Programming a Biofilm-Mediated Multienzyme-Assembly-Cascade

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Biotechnology and Biological Transformations

Programming biofilm-mediated multi-enzyme assembly cascade system for biocatalytic production of glucosamine from chitin Jingjing Bao, Nian Liu, Qing Xu, Liying Zhu, He Huang, and Ling Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02142 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Programming biofilm-mediated multi-enzyme assembly cascade system for

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biocatalytic production of glucosamine from chitin

3

Jingjing Bao1, Nian Liu2, Liying Zhu3, Qing Xu4, He Huang4, Ling Jiang2,*

4 5

1

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Nanjing 210009, People’s Republic of China; 2College of Food Science and Light

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Industry, Nanjing Tech University, Nanjing 210009, People’s Republic of China;

8

3

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210009, People’s Republic of China; 4College of Pharmaceutical Sciences, Nanjing

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College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

College of Chemical and Molecular Engineering, Nanjing Tech University, Nanjing

Tech University, Nanjing 210009, People’s Republic of China

11 12

*

Corresponding author: Ling Jiang, [email protected]

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ABSTRACT: Chitin is used as an essential raw material for the production of

14

glucosamine (GlcN). In this study, we adopted three key enzymes isolated from

15

Thermococcus kodakaraensis KOD1 that catalyze the sequential conversion of

16

α-chitin into GlcN, and developed a Multi-enzyme Assembly Cascade system (MAC

17

system) immobilized in a bacterial biofilm, which enabled a multi-step one-pot

18

reaction. Specifically, SpyTag/SpyCatcher and SnoopTag/SnoopCatcher pairs

19

provided covalent and specific binding force to fix enzymes to the biofilm one by one

20

and assemble enzyme cascades close. The MAC system showed a great catalytic

21

activity, converting 79.02%

22

which was 2.09 times of GlcN catalyzed by mixture of pure enzymes. The system also

23

exhibited good temperature- and pH stability. Notably, 90% of enzyme activity was

24

retained after six rounds of reuse and appreciable activity remained after 17 rounds.

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KEYWORDS:

26

SnoopTag/SnoopCatcher, multi-enzyme assembly

Chitin,

3.61% of α-chitin into GlcN with little by-products,

glucosamine,

biofilm,

2


SpyTag/SpyCatcher,


27

INTRODUCTION

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Glucosamine (2-amino-2-deoxy-D-glucose, GlcN), which widely exists in all

29

organisms, is an essential precursor in synthesizing cell wall constituents such as

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peptidoglycans and lipopolysaccharides.1,2 It also acts as an alternative carbon source

31

for bacterial growth.3 GlcN has been used as a good additive in diverse fields such as

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food,4,5 pharmaceutical,6 cosmetic7 and agricultural industries.8 Especially, it is

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marked as an nutritional supplement for treating osteoarthritis.9 The approval of

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glucosamine as a medical product in countries in Europe, Asia and Latin America has

35

led to increasing needs for glucosamine preparations with good yields and quality.

36

Chitin is the polymerization of acetylated forms of GlcN,10-12 and α-chitin, mainly

37

extracted from shellfish, is an important raw material in producing GlcN.13,14

38

Currently, acid hydrolysis processes are most available for producing GlcN in

39

industrial scale. Chitin powders are refluxed with high concentration acid solution at

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approximately 100 °C.15 Although the reaction is simple and takes place easily in

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aqueous acid, the great threat it poses to the environment has hampered its

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development. In this context, microbial approaches are becoming more attractive.

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Researches on producing glucosamine by microbial fermentation processes have been

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carried out for decades, but substrates are normally glucose,16-19 and no fermentation

45

using α-chitin as starting substrates has been reported. Isolated enzymes, though, are

46

good alternatives in using α-chitin as the substrate to produce GlcN. Different sorts of

47

chitinolytic enzymes isolated from different sources are involved in the degradation of

48

chitin, such as endo-chitinases (EC 3.2.1.14),20,21 exo-chitinases (EC 3.2.1.200),22,23 3


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3.2.1.52),24,25

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β-N-acetylhexosaminidases

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deacetylases (EC 3.5.1.33),26,27 and so on. Challenges are, in particular, the high costs

51

of enzyme preparations, low qualities and low yields of products, with mixtures of

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various molecular weight and degree of deacetylation.28

(EC

and

N-acetyl-D-glucosamine

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Enzyme immobilization, a traditional and promising method in biocatalysis,

