High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid

Accepted Manuscript High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid Xuejia Shi, Jingcong Xie, Shiyong Liao, Tao Wu, Lin-Guo Zhao, Gang Ding, Zhenzhong Wang, Wei Xiao PII: DOI: Reference:

S0960-8524(17)30760-5 http://dx.doi.org/10.1016/j.biortech.2017.05.105 BITE 18131

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

27 March 2017 15 May 2017 16 May 2017

Please cite this article as: Shi, X., Xie, J., Liao, S., Wu, T., Zhao, L-G., Ding, G., Wang, Z., Xiao, W., High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.105

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1

High-level expression of recombinant thermostable β-glucosidase in Escherichia

2

coli by regulating acetic acid

3 4

Xuejia Shi1, +, Jingcong Xie1, +, Shiyong Liao 1, Tao Wu1, Lin-Guo Zhao1,*, Gang Ding2,

5

Zhenzhong Wang2, Wei Xiao 2

6 7

1

8

Nanjing 210037, China 2

9 10 11

College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road,

Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang

222001, Jiangsu Province, China +

These authors equally contributed to this work

12

*

13

Forestry University, Nanjing, 210037, China. Phone: +86-025-85428300. E-mail:

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[email protected].

15 16 17 18 19 20 21 22

Corresponding authors for Linguo zhao at College of Chemical Engineering, Nanjing

23 24

Abstract In the fermentation progress, fermentation parameters including the feed rate,

25

induction temperature, and induction pH evidently regulate the accumulation of acetic

26

acid generated by recombinant E. coli in the medium. The production of thermostable

27

β-glucosidase (Tpebgl3) was increased by optimizing the parameters mentioned step

28

by step. The optimal conditions were obtained with the highest enzyme expression

29

(560.4 U/mL) and the maximum DCW (65 g/L) at the pre-induction specific growth

30

rate of 0.2 h-1 followed by a post-induction specific growth rate (0.18 h-1); induction

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temperature is 39°C; the pH is 7.2; the concentration of acetic acid was maintained all

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along below 0.9 g/L. Results show it is necessary for the synthesis of Tpebgl3 to

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regulate the accumulation of acetic acid at the premise of feeding to meet the normal

34

growth of E. coli. The production of Tpebgl3 by recombinant E. coli is the highest

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reported to date.

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Keywords: Acetic acid; Fermentation; Optimal condition; β-glucosidase.

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1. Introduction

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Tpebgl3 is a thermostable GH3 β-glucosidase from Thermotoga petrophila DSM

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13995. The ginsenosides Rb1 or Rd was transformed to minor ginsenoside 20(S)-Rg3

40

by using Tpebgl3 (Xie et al., 2015a). In many general respects, it does not resemble

41

many regular β-glucosidases. This high thermal stability of enzyme has great

42

properties as bio-catalysts for industrial bioconversion. Firstly, it is easy to obtain the

43

relatively pure product with the heat treatment than the purification of other enzymes.

44

Secondly, the high reaction temperature can prevent microbial contamination and

45

reduce the viscosity, promoting high reaction velocities and hydrolysis rate (Haakana

46

et al., 2004). Finally, these enzymes are propitious to more large-scale commercial

47

production than the enzymes from mesophilic sources. In particular, the recombinant

48

enzyme shows its advantages because of its high selectivity and productivity (Quan et

49

al., 2012; Ten et al., 2015). For example, Cellulase-12T was used to transform ginseng

50

saponin glycosides from white ginseng extract (Chang et al., 2009). However, the cost

51

of preparation of the recombinant enzyme has limited its widespread application in the

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industrial transformation from major ginsenosides into minor ginsenosides with higher

53

activity. It has been an harder task to find a thermophilic recombinant enzyme which

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can transformed major ginsenosides to ginsenoside 20(S)-Rg3. Owing to the optimal

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expression condition on the flask level was obtained and the highest enzyme activity of

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Tpebgl3 reached 21 U/mL (Xie et al., 2015a), there remains a gap to be overcome

57

before it could be utilized on a large scale. Therefore, to reduce the cost of the

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production of the recombinant enzyme, fed-batch fermentation become a better choice

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for large-scale preparation of enzyme protein Tpebgl3. It can overcome the conundrum

60

of the poor expression of extracellular secretion because of the defect of the

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recombinant E. coli itself.

