); lane 5, solublefraction of PPA; lane 6, purified PPA (20.5 KDa)

cell lysate in successful in vitro PAPS production(Figure S2) and the need to balance the expression of

the three enzymes involved in vitro PAPS synthesis. ThePAPS ePathbrick system was used to evaluate the impactof the expression levels of these three enzymes on PAPSproduction. Optimal PAPS production required the threegenes (PPA, APSK, ATPS) to be placed in a pseudo-operon configuration (He 2017). The reactions were car-ried out without supplementing ATP after cell lysis. Theoptimal strains contained plasmid pETM6_PSPK, whereAPSK was most highly expressed, followed by PPA, andthen ATPS (Fig. 3b). An optimal strain should be able tocovert most of the ATP to PAPS. Based on these results,two recombinant E. coli strains were engineered; namely,(1) E. coli BL21 Star™ (DE3) strain expressing PPA and(2) E. coli BL21 Star™ (DE3) strain expressing APSKand ATPS. These two strains were used for scale-upfed-batch fermentation experiments.

High cell density non-autoinduction fed-batch fer-mentation of PAPS synthesis enzyme

High cell density, fed-batch fermentation of PAPS synthesisenzymes without autoinduction may be broadly classified intothree growth phases: (1) seed inoculation and cell growth inbatch media; (2) initiation of feed and growth of cells at theexponential growth rate; and (3) induction with IPTG. Twostrains expressing the three PAPS synthesis enzymes wereexpressed using the same fermentation protocol. The fermen-tation profiles for the two strains are shown in Figure S3a(E. coli BL21 Star™ (DE3) strain expressing PPA), andFigure S4a (E. coli BL21 Star™ (DE3) strain expressingAPSK and ATPS). The yield and MW of the enzymes fromsoluble fraction were analyzed and quantified. Enzymes werepresent in both soluble and insoluble fractions (Figures S3band S4b). The high cell density fed-batch fermentation of

Fig. 3 In vitro PAPS synthesis reaction with 6 different molar ratios ofATPS, APSK, and PPA. a Pure enzyme with the same molar ratio frompETM6 pseudo-operon configuration with 0.09mMof ATP. b The crudelysate contained six different pETM6 plasmids with genes in differentorder. The intensity of the protein expression is indicated by the color:black is the highest, dark gray is the medium, and light gray is the lowest

Table 3 Fermentation and enzyme yield (see Figure S1 for schematic diagram and experimental plan; see Table S3 for calculations; # semi-quantitativeSDS-PAGE analysis)

Strains ASTIV PPA ATPS and APSKFermentation strategy Fed-batch lactose induction IPTG induction (0.5 mM) IPTG induction (0.5 mM)

Total fermentation time 23 to 27 h 26 h 24 h

Total induction time 19 h 21 h 19 h

Harvest OD600 57 40 34

Soluble enzyme yield# (mg/g cell paste) 5.75 14.45 ATPS: 4.65APSK: 6.44

Volumetric enzyme yield in soluble fraction(mg/L fermentation broth)

555 908 ATPS: 199APSK: 276

Volumetric total enzyme yield in solubleand insoluble fraction (mg/L fermentation broth)

1153 1383 ATPS: 579APSK: 419

% Enzyme in soluble fraction 48 % 66 % ATPS: 34%APSK: 66%

Enzyme purification and utilization method Ni-NTA resin purification Soluble fraction Soluble fraction

strain expressing PPA resulted in a volumetric yield of 1380mg/L of fermentation broth (Table 3); and 34% of the PPAenzymes accumulated in the insoluble fraction (misfoldedproteins or inclusion bodies) and the volumetric yield ofPPA in the soluble fraction (correctly folded active proteins)was 910 mg/L (Table 3). The high cell density fed-batch fer-mentation of strain expressing both APSK andATPS varied involumetric yield and the percentage of enzyme in the solublefractions (Figure S4b and Table 3). The high cell density fed-batch fermentation of APSK resulted in a volumetric yield of420 mg/L of fermentation broth (Table 3) and 34% of theAPSK enzymes accumulated in the insoluble fraction andthe volumetric yield of APSK in the soluble fraction was280 mg/L (Table 3). The high cell density fed-batch fermen-tation of ATPS resulted in a volumetric yield of 580 mg/L offermentation broth (Table 3) and 66% of the ATPS enzymesaccumulated in the insoluble fraction and the volumetric yieldof ATPS in the soluble fraction was 200 mg/L (Table 3).Current PAPS synthesis involves the quantification of thePAPS enzymes from the soluble fraction and directly addingthe filtered soluble fractions (that contain the PAPS synthesisenzymes) into the PAPS synthesis reaction mixture.