54

outperforms isolated enzyme in many aspects such as contamination and separation of

55

products, as well as stability and reusability of enzymes.29,30 However, the process

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remains challenging due to issues of compatibility between carriers and target

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enzymes,31 which makes it rather difficult to achieve the multi-enzyme

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immobilization goal. Engineerable and adaptable platforms and linking materials are

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necessary for tethering enzymes into a cascade. Cascade biocatalysis that enable

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one-pot reactions in a stepwise manner has been successfully used in the synthesis of

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specialty chemicals and drug precursors, as well as conversion of biomass into fuels

62

and desired products.32 In the past decades, the study of biofilms has enhanced our

63

understanding of bacterial systems as a genetically programmable foundry.33-35

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Inspired by the features of self-generated extracellular biofilms, a cell-surface-based

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immobilization strategy directed at products has emerged. Engineered biofilms

66

anchored with target enzymes can serve as programmable and modular extracellular

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biosynthesis materials and an enzyme-mediator platform to realize cascade

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biocatalysis strategies.36 In this way, microbial biofilms with naturally immobilized

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biomolecules are a possible solution for continuous bioprocesses, as an elegant,

70

powerful, and cheap method of cell immobilization, without the necessity of any 4



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added polymers or chemicals37. The enhanced overall robustness of bacteria in

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biofilm formation makes them attractive living biocatalysts for challenging

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conversions in harsh environments. In cell-based biocatalysis, biofilm forms of

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bacteria outperform planktonic cells not only in that they exhibit a superior tolerance

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to physical and chemical insults, and other harsh reaction conditions, but also in that

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the unwanted diffusion of intermediates is limited.38 In addition, covalent binding is

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often used to strengthen the anchoring between the enzymes or between the enzyme

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and the carrier. Many peptide/protein pairs establish specific interactions with their

79

partners through covalent bonds.39 Among them, the SpyTag/SpyCatcher tagging

80

pair40 and the SnoopTag/SnoopCatcher tagging pair41 are fully orthogonal.42 They can

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react with their partners irreversibly and spontaneously under a wide range of

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temperature, pH and organic solvents conditions with little breakup and no cross

83

reactions.

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In this paper, we presented a strategy of constructing a Multi-enzyme Assembly

85

Cascade (MAC) system on the cell surface on the basis of extracellular biofilm and

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two peptide/protein pairs to produce glucosamine directly from α-chitin. In the MAC

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system, curli protein, part of the biofilm structure in Escherichia coli, served as a

88

bridge between biofilms and target enzymes, and Spy and Snoop pairs were used to

89

realize the specific and covalent linkage (Figure 1). Three enzymes involved in the

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chitin degradation pathway in Thermococcus kodakarensis KOD1 were adopted as the

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target enzymes in our work, with the cascade consisting of chitinase (Tk-chiA),20

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exo-β-D-glucosaminidase (Tk-glmA)44 and deacetylase (Tk-dac).27 The MAC system 5


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successfully produced GlcN with little by-products and had an excellent performance

94

with high conversion rate of substrates, pH- and temperature-stability and good

95

reusability. We suggest that the strategy fills a gap in the utilization of chitin and the

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production of GlcN, and also provides a novel perspective for constructing

97

multi-enzyme assembly system for continuous reactions.

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MATERIALS AND METHODS

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Bacteria and reagents. Chemical reagents were purchased from J&K Scientific

100

Ltd. (Beijing, China), Sinopharm Chemical Reagent Ltd. (Beijing, China), and

101

Thermo Fisher Scientific (Guangzhou, China). Especially, α-chitin was purchased

102

from Aladdin Bio-chem Technology (Shanghai, China). Other chemical solvents were

103

all purchased from standard commercial sources.

104

Bacterial strains, plasmids and primers used in this work were listed in Tables S1

105

and S2. Genes were fully synthesized by GENEWIZ (Suzhou, China). The CsgA

106

deletion mutant PHL628 (MG1655, malA-Kan ompR234 ∆CsgA) was kindly provided

107

by Professor Chao Zhong (Shanghai Tech University, Shanghai, China).

108

Scanning electron microscope/Transmission electron microscope (SEM/TEM)

109

observation. A JEOL 1200 TEM (JEOL, Japan) was used for TEM analysis. Five μL

110

of cell sample was dipped onto copper grids, then washed in Millipore H2O and

111

stained with 1% phosphotungstic acid.

