In situ Biocatalytic Production of Trehalose with Auto-Induction

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In situ Biocatalytic Production of Trehalose with Auto-Induction Expression of Trehalose Synthase Xincheng Yan, Liying Zhu, Yadong Yu, Qing Xu, He Huang, and Ling Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06031 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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Journal of Agricultural and Food Chemistry

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In situ Biocatalytic Production of Trehalose with Auto-Induction Expression of

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Trehalose Synthase

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Xincheng Yan1,2#, Liying Zhu3,#, Yadong Yu4, Qing Xu4, He Huang4, Ling Jiang1,*

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1

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210009, China

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2

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Nanjing 210009, China

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3

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210009, China

College of Food Science and Light Industry, Nanjing Tech University, Nanjing

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

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

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4

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China

College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009,

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#

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*

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Ling Jiang, [email protected]

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These authors contributed equally to this work.

Corresponding author:

1

ACS Paragon Plus Environment


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ABSTRACT: We developed an in situ biocatalytic process that couples

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auto-induction expression of trehalose synthase (TreS) and whole-cell catalysis for

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trehalose production. With lactose as the auto-inducer, the activity of recombinant

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TreS in recombinant Escherichia coli was optimized through a visualization method,

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which resulted in a maximum value of 12033 ± 730 U/mL in pH-stat fed-batch

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fermentation mode. Meanwhile, the permeability of the auto-induced E. coli increased

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significantly, which makes it possible to be directly used as a whole-cell biocatalyst

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for trehalose production, whereby the by-product glucose can also act as an extra

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carbon source. In this case, the final yield of trehalose was improved to 90.5 ± 5.7%,

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and remained as high as 83.2 ± 5.0% at the 10th batch, which is the highest value

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achieved using recombinant TreS. Finally, an integrated strategy for trehalose

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production was established, and its advantages compared to the traditional mode have

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been summarized.

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KEYWORDS: Escherichia coli, trehalose, recombinant TreS, auto-induction,

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whole-cell catalyst, in situ biocatalysis

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INTRODUCTION

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Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a stable, colorless,

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odor-free, and non-reducing disaccharide, in which two glucose moieties are linked by

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an α-1,1-glycosidic bond. Trehalose is widespread in nature and is found at high

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concentrations in various organisms which can naturally survive dehydration, even

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when 99% of their body water is removed, a suspended-animation state termed

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anhydrobiosis.1 Many studies have proved that trehalose not only serves as a source of

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energy, but also acts as an active protectant of DNA, enzymes, and other proteins,

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cellular membranes, resist diverse chemical and physical stresses, such as desiccation,

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extreme temperatures, and high doses of ionizing radiation, in addition to

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dehydration.2,3 It is the high water-holding activity that makes trehalose applicable to

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the development of additives, stabilizers, humectants, and sweeteners that are quite

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useful in the fields of food, agriculture, cosmetics, and pharmaceuticals.4 At present,

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trehalose is mainly produced via extraction from plants and brewery-spent yeast,

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microbial fermentation and enzymatic conversion.5,6 The commercial production of

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trehalose began in the 1990s by Hayashibara Biochemical Laboratories, and

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enzymatic conversion remains the main way of trehalose production to this day.7 So

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far, at least 5 different trehalose biosynthetic pathways have been found in various

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organisms.8,9 Among the established enzymatic trehalose production methods, the

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trehalose synthase (TreS, EC 5.4.99.16) pathway has become appealing for industrial

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applications due to the one-step formation of trehalose from the inexpensive and

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easily obtainable substrate maltose. This reaction proceeds by an intramolecular 3



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rearrangement of the α-1, 4 into an α-1, 1 glycosidic bond, which represents a simple,

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fast, and low-cost method.10 Up to now, dozens of treS genes from different species

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have been reported in the literature and heterologously expressed in the gram-negative

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bacterium Escherichia coli using lac-derived promoters, which are usually induced by

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the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).11-18 However, the

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relatively high cost of IPTG and the inhibition of bacterial growth during strong and

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sudden induction with IPTG can have a huge impact on the high-throughput,

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economical production of recombinant TreS protein. Furthermore, since TreS is an

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intracellular enzyme, it is usually necessary to break the cells by ultrasonication and

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subsequently purify the enzymes from the crude extract before the enzymatic reaction.