62

Fed-batch fermentation is a batch culture continuously or consecutively fed using

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substrate to achieve high densities of E. coli and desired protein production (Huber et

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al., 2009; Wetzel et al., 2016; Wilming et al., 2014). It is essential to optimize the

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fermentation parameters affecting the growth of recombinant Escherichia coli,

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including glucose feeding rate, induction temperature, induction pH, etc. Glucose is a

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cheaper and utilizable carbon for the growth of E. coli, and thus, it is a superior carbon

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source to others. Plethoric glucose promotes the production of metabolic byproducts in

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E.coli under aerobic conditions (Jean et al., 2012). The most common byproduct acetic

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acid is synthesized by phosphotransacetylase (PTA)/acetate kinase (ACKA) and by

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pyruvate oxidase (POXB) (Valgepea et al., 2010), The emergence of this phenomenon

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is because of the imbalance between carbon poured into the central metabolic system

73

and the limited capacity of the cellular respiration or tricarboxylic acid cycle (Cheng et

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al., 2012; Glazyrina et al., 2012; Krause et al., 2016; Shin et al., 2009a). Accumulation

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of acetic acid, which usually occurs in the fast growing period of E. coli and especially

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during the induction stage of the desired protein synthesis, inhibits growth and product

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formation (Shiloach and Rinas, 2009; Wang et al., 2011; Weicai et al., 2000;

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Xiao-Xuan et al., 2006). Thus, it is extremely important for Tpebgl3 production to

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construct a feeding strategy to avoid underfeeding or overfeeding. In addition, with

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dissolved oxygen ensured constant, induction temperature and induction pH act huge

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impact on accumulation amount of metabolic acetic acid at the post-induction of high

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density fermentation process (Shiloach and Rinas, 2009; Wang et al., 2011; Weicai et

83

al., 2000). Accelerate the metabolism and the growth of cells with high temperature

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conditions, resulting in a large number of acetic acid formation (Cen et al., 2011;

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Xiao-Xuan et al., 2006). Furthermore , the pH at the post –induction period is key

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factor for reducing concentration of acetic acid by adding NH4OH to form ammonium

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salt. However, high concentration of NH4 + has negative influence on energy efficiency

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and the growth of E. coli (Yang et al., 2010). Until now, there is fewer study on the

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effect of acetic acid for the enhanced production of heat-resistant β- glycosidase.

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In this study, the aim was to control amount of acetic acid through determined

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suitable feeding rate, induction temperature, and induction pH for the Tpebgl3

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production. And then the volume of Tpebgl3 on fermentation level was successfully

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amplified. Acetic acid generated by E. coli itself could be indirectly adjusted at

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constant dissolved oxygen condition. A different discipline was discovered and could

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regulate the accumulation of the acetic acid to increasing the desired protein expression,

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and then obtain a high productivity of the target protein.

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2. Materials and methods

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2.1. Bacterial strains, plasmids, and materials

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The study used the recombinant E. coli BL21 (DE3) harboring plasmid Tpebgl3/

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pET20b (+), in which the Tpebgl3 gene encoding thermostable β-glucosidase was from

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Thermotoga petrophila DSM 13995 and has been described elsewhere (Xie et al.,

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2015b).