112

A JEOL JSM-7600F (JEOL, Japan) scanning electron microscope was used for

113

SEM analysis. Cell samples were mixed with 2% paraformaldehyde/2.5%

114

glutaraldehyde in PBS (pH 7.0) and incubated for 12 h at 4 °C, then washed in 6



115

Millipore H2O, and finally dehydrated with a gradient of increasing ethanol

116

concentrations. Samples were freeze-dried before gold sputtering and further analysis

117

under 15 kV accelerating voltage.

118

Preparation of colloidal chitin. In order to make the chitin more soluble, 10 g of

119

chitin was mixed with 500 mL of 85% phosphoric acid and stirred for 24 h at 4 °C.

120

Five liters of distilled water was used to dissolve the suspension, followed by

121

centrifugation at 12,000 × g for 10 min. The precipitate was washed with distilled

122

water until the pH reached 5.0 and finally the pH was adjusted to 7.0 with sodium

123

hydroxide buffer. The solution was centrifuged and washed with 3 L of distilled water

124

for desalting. The precipitate was re-suspended in distilled water to a final

125

concentration of 2% (w/v).20

126

Protein expression and purification. The CsgA deletion mutant was transformed

127

with pET-28a(+) plasmids encoding CsgA-SpyTag (an empty pET-28a(+) plasmid as

128

a control group). Seed cultures were initially grown in LB medium with kanamycin

129

(Kan, 50µg/mL) for 12 h at 37 °C. Then, the bacteria were collected by

130

centrifugation and resuspended in M63 medium, supplemented with 1 mM MgSO4

131

and 0.2% w/v glucose. The bacteria were grown at 30 °C and 180 rpm, followed by

132

induction with 0.3mM isopropy-β-D-thiogalactoside (IPTG) for 20 h. Subsequently,

133

the cultures were incubated at 30 °C for 24 h statically to facilitate amyloid

134

aggregations. The cell pellets collected by centrifugation were washed with 50 mM

135

pH 7.0 phosphate buffer and subsequently resuspended for further analysis and

136

immobilization. E. coli BL21 (DE3) cells (Vazyme Biotech Co., Ltd, China) were 7


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transformed with recombinant plasmids expressing other target enzymes, and details

138

were shown in supplementary information.

139

Enzymatic activity assay. The activity of Tk-chiA enzyme was measured by a

140

fluorometric assay with 4-methylumbelliferyl-β-D-N,N’,N’’-triacetyl chitotrioside

141

(GlcNAc3-4MU; Harveybio Co., Ltd, China) as the substrate. The fluorescence of

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liberated 4-methylumbelliferone (4MU) was measured using a SpectraMax M3 plate

143

reader with the excitation/emission wavelength at 350/440 nm. The reaction mixture

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(1 mL) containing 100 μL of 1 mM GlcNAc3-4MU and 0.3 μg of Tk-chiA in 50 mM

145

sodium acetate buffer was incubated for 20 min. One unit (U) of Tk-chiA corresponds

146

to the amount of enzyme which produces 1 μM of reducing sugar per minute under

147

the reaction conditions of our assay.

148

The

activity

of

Tk-dac

was

measured

by

using

4-methylumbelliferyl

149

N-acetyl-β-D-glucosaminide (GlcNAc-4MU) as the substrate. The reaction was

150

performed for 20 min in 250 μL of 10 μM GlcNAc-4MU, 50 μg Tk-dac and 50 mM

151

sodium acetate buffer, with excessive Tk-glmA as a coupling enzyme. One unit (U) of

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Tk-dac was defined as the amount of enzyme which produces 1 μM of GlcN per

153

minute.

154

The activity of Tk-glmA was measured by using 4-methylumbelliferyl

155

β-D-glucoside (4-MUG) as the substrate. The reaction mixture (1 mL) containing 500

156

μL of 10 μM 4-MUG, 40 μg of Tk-glmA and 50 mM sodium acetate buffer was

157

incubated for 20 min. One unit (U) of Tk-glmA was defined as the amount of enzyme

158

which hydrolyzes 1 μM of GlcN per minute. 8



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The catalytic activity of three enzymes after forming the tri-enzyme cascade was

160

measured via the production of intermediates. Pre-treated chitin was used for testing

161

enzyme activity of MAC system and analyses of GlcN, diacetylchitobiose (GlcNAc2)

162

and N-acetyl-D-glucosamine (GlcNAc) were carried out using High Performance

163

Liquid Chromatography (HPLC). The supernatant of the reaction mixtures was loaded

164

onto a Bio-Red Hercules HPX-87H HPLC column at 65 °C and eluted with 5 mM

165

H2SO4 at a flow rate of 0.6 mL/min. The products were identified using a differential

166

refraction detector (SHIMADZU Co., Ltd, Japan).