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During the purification procedure, the enzymatic activity of TreS will decrease

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significantly, and the resulting biocatalyst is difficult to recycle.

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A mentioned approach to solve this problem involves the development of the

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lactose-based auto-induction to produce recombinant proteins in E. coli.19 The

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principle of auto-induction is based on the diauxic response of E. coli when grown in

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multiple carbon sources such as glucose and lactose, which results in the induction of

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the lactose promoter upon depletion of glucose after E. coli has already produced a

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certain amount of biomass.20,21 This method of induction was found to be superior to

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the standard IPTG approach since it omits the need for biomass monitoring to ensure

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the correct timing of inducer addition. Auto-induction thus places the shift from

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growth to recombinant protein production under metabolic control of the expression

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host, and thereby minimizes the required dealing with cultures from inoculation until 4


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cell harvest, which is a major advantage for high-throughput experiments.22,23

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Furthermore, our preliminary experiment has proved that the high cell densities

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achieved by auto-induction can produce more target protein (Figure S1) and higher

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activity (Figure S2) than IPTG induction. However, the efficiency of auto-induced

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expression of heterologous protein in E. coli is determined by different parameters,

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and many factors such as the medium composition and cultural conditions will

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complicate the use of auto-induction.24,25

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The study of critical medium components through conventional one-factor

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procedures is time-consuming, and does not allow us to identify the effects of possible

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interactions between the individual factors. To overcome this defect, a visualization

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method (VM) was employed to study the effects of mutual interactions of media

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components and other process parameters.26 It has been proved that VM was an

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efficient method for searching optimum operation point based on the orthogonal test

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or uniform test.27-29 In the present work, the VM for producing trehalose via TreS

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expressed in E. coli as a whole-cell biocatalyst was for the first time established and

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also optimized by studying the components of the auto-induction medium.

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Furthermore, we found that the auto-induced E. coli can directly convert maltose into

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trehalose without the broken operation, while the by-product glucose generated by

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TreS was proposed to supply nutrient for cell growth (Figure 1), which further

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improved the final yield of trehalose from maltose even after the multiple cycles of

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coupled conversion process.

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MATERIALS AND METHODS 5



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Strains and culture conditions. The engineered E. coli strain BL21(DE3)

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(pET22b(+)-TreS), containing a new treS gene, was constructed previously by our

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laboratory and stored in glycerol cryopreserved tubes at -80 °C.15 The strain was

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activated in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin (Sangon

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Biotech Co., Shanghai, China) at 37 °C and 200 rpm overnight, and the resulting

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culture was used to inoculate auto-induction medium containing 100 µg/mL

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ampicillin for fermentation.19

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Materials and chemicals. All sugars, including maltose, trehalose and lactose,

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were purchased from Sigma Chemical Co. (St. Louis, MO). The industrial

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by-products whey and glycerol were both purchased from Lingfeng Co. (Shanghai,

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China). All other chemicals and reagents were from commercial sources and were of

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analytical grade. The basal auto-induction medium contained 5 g/L of glycerol, 20 g/L

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of lactose, 6 g/L of Na2·HPO4, 3 g/L of K2HPO4, 0.5 g/L of MgSO4·7H2O, 1 g/L of

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NH4Cl, 10 g/L of yeast extract, and 0.5 g/L of NaCl, as well as an initial glucose

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concentration ranging from 0 to 20 g/L. The supplementary auto-induction medium

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contained 150 g/L of glycerol, 50 g/L of whey (with lactose as the main ingredient

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apart from water), NH4Cl 20 g/L, (NH4)2SO4 19.6 g/L and 7.5 g/L of MgSO4·7H2O.

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All media were sterilized by autoclaving at 121 °C for 20 minutes.

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Optimization of fermentation for the production of recombinant TreS. The

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fermentation of E. coli with recombinant TreS was conducted in three different

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culture systems: (i) shake flask, (ii) 5-L fermenter, and (iii) 500-L fermenter.