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2.2. Media and feeding solutions The following media were used in the high density culture of E. coli : the seed

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medium - Tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L; the medium for

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fermentation - 4.2 g/L NaH2PO4·2H2O, 8.7 g/L K2HPO4·3H2O, 50.0 g/L yeast extract,

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30 g/L glucose, 5.5 g/L (NH4)2SO4, 2.5 g/L MgSO4·7H2O, 1.1 g/L EDTA, 1 mL/L bito,

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and 0.3 g/L foam; the addition medium for fermentation - 12.6 g/L NaH2PO4·2H2O,

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26.0 g/L K2HPO4·3H2O, 200 g/L yeast extract, 800 g/L glucose, 16.5 g/L (NH4)2SO4,

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20 g/L MgSO4·7H2O, 3.3 g/L EDTA, 3 mL/L bito, 0.3 g/L foam). Furthermore, the

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residual glucose in the bioreactor was fed back by allowing variation of the dissolved

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oxygen (DO) or pH value, and then the feed batch was initiated by using the

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exponential fed-batch and the two-stage feeding strategies (Gharibzahedi et al., 2014a;

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Wetzel et al., 2016). From the basic substrate mass balance, the feeding rate (F, ml/h)

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at a varied specific growth rate was given by: F=

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μ X V exp (μt)

/ ( − )

where µ is the specific growth rate (h-1), µset is the specific growth rate during

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pre-induction phase, µ´set is the specific growth rate in the post-induction phase, X0 is

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the initial cell concentration (g/L), V0 is the initial culture volume (L), YX/S is the

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theoretical cell yield on glucose, SF is the glucose concentration in the feeding solution,

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and t is the culture time (Gharibzahedi et al., 2014b).

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2.3. Culture conditions

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The seed culture was inoculated in a 500 mL shake flask with 150 mL of LB medium. This culture was shaken at 200 rpm for 10 h at 37 °C. The seed culture was

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then inoculated in the 7.5 L fermenter kept at 37°C and pH 7.0, and the work volume

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of the initial batch medium in the bioreactor was 3.0 L for cultivation. IPTG was added

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to the initial medium at a fixed rate to generate synthesis of the desired protein when

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the biomass of E. coli reached the desired growth level. The induction for expressing

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recombinant protein was started with an appropriate value (OD600 nm values of 55–60).

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The plasmid stability was guaranteed by adding 100 mg/L Ampicillin into cultures

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(Duan et al., 2013). The media were kept in aerobic conditions (DO ≥ 15% unless

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otherwise noted) with an air flow rate of 60 L/h and a stirring speed between 300 and

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1200 rpm. The parameters including temperature, pH, DO, and impeller rabbling speed

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were controlled by using New Brunswick Scientific (Edison, NJ) BioFlo 310. The

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culture pH was regulated by automatic addition of NH4OH 100% and HCL 25% v/v.

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2.4. Tpebgl3 activity assay

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Enzyme activity definition is that 1mmol p-nitrophenyl-β-D-glucopyranoside

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(pNPG) per minute is hydrolyzed to release 1 µmol pNP, which is defined as a unit of

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enzyme activity [U]. To measure the activity of crude Tpebgl3, a sample of culture

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broth (1 mL) was centrifuged at 12,000 g for 5 min, and then, the pelleted cell was

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suspended in citric acid-Na2HPO4 buffer (50 mM, pH 7.8) and disrupted by

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ultrasonication at 20 kHz for 10 min in an ice bath. The activity of crude enzyme from

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the intracellular extract was detected and represented as the units per mL of culture.

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The reaction mixture (200 µL) contained 20 mM pNPG, citric acid Na2HPO4 buffer

144

(50 mM, pH 5.0), and enzyme with appropriate diluted ratio. After incubation at 90°C

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for 10 min, the reaction was terminated by addition of 600 µL of 1 M Na2CO3 and

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measured by an absorbance microplate reader (SpectraMax 190).

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2.5. Determination of protein concentration

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The crude cell extracts, as described in section 2.4, were (70°C; 30 min) treated

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by heat shock and then cooled in an ice bath and centrifuged (12,000 g; 4°C; 30 min).

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The resulting supernatants were treated by an immobilized metal affinity column

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(Novagen, USA), and the purified enzyme protein was eluted with imidazole (1 M),

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NaCl (0.5 M), and Tris–HCl buffer (20 mM; pH 7.9). The result was measured by

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SDS-PAGE, and the protein bands were analyzed by a density scanning with an image

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analysis system (Bio-Rad, USA). The purified protein concentration was examined

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with the Bradford protein Assay Kit (Sangon Biotech, Shanghai, China).