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Enzymatic synthesis of GlcN from chitin. The 1 L reaction mixture containing 5

168

g chitin and 20 mg recombinant MAC system was incubated for 0 h, 1.5 h, 3 h, 4.5 h,

169

6 h, 9 h, 12 h and 18 h, and samples of the solution were taken for further analysis of

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GlcN and intermediates using HPLC. For the modified MAC system, the reaction was

171

carried out under same conditions except for the addition of tri-enzyme cascade being

172

23 mg.

173

Calculation of conversion rate of MAC system was based on the following formula:

174

α=

175

where mGlcN, mchitin is the weight of GlcN as the product and α-chitin as the

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substrate, while MGlcN and Mchitin is the relative molecular weight (MW) of GlcN

177

(179.17 g/mol) and α-chitin (221.21 g/mol) respectively. The relative molecular

178

weight of α-chitin is the MW of the repeating unit of α-chitin, assuming that the

179

deacetylation degree of α-chitin is 0.

180

× 100%

(1)

Reusability test of the MAC system. Repeated utilization of the MAC system was 9


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carried out in a 250 mL Erlenmeyer flask with 50 mL reaction mixture, under 14-hour

182

catalysis cycles. After each batch was finished, the mixture was centrifuged to collect

183

the bacterial biofilms with the anchored multi-enzyme cascades. Colloidal chitin was

184

added to the reaction mixture again for the following rounds of catalysis.

185 186

RESULTS AND DISCUSSION

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Design of the MAC system anchored in functional amyloid fibrils. Our strategy

188

aimed at building enzyme blocks based on tagging systems and extracellular matrix.

189

Curli proteins, encoded by the csgA gene, are major components of a complex

190

extracellular

191

overexpression of CsgA protein, monomers are secreted from the intracellular space

192

and assemble into fibers extracellularly. Additionally, SpyTag is a 13-amino acid short

193

peptide that forms a spontaneous and irreversible isopeptide bond upon encountering

194

its protein partner SpyCatcher (15 kDa), while 12-amino acid SnoopTag can form an

195

isopeptide bond with its partner protein SnoopCatcher (15 kDa). In the Spy pair, the

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reactive Asp117 of SpyTag attacks the carbon of Lys31 of SpyCatcher, catalyzed by

197

neighboring Glu77.40 Similarly, in the Snoop pair, Lys742 of SnoopTag reacts with

198

the reactive Asn854 of SnoopCatcher.41 Different ends of tags were recognized by

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their catchers specifically, making the two pairs fully orthogonal. The covalent

200

peptide interaction is an accurate and effective tool for bioconjugation and can be

201

used to expand the repertoire of accessible protein architectures.

202

matrix

produced

by

many

Enterobacteriaceae.43

After

the

A chitinase consortium from the hyperthermophilic archaeon Thermococcus 10



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kodakaraensis KOD1 can catalyze the conversion of α-chitin to GlcN with three

204

enzymes involved: Tk-chiA, Tk-glmA and Tk-dac (Figure 1a). The three enzymes

205

were expressed in sequence and fused with different Tag or Catcher of Spy and Snoop

206

pairs. Reengineered biofilms that displayed the SpyTag on the cell surface were

207

further constructed by fusing the SpyTag to the C-terminus of curli protein (Figure

208

S1). Five steps were involved for further immobilization of three enzymes (Figure

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1b). First, monomers of CsgA expressed by pET28a(+) CsgA-SpyTag (abr:

210

pET-CsgA) were secreted from the over-expressing cells and assembled in the

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extracellular space. The preparation of CsgA-SpyTag fusion proteins was shown in

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Table S3. The functional amyloid fibrils will enable the further site-specific enzyme

213

immobilization.