High cell density fed-batch autoinduction fermenta-tion of ASTIV

High cell density fed-batch autoinduction fermentation ofASTIV can be broadly classified into two growth phases: (1)seed inoculation and cell growth in a batch media and (2)initiation of feed and growth of cells at exponential growthrate (Fig. 4a). The yield and MW of the enzymes from solublefraction were analyzed and quantified. Results demonstratethe presence of ASTIV in both soluble and insoluble fractions(Fig. 4b). The high cell density autoinduction fed-batch fer-mentation resulted in a volumetric ASTIV yield of 1150mg/Lof fermentation broth (Table 3). However, approximately 52%of the enzyme accumulated in the insoluble fraction (inactiveenzyme) and the volumetric yield of ASTIV in the solublefraction (active enzyme) was 560 mg/L and most of the solu-ble ASTIV was immobilized using Ni-NTA resin (Figs. 4b, 5,and Figure S5).

Utilization of PAPS towards generation of definedoligosaccharides, using HS-6OST3 sulfotransferaseand ASTIV regeneration system

PAPS produced using the PAPS synthesis enzymes can besuccessfully used in the synthesis of defined HS polysaccha-rides in the presence of a ASTIV-coupled PAPS regenerationsystem. To this end, the ASTIV was prepared as a His-taggedfusion protein. The HS biosynthetic enzyme, 6OST-3 was

a

b

Fig. 4 High cell density fermentation of ASTIV and production ofsoluble enzymes. a Fermentation was performed in 1-L Applikonbioreactor, equipped with a BugLab cell growth probe. The primary y-axis denotes cell growth, using arbitrary Buglab units (black line). Thesecondary y-axis denotes pH (set at 6.9 ± 0.1, dashed gray line),temperature (°C, gray line), and offline OD600 measurement (blackspots/dots). The spots are offline OD600 measurements (usingspectrophotometer). The OD600 is shown in secondary Y-axis. The cellgrowth was monitored using BUGLAB) and the units are arbitraryBUGLAB units (straight line, primary Y-axis). The graph demonstratesthat the BUGLAB accessory could be successfully used to assess the cellgrowth and is comparable with the offline OD600 measurements. bFollowing fermentation, the cells were harvested and analyzed forinsoluble and soluble enzymes. Lane 1, protein ladder; lane 2, enzymesin the undiluted insoluble fraction; lane 3, enzymes in the undilutedsoluble fraction; lane 4, enzymes in the 1/10 diluted insoluble fraction;lane 5, enzymes in the 1/10 diluted soluble fraction; lane 6, 62.5 μg/mLBSA standard; lane 7, 125μg/mLBSA standard; lane 8, 250 μg/mLBSAstandard; lane 9, 500 μg/mL BSA standard; lane 10, 1000 μg/mL BSAstandard. For each lane, 12 uL of sample was loaded. Results demonstratepresence of the 37 KDa ASTIV enzyme in both soluble and insolublefractions

prepared as a maltose binding protein (MBP) fusion. Theseproteins were immobilized on Ni-NTA Sepharose and amy-lose resins, respectively. NSH was treated with immobilized6OST3 overnight at 37 °C. The sulfation reaction was coupledwith PAPS recycling system consisting of (1) ASTIV, (2)PNPS as a sacrificial sulfo-donor, and (3) PAPS. After thereaction was complete, the reaction mixture was filtered toremove the beads and desalted using 1 KDa MWCO centrif-ugal ultrafiltration. The purified mixture was lyophilized forfurther disaccharide analysis and NMR analysis. The disac-charide compositions of NSH and NSH treated with 6OST3are shown in Table 4. After treatment with 6OST3, in pres-ence of PAPS recycling system, NSH affords 6-O-sulfated,N-sulfoheparosan (NS6S). The formation of NS6S was verifiedusing 1D 1H-NMR (Fig. 6).