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2.6. Acetic acid assay

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The concentration of acetic acid of fermentation was investigated by GC, the

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sample was obtained through extraction of supernatant fermentation broth (1mL)with

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adding 0.2mL of 50% sulfuric acid and 1mL ether in a 5mL tube after centrifuging

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(1000g) for 1min (Weicai et al., 2000). In the GC method for the detection of acetic

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acid, the column temperature was started with an initial temperature of 70°C for 3 min,

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then it was raised to 230°C with an incremental rate of 8°C per minute, and it was held

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for 3 min (30 m × 0.25 mm × 0.25 µm, Agilent); the flow rate of the carrier gas (N2)

164

was 2 mL/min; the temperature of the front detector (FID) was 300°C; the flow rate of

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the tail gas was 25.00 mL/min; the H2 flow rate was 30.00 mL/min; the air flow rate

166

was 400 mL/min (Kai et al., 1998).

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3. Results and discussion

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3.1 Effects of feeding strategies on the accumulation of acetic acid in producing

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Tpebgl3

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3.1.1. Exponential fed-batch culture

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In the exponential fed-batch culture, the specific growth rate of E. coli was

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determined by the feed rate under limited nutrient (Zheng et al., 2009). Excess glucose,

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as the carbon resource, usually forces E. coli to generate acetic acid by PTA-ACKA

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and POXB synthesis pathways with the µ exceeding the value of the threshold growth

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rate, but acetic acid is not investigated when the µ is lower than a certain threshold

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(Eiteman and Altman, 2006; Shin et al., 2009b). Though aerobic acetogenesis can

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generate extra ATP to support the faster growth of E. coli (Kayser et al., 2005), the

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excessive accumulation of acetic acid has an unbeneficial influence on the formation of

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Tpebgl3 by recombinant E. coli. Therefore, the best way to reduce the acetic acid

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formation and increase the biomass of E. coli is to adjust the specific growth rate,

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which is beneficial for increasing the expression of recombinant protein (Shiloach and

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Rinas, 2009). Figure 1 shows that the production of Tpebgl3 is increased at the specific

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growth rates of 0.1 h-1, 0.2 h-1, and 0.3 h-1. IPTG was used as the inducer in the

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exponential fed-batch fermentations. Among these, the maximum values of the actual

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specific growth rates were less than the values designed at the later stage of

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fermentation (given in Table 1). When the specific rate µset was 0.1 h-1, the acetic acid

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content began to decrease rapidly, and the final concentration was about 0 g/L after

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induction, it might be a reasonable explanation, that the concentration of supplemented

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carbon substrate was lower than a certain threshold. Indeed, it spent 16 h until the

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biomass of E. coli (0.1 h-1) reached the maximum and then biomass declined slowly

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which indicated the E. coli began to age and die. The analysis showed that the

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supplemental nutrients with a low feed rate at 0.1h-1 could not meet the demands of its

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own growth and the strain was starved on growth during the later stage of

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fermentation.

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When µset is 0.2 h-1(Fig. 1B), the acetic acid content begins to decrease gradually

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after induction. The results showed that the final concentration of acetic acid was 2.68

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g/L and the highest activity of Tpebgl3 reached 290 U/mL, both of these value were

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much higher than that ofμset under 0.1 h-1 and 0.3 h-1 and 14.5 times higher than that

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of shaking flask culture (Xie et al., 2015b), the dry cell weight (DCW) was about 43.7

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g/L. Though the concentration of acetic acid was maintained at a relative high level, it

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slowed down the growth of E. coli and indirectly promoted E. coli to increase the

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Tpebgl3 production. When µset was 0.3 h-1 (Fig. 1C), the content of acetic acid in the

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fermentation broth was above 3 g/L at the post-induction phase. The supplementation

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of carbon source in the aerobic fermentation exceeded the demands of cell growth,

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which resulted in excessive acetic acid produced by E. coli in the culture and then both

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of cell growth and Tpebgl3 production were inhibited. The results indicated that the

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acetic acid generation had an extremely detrimental influence on the cell growth and

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the production of recombinant β-glucosidase protein by E. coli, and the exponential

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fed-batch with different specific growth rates could evidently regulate the

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accumulation of acetic acid.