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SpyCatcher-glmA-SnoopTag (abr: pET-glmA) and linked to membranes through a

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covalent interaction between SpyCatcher expressed from pET-glmA and SpyTag

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from pET-CsgA. Subsequently, we constructed pET22b(+) SnoopCatcher-SpyCatcher

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(abr: pET-Catcher) to avoid fusing large-size amino-acid sequences to the termini of

218

target enzymes, which may affect enzyme activities due to steric clashes. Afterwards,

219

the N-terminus of proteins expressed by pET22b(+) SpyTag-dac-SnoopTag (abr:

220

pET-dac) was linked to the C-terminus of pET-Catcher through the Spy pair, and the

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C-terminus of pET-dac was linked to the second pET-Catcher through the Snoop pair.

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Ultimately, proteins expressed by pET22b(+) SpyTag-chiA-SnoopTag (abr: pET-chiA)

223

were combined with the system through Spy pair. Finally, we achieved a

224

multi-enzyme-displaying engineered biofilm that could be used to catalyze three-step

Then,

pET-glmA

was

expressed

11


by

pET22b(+)

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225

transformations.

Sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis

226

(SDS-PAGE) analysis of five proteins expressed from recombinant plasmids were

227

shown in Figure 2a-e.

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This novel MAC system for the efficient immobilization based on the engineered

229

biofilms could be combined with metabolic engineering approaches for manipulating

230

target pathways. The modularity and flexibility of linking strategy enables

231

high-efficiency multi-step reactions in one pot in the future. More importantly,

232

compared with building enzyme cascades by fusing amino acid frameworks of

233

multiple enzymes into a whole, our post-expression modification had little impact on

234

expressions of proteins and enzyme stoichiometry, so that enzymes can maintain their

235

catalytic activities.

236

Characterization of amyloid fibrils formation. Amyloid fibrils are ordered

237

aggregates of proteins that are fibrillar in structure.43 SEM and TEM were carried out

238

to analyze the ultrastructure of curli nanofibers. A biomaterial with obvious amyloid

239

fibrils was observed wrapping around the cell surface of recombinant E. coli when

240

pET-CsgA was expressed, which was absent in the control strain (the CsgA deletion

241

mutant PHL628) expressing no curli (Figure 3a-f). Wirelike amyloid nanofibers were

242

found to adhere to the cells with a diameter of approximately 4‒7 nm (Figure 3f),

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which was in consistence with previous report.45 The Congo red binding assay and the

244

Thioflavin T fluorescence assay46 were also carried out to confirm the formation of

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curli fibers. Difference between control group and experimental group further

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confirmed the presence of curli fibers and β-sheet structure in engineered biofilms 12



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(Figures S2, S3). The biomaterial provided a good foundation for later combination

248

strategy, which was inherent in bacteria and possessed large surface areas.

249

Construction of the MAC system. We proposed to develop a new bioprocess for

250

the direct synthesis of GlcN from α-chitin using engineered biofilms displaying

251

multi-enzyme-assembly. Since tagging systems are self-catalyzed, and peptide and

252

protein can bind each other spontaneously, they were simply contacted by being

253

incubated together at room temperature to establish multi-enzyme system. Previous

254

studies have shown that both the core of curli fibers and former anchored enzymes

255

can affect the linkage of free enzyme to neighboring peptide sites,35 which means that

256

peptide domain must remain accessible for the following linkage. To ensure that

257

sufficient isopeptide bonds can be formed to immobilize enzymes, excessive protein

258

was added. Firstly, the pET-glmA fusion protein was incubated with CsgA-SpyTag

259

biofilm concentrated from fermentation broth of cells grown for 6‒72 h at 180 rpm at

260

room temperature. After incubation, free enzymes were separated by centrifugation.

261

The collected cell pellets with the pET-glmA fusion protein were washed twice with

262

50 mM pH 7.4 phosphate buffer for further linking and analysis. The rest of the

263

enzymes in MAC system were added one by one in the same way.

264

The conjugation time of five steps for assembly was adjusted from 6 to 60 h

265

respectively, and free enzyme that remained unattached was separated by

266

centrifugation. The optimal incubation time for immobilization of each step was

267

different (Figure S4), and each step reached the maximum linking percentage ~70%.

268

The loading of immobilized multi-enzyme-assembly can reach 0.9 M protein 13


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/MCsgA-SpyTag, as calculated via the product of the maximum linking efficiency

270

and the initial weight of added enzymes. These results confirmed that SpyTag peptide

271

can be fused to CsgA and maintain its functionality as a site-specific covalent

272

immobilization tag after extracellular assembly into curli nanofibers. Furthermore,

273

residual enzymes tagged with peptide/protein could be effectively fixed onto the

274

resulting CsgA-SpyTag modified biofilms as long as we added them individually to

275

avoid cross-linking.