Discussion

The scalable production of enzymes using high cell densityfermentation generally focuses on the cost-effective produc-tion of active biocatalysts. Upstream fermentation parameters,such as media design, can impact downstream purificationand utilization of the enzymes. The current work demonstratestwo different enzyme fermentation and utilization strategies:

(1) fed-batch fermentation of PAPS biosynthetic enzymeswithout autoinduction, followed by utilization of the enzymespresent in the soluble fraction; and (2) autoinduction fed-batchfermentation of ASTIV, followed by utilization of purifiedenzymes in a PAPS regeneration system. In both strategies,the first step requires minimizing feed and reagents in thefermentation. For example, trace metals were omitted andthe batch medium was prepared using tryptone, yeast extract,and glycerol. The batch medium for autoinduction was de-signed to use ammonium chloride, sodium chloride, phos-phate buffer, and lactose. Minimizing the number of ingredi-ents ensures better fermentation control, reduces errors relatedto media formulation, scale-up, and technology transfer, andreduces the costs and inventory management. Fed-batch fer-mentation without autoinduction for the production of thePAPS synthesis enzymes, PPA, APSK and ATPS, relies onstandard IPTG induction. This fermentation strategy is robustand has been widely used towards the production of manyother enzymes with minor modifications for IPTG concentra-tion, duration of induction, and induction temperature. A fed-batch fermentation strategy using lactose autoinduction for theproduction of ASTIV was inspired based on the shake flaskexperiments. Protein expression in shake flasks using com-plex or defined media supplemented with phosphate buffer,glucose, glycerol, lactose, and trace metals has been reported(Studier 2005). The fed-batch fermentation strategy forASTIV utilized this strategy with modifications includingthe elimination of glucose and trace metals and an optimizedratio of tryptone and yeast extract.

Enzyme fermentations are best evaluated using the volu-metric yield of active enzymes required to generate the desiredproduct. The current study utilizes two approaches of enzymeutilization: (1) purification of enzymes using specific resin andusing immobilized enzymes in the reaction of ASTIV-coupledPAPS regeneration system and (2) directly adding the solublefraction to the reactionmixture in PAPS synthesis. The currentHS polysaccharide synthesis involves purification of ASTIVfrom the soluble fraction using a Ni-NTA Sepharose resin,and the immobilized enzymes utilized for the ASTIV-coupled PAPS regeneration system. The high cell densityautoinduction fed-batch fermentation of the strain expressingASTIV resulted in a volumetric yield of 560 mg/L of fermen-tation broth, which comprises 48% of the total enzymesexpressed in the soluble and insoluble fraction (Table 3).The current approach for PAPS synthesis involves quantifica-tion of the PAPS enzymes from the soluble fraction and di-rectly adding filtered soluble fractions containing PAPS syn-thesis enzymes in the PAPS synthesis reaction mixture. Afterthe fermentation to produce PAPS biosynthetic enzymes, theenzyme yield was calculated using a semi-quantitative SDS-PAGE (Table 3). The PAPS biosynthetic enzymes wereexpressed in two strains, E. coli BL21 Star™ (DE3) strainexpressing PPA and E. coli BL21 Star™ (DE3) strain,

Fig. 5 Purified ASTIV on Ni-NTA Sepharose resin. Lane 1, proteinladder; lane 2, 250 μg/mL BSA standard; lane 3, 125 μg/mL BSAstandard; lane 4, 62.5 μg/mL BSA standard; lane 5, purified ASTIV(37 KDa) enzyme

Table 4 Disaccharide compositional analysis of NSH and NS6S

0S NS 6S 2S NS6S NS2S 2S6S TriS

NSH 0.8 99.2 0.0 0.0 0.0 0.0 0.0 0.0

NS6S 0.7 13.1 0.0 0.0 86.2 0.0 0.0 0.0

expressing APSK and ATPS. The high cell density fed-batchfermentation of the strain expressing PPA resulted in a volu-metric yield of 910 mg/L of fermentation broth, which com-prises 66% of the total enzymes expressed in the soluble andinsoluble fraction (Table 3). Fermentation of the strain co-expressing APSK and ATPS demonstrated variable expres-sion of APSK and ATPS. The high cell density fed-batchfermentation for APSK resulted in a volumetric yield of 280mg/L of fermentation broth with 66% of the total enzymesexpressed in the soluble (active) and insoluble (inactive) frac-tion (Table 3). The high cell density fed-batch fermentation ofATPS resulted in a volumetric yield of 200 mg/L of fermen-tation broth, which comprises 34% of the total enzymesexpressed in the soluble and insoluble fraction (Table 3).The expression of APSK and ATPS are desirable featuresbecause, preliminary experiments showed that decreasedATPS expression increased APSK expression favor PAPSproduction (Fig. 3).