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(i) shake flask: E. coli cells were cultivated overnight and the resulting culture was 6


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used to inoculate shake flasks containing fresh auto-induction medium with 100

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µg/mL ampicillin at 37 °C for 16 h in an orbital shaker set at 200 revolutions per

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minute (rpm). In addition, a three-level, four-factor, orthogonal design was used to

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optimize the fermentation conditions, and the enzyme activity was used as the index

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(Table 1). For each of these treatments, two replicates were used for statistical

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analysis.

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(ii) 5-L fermenter: A fermentation in pH-stat fed-batch mode was carried out in a

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5-L fermenter (FUS-5 L, Guoqiang, Shanghai, China)with the operating conditions as

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follows: 2 volume/culture volume/min (vvm) of aeration, 300 rpm of agitation speed,

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and 37 °C. The fermentation was started in batch mode with 1 L basic culture medium

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(initial pH 7.0). When the culture pH began to drop, fed-batch operation was activated,

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and the culture pH was controlled at 7.0 ± 0.1 by the addition of the supplementary

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medium. In the constant fed-batch fermentation mode, the supplementary medium

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was added to the fermenter at a flow rate of 1.0 mL/min. The pH was kept at 7.0 by

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the addition of 25 wt % ammonia liquorwith an online sensing and dosing system.

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Both fed-batch fermentation modes were terminated when a final culture volume of

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2 L was reached.

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(iii) 500-L fermenter: Fermentations on a larger production scale were conducted in

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a 500-L fermenter system (FUS-500 L, Guoqiang, Shanghai, China) with a working

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volume of 300 L, operated in the same pH-stat fed-batch culture mode as the 5-L

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fermenter system. During the large-scale fermentation process, the OD600 value,

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dissolved oxygen and enzyme activity of the culture broth were determined and 7



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recorded at the regular time.

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Visualization method. The visualization software (version 1.0, Wuhan University

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of Technology, Wuhan, Hubei, China) was used to optimize the experimental results

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from the orthogonal experiment.26,28 The multidimensional space was connected by a

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mapping plane comprising a systemic network in the mapping model (Figure 2).

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From the m-dimensional inputs vector, x, to the mapping plane, z1 and z2:

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Z1 =W1XT, Z2 =W2XT

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From the mapping plan to the outputs vector, y:

(1)

(2)

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where X = [1, x1, x2,..., xm], wj = [w0j, w1j,..., wmj], j = 1,2, Y = [y1, y2,..., yl], P = [1,

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f1(z1, z2), f2(z1, z2),..., fp (z1, z2)], V = [v1,v2,...,vl]l× p, vk = [vk0, vk1,vk2,...,vk( p+l)], k = 1,

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2,...,l, where w1, w2 and v are the weight vectors of the systemic network, while P is a

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nonlinear extending vector used to enhance the mapping effect. In this study, we

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simply assume that P = [1, f1(z1, z2), f2(z1, z2), . . . , fp(z1, z2)] =[1, z1, z2, z12, z22, z1z2].

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In the mapping process, the contours of the objective functions can be produced

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according to the sample value in multidimensional space mapped to a plane. And then

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the optimized region on the mapping plane can be easily located based on the contour

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distribution of different objective functions. Next, the optimal point can be inversed to

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the multidimensional space where it is represented by the original variables for

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practical use.

8


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ac ab

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β=

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where c is any point of the line through points a and b, β is the step size and its

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value equals the ratio of the distance between points a and c to the distance between

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points a and b, 0≤ β ≤1 represents interpolation, and β ≥ 1 represents extrapolation.

(3)

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According to the formula (3), the optimization direction and the step size can be

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selected. Finally, we can thus obtain the corresponding optimal point in the original

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multi-dimensional space from the optimal point in the mapping plane.

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Production of

trehalose

by

whole-cell biocatalysis.

During

fed-batch

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fermentation in pH-stat mode in the 5-L fermenter, the addition of feeding medium

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was stopped when the total broth volume reached 2 L. Meanwhile, 1 L of phosphate

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buffer (pH 7.0) containing maltose (45 wt %) was supplied to the fermenter at a flow

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rate of 3 mL/min (the final concentration of maltose was 15 wt %). Next, fermentation

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and biocatalysis were conducted concomitantly at 37 °C. The pH was kept at 7.0 by

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the addition of NH4·OH via an online sensing and dosing system. The same approach

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was taken in the 500-L fermenter with the operating volume amplified in equal

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proportion.