276

Enzymatic activity of three enzymes tagged with peptide/protein. We first

277

evaluated reaction conditions required by each enzyme to determine the optimal pH

278

and temperature for the multi-enzyme system. Previous reports showed that the

279

stability of three enzymes from Thermococcus kodakaraensis KOD1 lies in a

280

temperature range of 37‒100 °C and pH range of 4‒9.20,27,44 Figure S5 illustrated that

281

pH 7 and 75 °C were the optimal reaction conditions for the multi-enzyme cascade,

282

whereby 80% of activities remained at pH 5‒9 and temperature 65‒85 °C, which

283

indicated that our system possessed good stability at a wide pH- and temperature

284

range. Further catalytic reactions were carried out under pH 7 and 75 °C. Notably, the

285

relatively high reaction temperatures are appreciated in industrial process, thus

286

enzyme activities can be maintained even when reaction temperatures can not be

287

monitored precisely.

288

The activity of pET-chiA, pET-dac, and pET-glmA was measured by using

289

fluorescently labeled substrates, as described in Materials and Methods. The kinetic

290

properties shown in Table 1 suggested that pET-dac may possess relatively low 14



291

catalytic activity in the chitinase consortium.

292

To test whether fusing peptides or proteins to target enzymes may influence the

293

catalysis efficiency, we compared reaction profiles of wild-type and fusing enzymes

294

tagged with peptide/protein in the termini. Results shown in Figure S6 validated that

295

the fusion of peptides or proteins had little effect on the enzyme activity. This is likely

296

due to the fact that small modifications on the termini of enzymes did not impair the

297

refolding of proteins, thus no enzymatic activity losses were observed.

298

Utilization of the MAC system for the synthesis of GlcN from chitin. To test the

299

catalytic activity of the MAC system, 5 g of α-chitin was mixed with 20 mg of the

300

tri-enzyme mixture (total weight) to a 1 L reaction mixture (under 75 °C and pH 7) for

301

18 h. Judging by the time-profiles of GlcNAc2, GlcNAc and GlcN concentration

302

produced by MAC system (Figure 4a), the amount of GlcNAc as an intermediate was

303

relatively high in the first 9 hours, indicating that the catalytic activity of deacetylase

304

is a rate-limiting step for the entire catalysis process. Notably, differences in catalytic

305

activities of enzymes are unavoidable in three-step sequential biocatalysis, which lead

306

to high metabolic loads of intermediates and affect yields of cascade reactions. In

307

order to increase the degrading rate of GlcNAc, we further optimized our strategy by

308

repeating the immobilization of pET-dac in the enzyme cascade, given that total

309

enzymatic activity can be enhanced by increasing the amount of enzymes (Figure 4c).

310

After employing a flexible design of immobilization strategy, the optimized system

311

led to a higher GlcN yield and higher GlcNAc conversion efficiency (Figure 4b),

312

with the production of GlcN reached to 79.02%

3.61%. The conversion rate was

15


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313

calculated based on Formula 1. The concentrations of substrate, intermediate and final

314

products with both original and modified MAC system after 18 hours were further

315

displayed by HPLC analysis, with the standard substances as a reference (Figures S7,

316

S8). These results illustrated that rationally designed strategy can be used to optimize

317

the output of multi-enzyme catalysis systems. We further compared reactions

318

catalyzed by the optimized tri-enzyme system to the mixture of three pure enzymes

319

that were not assembled into a cascade. The production of GlcN catalyzed by the

320

traditional consecutive conversion of substrates in three separate steps was 1.53 g, and

321

the concentration of GlcN was relatively stable after 9 hours of reaction (Figure 5a).

322

In comparison, the yield of GlcN from modified MAC system was 1.68 times of

323

original MAC system and 2.09 times of mixture of pure enzymes. The higher reaction

324

rates of multi-enzyme-assembly implied that faster mass conversion of chitin to GlcN

325

occurred in the case of engineered biofilms. It is very likely that the extracellularly

326

immobilized multi-enzyme cascade provided a more stable and favorable

327

environment for capturing the substrates and preventing the diffusion of intermediates,

328

thus enabling a more efficient enzymatic reaction compared with free enzymes in

329

solution. In addition, the average distance of immobilized enzymes was much smaller

330

than that of free enzymes, which could facilitate the mass transfer of substrates and

331

products in this sequential process. The productivity of glucosamine in our work was