We hypothesize that enzymes in the insoluble fraction con-sist primarily of unfolded, partially processed, or inactive en-zymes and that these enzymes may be scavenged with addi-tional purification steps, including enzyme refolding. Theremay be various factors that impact the accumulation of en-zymes in the insoluble fraction. In analyzing PAPS biosyn-thetic enzymes and their fermentations, we speculate thatsmaller enzymes can easily fold and accumulate in the soluble

(active) fraction (Palmer and Wingfield 2012). Both APSK(25 KDa) and ATPS (57 KDa) are produced in the samefermentation. However, there are differences in the volumetricyield of APSK (66% soluble) and ATPS (34% soluble).Similarly, PPA is smaller (21 KDa) and 66% of the enzymeis in the soluble fraction. The production of enzymes is oftennot straightforward and many factors can impact the produc-tion of the volumetric yield of active enzymes, including (1)expression of chaperones, (2) post-translational modifications,and (3) strain metabolism (Belval et al. 2015; Brylski et al.2019; Ellis 2013; Jhamb and Sahoo 2012; Thomas et al.1997). In this context, we have observed with other enzymesthat under similar fermentation conditions, and either induc-tion at 30 °C for 4 h or higher cell growth rate during induc-tion, the majority of enzymes expressed accumulate in theinsoluble fraction. Additionally, the carbon and nitrogen mo-lar ratio during fermentation can impact enzyme production.Future research is geared towards evaluating strategies to-wards improving soluble enzymes utilization, including strainengineering, fermentation optimization, and purification ofenzymes from insoluble fractions.

In conclusion, the paper demonstrated that the ePathbrickplatform that had previously been used for metabolic engineer-ing, could be successfully utilized to study in vitro enzymaticsynthesis conditions. This application of ePathbrick platformcould be applied for one-pot synthesis of polysaccharides, such

Fig. 6 1D 1H-NMR of NSH before and after 6OSTs treatment (NS6S). Assignment, A, glucosamine; G, glucuronic acid; *, residual PAPS and PAP

as heparin and heparan sulfate. Heparin and heparan sulfate aresulfated polysaccharides with tissue-specific sulfation domains.Tissue-specific sulfotransferases (and their isoforms) dictate thesulfation patterns on heparin and heparan sulfate. TheePathbrick tool could assemble seven-gene pathways (Xuet al. 2012) and could be utilized to evaluate effect ofsulfotransferase expression ratio on sulfation patterns on hepa-rin and heparan sulfate. In parallel, we have successfully dem-onstrated a fed-batch fermentation protocol (IPTG induction ornon-autoinduction protocol) that could be utilized for expres-sion of various enzymes, including sulfotransferases (e.g.,6OST-3). The protocol has been successfully utilized for fer-mentation, scale-up (100-L) and technology transfer (to indus-trial partners) of heparin biosynthetic enzymes (e.g., 2OST, C5epimerase, 6-OST-1, 6OST-3, and 3OST-1). The media andfeed composition are similar for all these fermentations, withvariations in induction parameters (e.g., IPTG concentration,induction temperature, and induction time). Adapting all thebiosynthetic enzymes in similar fermentation protocol enablesus to control inventory management and cost. In addition, thepaper successfully demonstrates an autoinduction microbialfed-batch fermentation strategy towards the production of theenzyme ASTIV. The successful fermentation strategies and re-duction in cost of the production of enzymes (related to up-stream) has enabled the successful pilot scale production andutilization of the enzymes.

Authors’ contributions RJL and JSD provided the funding for this study.RJL and PD planned the study. PD, RJL, and JSD wrote the manuscript.PD performed the fermentations. WH andMAGK designed and preparedthe recombinant organisms. LF was responsible for the studies.

Funding information This work was supported by the US NationalScience Foundation award CBET-1604547 awarded to MAGK and byNational Institutes of Health grants CA231074 and DK111958 to RJL.

Compliance with ethical standards This article does notcontain any studies with human participants or animals performed byany of the authors.

Conflict of interest The authors declare that they have no conflict ofinterest.

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Affiliations

Payel Datta1,2 & Li Fu 1,2 &Wenqin He1,2 &M. A. G. Koffas1,2,3 & J. S. Dordick1,2,3,4 & R. J. Linhardt1,2,3,4

1 Department of Chemical and Biological Engineering, Center forBiotechnology and Interdisciplinary Studies, Rensselaer PolytechnicInstitute, Troy, NY 12180, USA

2 Department of Chemistry and Chemical Biology, Center forBiotechnology and Interdisciplinary Studies, Rensselaer PolytechnicInstitute, Troy, NY 12180, USA

3 Department of Biomedical Engineering, Center for Biotechnologyand Interdisciplinary Studies, Rensselaer Polytechnic Institute,Troy, NY 12180, USA

4 Department of Biology, Center for Biotechnology andInterdisciplinary Studies, Rensselaer Polytechnic Institute,Troy, NY 12180, USA

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