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Analytical methods. Cell density was measured using a spectrophotometer

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(25UV/VIS Lambda, Perkin Elmer, Norwalk, CT) at a wavelength of 600 nm (OD600).

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Cell viability was determined by measuring the fluorescence of reduced resazurin,

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which is proportional to the viable cell number, using a SpectraMax M3 microplate

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reader (Molecular Devices, Sunnyvale, CA) with the excitation and emission

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wavelengths set to 544 and 590 nm, respectively.30 The enzyme activity of TreS was 9



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assayed by measuring the trehalose produced from maltose. The reaction mixture

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consisting of 800 µL of 150 mM maltose solution in 50 mM sodium phosphate buffer

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(pH 7.0) and 200 µL of crude enzyme solution in a final volume of 1 mL was

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incubated at 30 °C for 30 min. After this, the reaction mixture was incubated at

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100 °C for 10 min to stop the reaction. Quantitative analysis of the trehalose

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concentration was conducted on an high-performance liquid chromatography (HPLC)

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system (Dionex, Sunnyvale, CA) equipped with an NH2 column (250 mm × 4.6 mm,

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Sepax, Newark, DE) and a refractive index detector (RID). The column was

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maintained at 35 °C and was eluted isocratically with a mobile phase consisting of

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acetonitrile and ultrapure water (Millipore Milli-Q, Bedford, MA) at a volume ratio of

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75:25 and a flow rate of 1.0 mL/min. One unit (U) of enzyme activity was defined as

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the amount of enzyme that catalyzed the formation of 1 µmol of trehalose per minute

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under the described assay conditions.

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RESULTS AND DISCUSSION

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Optimization of auto-inducted E. coli for the expression of TreS in shake flasks.

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Auto-induction can be used to obtain high cell densities and good overproduction of

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proteins in a simple and low-cost operation.31 The protein expression efficiency in E.

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coli is decided by various parameters, and interactions among them will make it

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complicated to identify the most contributing one to the improvement of protein

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expression. Since the optimal implementation of auto-induction relies on both the

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induction conditions and the composition of the auto-induction medium, especially

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the suitable proportions of different carbon sources, the present study focused on the 10


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optimization of relevant culture parameters for the heterologous expression of TreS in

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E. coli using a visualization method. According to the literatures, the medium

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composition, time of induction, and the environmental factors such as temperature

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and pH, all of which have intense effects on the implementation of the auto-induction

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process.19,24,25 Since the presence of glucose inhibits transcription of the recombinant

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genes from lactose-inducible promoter, the concentration of glucose actually

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determines the lactose induction time.20 On the basis of the results of our previous

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study,27 the L9 (34) orthogonal experimental design was used in this study, and four

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independent elements were therefore selected: A, temperature; B, concentration of

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glucose; C, pH; and D, concentration of glycerol. Table 1 lists the details of the

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orthogonal experimental design, and the results are given in Table 2.

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According to the range analysis of the orthogonal experimental design, the strength

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of the effect of individual factors on the enzyme activity was in the order:

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concentration of glycerol > temperature > pH > concentration of glucose (Table 2).

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The following results were obtained: (a) 5 g/L of glycerol gave a higher TreS activity

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than 10 or 15 g/L; (b) 37 °C gave a higher TreS activity than 32 or 27 °C; (c) the TreS

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activity was higher at pH 7.0 than at pH 5.5 or 6.0; (d) 10 g/L of glucose gave a better

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induction time, which resulted a higher TreS activity. The optimum reaction

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conditions for the production of recombinant TreS in shake flasks were thus identified

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as temperature of 37 °C, pH 7.0, 10 g/L of glucose and 5 g/L of glycerol. Under these

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conditions, TreS activity reached up to 6113 U/mL. The results of shake-flask

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fermentations showed that the concentration of glycerol exhibited the greatest 11



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influence on TreS activity, which was probably due to a direct effect on the cell grow

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and an indirect effect on the environmental pH in the culture broth.32 Excessive

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feeding of glycerol can cause a severe decrease of pH due to the formation of acidic

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metabolites, while insufficient supply of glycerol may lower the synthesis of TreS.