332

about 100 times of that catalyzed by an enzyme cascade consisting of a deacetylase

333

from Cyclobacterium marinum and a β-N-acetylhexosaminidase from Zobellia

334

galactanivorans,47 even higher than those monoenzyme–catalyzed reactiones in the 16



335

whole pathway of chitin degradation.48,49 For example, the conversion rate of chitin to

336

GlcNAc catalyzed by endo-chitinase isolated from Aeromonas hydrophila H-2330

337

was 66 %, with the 70% deacetylated chitin as the substrate.48 For reactions catalyzed

338

by crude enzymes from Aeromonas sp. GJ-18, the yield of GlcNAc from pretreated

339

chitin was 62.8 % , with 4.1 % of GlcNAc2 as the by-product.49 We assumed that the

340

reason why cascades consisting of homologous enzymes outperformed those

341

consisted of enzymes isolated from different organisms may be the inherent

342

coordination among homologous enzymes. Some inherent substrate channellings may

343

exist in facilitating the transferring of substrates when homologous enzymes work

344

together in vivo and the spatial proximity in enzyme cascades allows the formation of

345

these channellings in vitro, which was consistent with the experimental results

346

reported by Wheeldon et al..50

347

Reusibility test was carried out to test the performance of MAC system anchored in

348

engineered biofilms. Bacteria with engineered biofilms were collected by

349

centrifugation for batches of reuse. In the repeated utilization of MAC system, 90% of

350

catalysis activity remained for the first 6 batches (shown in Figure 5b). Though the

351

relative activity decreased drastically from the seventh reuse, it can maintain more

352

than 60% of the initial productivity after 17 rounds of reaction. We proposed that curli

353

fibers adhered to engineered biofilms may protect cells from harsh environment and

354

help maintaining cellular morphology, thus enhancing the stability of cells.

355

In summary, a new multi-enzyme assembly cascade system has been developed

356

using curli fibers and dual specifically linking pairs. The MAC system was 17


Page 18 of 33

Page 19 of 33


357

successfully used in the multi-enzymatic synthesis of GlcN as the main product from

358

α-chitin. Three enzymes immobilized in this study can be optionally substituted by

359

any other interesting enzymes, since our post-expression enzyme linking system

360

won’t affect the expression of proteins. Designing cascade reactions are the grand

361

challenge, as well as the future of synthesizing chemicals. Some engineered pathways

362

suffered from flux imbalances, in which cases the accumulation of intermediates was

363

the great bottleneck. Herein, we provided a novel way of immobilizing multi-enzymes,

364

in which case rational design of the immobilization strategy can be used to tackle the

365

substrate- and reaction-intermediate mass transfer bottleneck, and high metabolic

366

loads of intermediates, and further adjust the output of catalytic bioprocess.

367

Protein/peptide pairs performed as ideal tools for building production lines. Also, the

368

biocompatibility of biofilms makes them an ideal platform for setting up a nano

369

factory in the future envisioned biorefinery.

370 371

ACKNOWLEDGEMENTS

372

This work was supported by the National Key R&D Program of China

373

(2017YFC1600404), the National Science Foundation of China (U1603112), the

374

Natural Science Foundation of Jiangsu Province (BK20171461), the Environmental

375

Protection Project in Jiangsu Province (2016053), and the Jiangsu Synergetic

376

Innovation Center for Advanced Bio-Manufacture (XTE1838).

377

SUPPORTING INFORMATION

378

Supplementary information for this paper is available in the online version of the 18



Page 20 of 33

379

paper. Construction of five recombinant plasmids, Figure S1. Congo red binding assay

380

to test the existence of fibrils, Figure S2. ThT binding fluorescence assay to test the

381

existence of fibrils, Figure S3. The linking efficiency of five steps in enzyme

382

immobilization, Figure S4. Relative activities of three enzymes under different pH

383

and temperature conditions, Figure S5. Comparison of wild type enzymes and tagged

384

enzymes in product formation for three enzymes, Figure S6. Analysis of products of

385

the enzymatic reactions by HPLC, Figure S7. Standard curves of different substances

386

relating to the catalysis process of chitin to glucosamine by HPLC, Figure S8. E. coli

387

strains and plasmids used in this work, Table S1. Primers used in this work, Table S2.

388

The preparation of CsgA-SpyTag fusion proteins, Table S3.