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3.1.2. Two-stage glucose feeding strategy

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A modified two-stage glucose feeding strategy based on both the specific growth

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rate and the amount of glucose residues at the pre- and post-induction phase was

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applied to control the cell growth and change the feed rate of the carbon resources

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(Chen et al., 2013; Gharibzahedi et al., 2014a). The strategies were thus evaluated to

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determine the most suitable nutrients flow to control the accumulation of acetic acid,

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so as to guarantee the requirements for the fast growth of the cells in the early stage

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and protein overexpression in the later stage. The glucose feeding rate was increased

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exponentially during the pre-induction phase, according to the exponential feeding

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method (Duan et al., 2013), the specific growth rate of the cultures was 0.2 h-1 before

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induction. When the OD600nm reached 55–60, the post-induction phase began with

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inducing IPTG, and the feeding rate was changed based on the gradient-decreasing

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method (Fig. 2). The main purpose of the induction was to express the recombinant

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protein efficiently and to control the accumulation of acetic acid. From Fig. 2, the

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accumulation of acetic acid increased with the gradient-increase of µ'set (Fig. 2) at the

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late stage of induction. The results showed that the accumulation of acetic acid could

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be well controlled by the exponential feeding strategies with different pre-and

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post-induced specific growth rates in the microbial fermentation process. When µ'set is

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0.14 h-1 (Fig. 2A), the DCW and content of acetic acid in the later stage of

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fermentation decreased rapidly, It could be a principal cause that limited carbon source

231

could not support the normal growth of E. coli. At the post-induction specific rate of

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0.16 h-1 (Fig. 2B), the concentration of acetic acid generated by E. coli was below 1.0

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g/L in the post-induction stage, though the DCW reached 51 g/L, the highest enzyme

234

activity was only 270 U/mL. When the post- induction specific rate was 0.18 h-1 (Fig.

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2C), E. coli continued to excrete acetic acid, and its concentration was maintained at

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around 1.7 g/L, the DCW of E. coli only approached appropriately 44.0 g/L and

237

decreased by 7 g with the post-induction specific rate of 0.16 h-1, but the highest

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enzyme activity was 353 U/mL, which is 1.31 fold higher than that with the

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post-induction specific rate of 0.16 h-1 and 63 U/mL higher than that with the

240

post-induction specific rate of 0.20 h-1 (Fig. 1C). The highest enzyme activity of the

241

condition (µ'set = 0.18 h-1) was compared with that of shaking flask culture by 17.7

242

folds. This potentially indicates that the accumulation of acetic acid inhibited the

243

growth and Tpebgl3 production at the premise of feeding to meet the normal growth of

244

E. coli. Meanwhile, as shown in table 1, the maximum values of the actual specific

245

growth rates at the post-induction and the specific productivity were critical parameters

246

reflecting the optimization of feeding strategies. The results of SDS–PAGE analysis on

247

pure Tpebgl3 was shown in Fig. 3. The specific productivity at a µ'set of 0.18 h-1 was

248

much higher than the other conditions.

249

3.2. Effect of progress parameters on the accumulation of acetic acid on

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producing Tpebgl3

251

3.2.1. Induction temperature

252

In general, Induction temperature is a key parameter of acetic acid regulation by

253

controlling the growth rate and metabolic velocity of recombinant E. coli. Studies

254

showed low temperatures usually prevent misfolding of recombinant proteins and

255

increase the correct expression in E. coli (Lingqia et al., 2015). Therefore, experiments

256

were performed to increase Tpebgl3 production with different induction temperatures

257

based on the previous condition of the optimism feeding strategy. The temperatures

258

were set at 35°C, 37°C, 39 °C, and 41°C in the post-induction phase with 37°C in the

259

pre-induction stage. As shown in Fig. 4, the low induction temperatures were

260

unbeneficial for thermostable enzyme Tpebgl3 production, this was contrary to the