389

AUTHOR CONTRIBUTIONS

390

J.B. performed the experiments, collected and analyzed the data, and drafted the

391

manuscript; N.L. and L.Z. assisted in conceiving and designing the experiments, and

392

revised the manuscript; Q.X. and H.H. performed the molecular dynamics simulation

393

and analyzed the data; L.J. contributed reagents, materials and analytical tools. All

394

authors read and approved the final manuscript.

395

NOTES

396

The authors declare no competing financial interest.

397

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Page 26 of 33

Page 27 of 33


FIGURE LEGENDS Table 1. Kinetic properties of pET-chiA, pET-dac, pET-glmA. Figure 1. (a) The metabolic pathway of chitin degradation in Thermococcus kodakarensis KOD1, and (b) five steps involved in the immobilization process of MAC system. Figure 2. SDS-PAGE analysis with Coomassie staining for five proteins. Figure 3. SEM and TEM images of curli fibers expressed by recombinant plasmid pET-CsgA and the control strain (PHL628) expressing no curli nanofibers. Figure 4. Production of GlcN and intermediates catalyzed by original MAC system and modified MAC system, and the strategy for modification. Figure 5. Catalytic activities of producing GlcN from chitin by mixture of recombinant enzymes, and the relative activity of MAC system after multiple cycles of reuse.

26



Page 28 of 33

Figure graphics

Figure 1. (a) The metabolic pathway of chitin degradation in Thermococcus kodakarensis KOD1. Tk-chiA produces GlcNAc2 from chitin, followed by Tk-dac site-specifically deacetylating GlcNAc2 to GlcN-GlcNAc. Subsequently, Tk-glmA hydrolyzes GlcN-GlcNAc to GlcN and GlcNAc, followed by a second deacetylation step of the remaining GlcNAc to form GlcN by Tk-dac. (b) Five steps were involved in the immobilization process of MAC system.

27


Page 29 of 33

Figure


2.

SDS-PAGE

analysis

SpyCatcher-glmA-SnoopTag,

(c)

with

Coomassie

staining

SnoopCatcher-SpyCatcher,

(d)

for

(a)

CsgA-SpyTag,

(b)

SpyTag-dac-SnoopTag,

(e)

SpyTag-chiA-SnoopTag. Note: The normal loading amount of SDS-PAGE was not sufficient for curli, so we doubled the loading amount (a, lane 2).

28



Page 30 of 33

Figure 3. SEM and TEM images showed the micro structure of curli fibers expressed by recombinant plasmid pET-CsgA (b, d, f) and the control strain (PHL628) expressing no curli nanofibers (a, c, e).

29


Page 31 of 33


Figure 4. Production of GlcN and intermediates via three-step sequential catalysis reactions. (a) Time courses of products catalyzed by the original MAC system, shown in Figure 1b. (b) Time courses of products catalyzed by modified MAC system, which involved the immobilization of pET-dac for twice. (c) The optimized MAC system was constructed by repeating step 3 and step 4 in Figure 1b. Free termini that were accseeible to next linking step remined to be SnoopTag.

30



Page 32 of 33

Figure 5. (a) Catalytic activities of producing GlcN from chitin by mixture of recombinant enzymes, and MAC system. We attempted to correlate the production of GlcN with the overall catalytic efficiency of such system. The reaction system of control group consisted of same amount of three enzymes. (b) The relative activity of MAC system after multiple cycles of reuse. Each batch of reaction was 14 h. The relative activity percentage equaled the ratio of GlcN produced from every batch to that of first batch.

31


Page 33 of 33


Tables Table 1. Kinetic properties of pET-chiA, pET-dac, pET-glmA *. Enzymes a

*

Substrate

Enzyme activity (U/mg) b

kcat/Km (M-1 S-1)

Km (mM) 07

1.028×104

pET-chiA

GlcNAc3-4MU

189.4

3.0

6.42

pET-dac

GlcNAc-4MU

98.6

2.6

22.8

9.683×102

pET-glmA

4-MUG

135.3

38.7

8.354×104

Each experiment was carried out for three independent times. The reaction condition was pH 7 and

75 °C. a

Enzymes were expressed by recombinant plasmids, tagged with peptide/protein.

b

The enzymes activity unit (U) of pET-chiA, pET-dac and pET-glmA was defined as the amount of

enzyme that produces 1 M of GlcNAc2 per minute, 1 µM of GlcN per minute and 1 µM of GlcN per minute under this assay conditions, respectively.

32


Programming a Biofilm-Mediated Multienzyme-Assembly-Cascade

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