261

previous reported results. Both of production of Tpebgl3 and DCW increased with the

262

induction temperature increasing, the results reflected badly the internal relations

263

between concentration of acetic acid and production of Tpebgl3. When the cells were

264

cultured at 39 °C, the concentration of acetic acid below 1.4 g/L was the lowest level

265

compared with others in the post-induction phase. The highest enzyme activity reached

266

444.0 U / mL at induction temperature of 39°C, which was 1.9 fold higher than that

267

observed at 35°C (Fig. 4B), the DCW also approached maximum 47.9 g/L parallel with

268

other induction temperatures. The enzyme activity of Tpebgl3 at 39°C was also much

269

higher than those noted at 37°C and 41°C, indicating the effects of induction

270

temperatures on the growth of the E. coli were obvious. However, the Tpebgl3 activity

271

at 41°C was decreased owing to the high temperature that accelerated metabolism shift

272

of cell and rapid accumulation of acetic acid at constant dissolved oxygen levels (Fig.

273

4A), thereby the growth of E. coli and the expression of Tpebgl3 were inhabited. These

274

results suggested that appropriate induction temperature usually controls the

275

accumulation of the acetic acid and increases productivity of thermostable enzyme in

276

the fermentation progress.

277

3.2.2. Induction pH

278

Many reports pay less attention to the effects of acidity or basicity on the

279

accumulation of acetic acid generated by E. coli in the high density culture progress

280

(Cheng et al., 2013; Stancik et al., 2002). The effects of pHs were complex, it was

281

associated with others conditions including DO, growth phase and metabolism shifts of

282

E. coli itself (Yong et al., 2010). The acidity or basicity of fermentation broths usually

283

was misdiagnosed as simply guaranteeing optimism acidity or basicity condition

284

suitable to the growth of bacteria and ignored the fundamentality of pH on the

285

regulation of acetic acid formation. The most suitable pH of acetic acid regulation was

286

obtained by adjusting the pH in the post-induction phase and the pre-induction pH (pH

287

7.0) wasn't changed. Induction pH was changed based on the gradient-decreasing

288

method. As show in Fig. 5. Results showed that concentration of acetic acid was

289

further controlled to a very low level when the induction pH is 7.2. The concentration

290

of acetic acid was maintained all along below 0.9 g/L, and it was at the lowest level

291

compared with others. The possible causations about decrease of acetic acid with pH

292

7.2 were that acetic acid in the medium was changed into ammonium salt and the

293

metabolic components inhibited acetic acid formation (Lin et al., 1996). On this

294

condition (pH 7.2), the highest activity of Tpebgl3 reached 560.4 U/mL. This

295

manuscript testified to the ability of pH to regulation of acetic acid in the later

296

induction stage and the activity of the GH3 β-glucosidase is the highest among the

297

production of β-glucosidases by recombinant E. coli in the fed-batch progress. The

298

highest enzyme activity of this condition (µset=0.2 h-1, µ'set = 0.18 h-1, 39°C, pH 7.2)

299

was compared with that of shaking flask culture by 26.7 folds. The DCW approached

300

maximum 65 g/L. However, E. coli generated amount acetic acid with pH 7.4, the

301

possible reason is that high pH induced the metabolic enzymes from E. coli partaking

302

in arginine and glutamate catabolic pathways that channel carbon into acids instead of

303

producing alkaline amines (Stancik et al., 2002). Meanwhile, the high pH is maintained

304

by cautiously adding NH4OH that is unbeneficial for protein formation. Overall, the

305

results indicated that the appropriate pH can not only accelerate the growth of bacteria

306

but it can regulate and control the formation of the acetic acid. Although the

307

accumulation of acetic acid were controlled at a fairly low level, the extended period in

308

the fermentation was harmful to the formation of Tpebgl3. It is necessary for the

309

growth of E. coli and metabolized products to control and reduce the accumulation of

310

acetic acid in E. coli high density fermentation progress.

311

4. Conclusions

312

In this study, the results showed the feed strategies, induction temperatures, and

313

induction pH evidently regulated the accumulation of acetic acid in the fermentation

314

progress. By optimizing the progress conditions (µset=0.2 h-1, µ'set = 0.18 h-1, 39°C, pH

315

7.2) step by step. The concentration of acetic acid generated by E. coli itself was

316

further reduced. The highest activity of Tebgl3 finally approached 560.4 U/mL and the

317

DCW reached maximum 65 g/L when the concentration acetic acid was controlled all

318

along below 0.9 g/L on the optimal fermentation.

319 320 321

Acknowledgements This work was supported by the National Key Research Development Program of

322

China (2016YFD0600805), the Jiangsu “333” project of cultivation of high-level

323

talents (Grant No. BRA2015317), the 11th Six Talents Peak Project of Jiangsu

324

Province (Grant No. 2014-JY-011) and the Priority Academic Program Development of

325

Jiangsu Higher Education Institutions (PAPD).

326

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Figure Legends

417

Fig.1 Effect of exponential fed-batch with different specific growth rates on

418

high-density fermentation of recombinant E. coli producing Tpebgl3 (A: the specific

419

rate of post-induction phase was 0.10 h-1; B: the specific rate of post-induction phase

420

was 0.20 h-1; C: the specific rate of post-induction phase was 0.30 h-1.).

421

Fig.2 Effect of fed-batch with two-stage specific growth rate on the high-density

422

fermentation of recombinant E. coli producing Tpebgl3 (A: the specific rate of

423

post-induction phase was 0.14 h-1; B: the specific rate of post-induction phase was 0.16

424

h-1; C: the specific rate of post-induction phase was 0.18 h-1. All of their pre-induction

425

specific rates were 0.2 h-1.).

426

Fig. 3. SDS–PAGE analysis of the heat treated and the pure Tpebgl3 from intracellular

427

fractions.(1-3) protein obtained by heat treatment fractions of E. coli ; (4-6) protein

428

obtained by purifying heat treatment protein by an immobilized metal affinity column.

429

Fig. 4 Effect of induction temperatures on high-density fermentation of recombinant E.

430

coli producing Tpebgl3 (A: the effects of induction temperatures on the enzyme

431

activity and DCW; B: the effects of induction temperatures on the formation of acetic

432

acid.).

433

Fig. 5 Effect of induction pH on high-density fermentation of recombinant E. coli

434

producing Tpebgl3 (A: the effects of induction pHs on the enzyme activity and DCW;

435

B: the effects of induction pHs on the formation of acetic acid.).

436

Table1

437

Comparison of parameters for Tpebgl3 production with varying feeding strategies in Exponential fed-batch culture

Two-stage glucose feed (µ'set,

(µset)

µset=0.20 h-1)

Style

Specific growth

0.10

0.20

0.30

0.14

0.16

0.18

specific

0.067 ±

0.170 ±

0.179 ±

0.081 ± 0.157 ±

0.109 ±

growth

0.003

0.005

0.004

0.002

0.002

773.9 ±

989.1 ±

rate (h-1) Maximum

0.001

rate (h-1) Volume of protein(m

811.0± 766.3 ± 65.3

19.3

3.8

1172.0 ± 937.3 ± 36.1

19.3

12.0

39.7 ±

44.0 ±

g/L) 34.6 ± DCW(g/L)

44.2±0.2

36.6±0.1

0.1

51.0 ± 0.8 0.3

0.1

20.4 ±

26.6 ±

Specific 22.4 ±

22.4 ±

productivit

20.9 ± 0.18 0.5

y(mg/g)

0.1

18.4 ± 0.5 0.6

0.3

438

439

Fig. 1

440

441

Fig. 2

442

443

Fig. 3

444

445

Fig. 4

446

447 448 449

Fig.5

450

Highlights

451

1. Effect of the acetic acid on Tpebgl3 production by E. coli was studied.

452

2. The acetic acid excretion was controlled by optimal fermentation conditions.

453

3. The Tpebgl3 activity reached 560.4 U/mL with acetic acid kept below 0.9 g/L.

454