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The optimal concentration of glycerol obtained from this study was in agreement with

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the reported preferable glycerol concentration for the production of other recombinant

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proteins (e.g., therapeutic proteins, polyvinyl alcohol hydrolase) in E. coli strains.32,33

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To further explore the optimum fermentation conditions for TreS expression from

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the orthogonal experimental results, the dimension-reducing mapping analytical

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method was applied and obtained with the contour of the objective function to predict

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the optimal operating point and optimization direction. The mapping diagram for the

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orthogonal experiment was based on a sample data set of nine points, with the solid

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black line representing the contour of enzyme activity (Figure 3). In this way, the

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optimal proportions of media components can be determined intuitively on the basis

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of the plane, while the optimal point in the plane can be mapped inversely to the

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original multidimensional space by means of an inversion mapping algorithm, where

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it is represented in terms of practical proportion data. By taking points 7 and 8 as

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reference, and using a step size of 1.1, a predicted point indicated by a red asterisk

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was obtained through extrapolation in the direction of the arrow, which indicated that

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the maximum value of TreS activity can achieve 6486 U/mL with the optimal

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experimental conditions of 37 °C, pH 7.0, 12 g/L of glucose and 5 g/L of glycerol.

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In order to test the validity of the optimized conditions obtained from the 12


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visualization software, an experiment was performed with the parameters suggested

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by the VM. The experimental values were quite close to the predicted values (6431 vs.

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6486 U/mL), which demonstrated that the VM can be used successfully to predict the

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enzymatic activity of TreS heterologously expressed in E. coli.

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Auto-induction expression of TreS in pH-stat fed-batch fermentation

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mode. Compared to batch and continuous fermentations, fed-batch fermentation

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usually comes with many advantages, including the achievement of high-cell-density

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cultures and the utilization of highly concentrated substrate, which can reduce

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hydraulic load and wastewater generated in the process.34 However, recombinant E.

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coli strains have an inherent tendency to accumulate acetic acid during the fed-batch

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fermentation process, which inhibits cell growth and the formation of recombinant

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proteins.35 Especially, cell growth and product formation is usually limited by

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catabolic repression inhibition when using glucose as the sole carbon source.36

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Combined with the optimized results from the shake-flask fermentation, the pH-stat

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fed-batch culture in a 5-L fermenter system was adopted to enhance the production of

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recombinant TreS in the case of using glycerol as the carbon source. The profiles of

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cell growth, as well as the production of TreS and acetic acid were compared with

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those in the constant-feeding fed-batch culture, and the results were shown in Figure

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4. As can be seen in Figure 4, more biomass and TreS production were achieved in

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the pH-stat culture than those in the constant-feeding culture, with a higher OD600

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value (31.2 ± 2.3 vs. 25.7 ± 1.8 in the constant-feeding fed-batch culture), and less

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acetic acid after 24 h of fermentation (2.6 ± 0.4 vs. 4.5 ± 0.9 g/L in the

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constant-feeding fed-batch culture). The maximum activity of TreS also increased

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1.24-fold in the pH-stat fed-batch culture over the constant-feeding fed-batch mode

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(12121 ± 680 vs. 9775 ± 530 U/mL). 13



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In addition, significant accumulation of glycerol was observed in the

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constant-feeding operation (Figure 4), which indicated that glycerol was overdosed.

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Excessive glycerol feeding may cause substrate inhibition, hindering the production

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of recombinant TreS. Moreover, a mass of base (NaOH) had to be added for pH

287

control because of the excess glycerol, which will handicap the TreS synthesis, since

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a previous study showed that fermentative production of recombinant β-glucosidase is

289

sensitive to the addition of acids or bases.37 Therefore, the pH-stat operation is a

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two-birds-with-one-stone strategy affording both better growth of cells and efficient

291

accumulation of TreS. Interestingly, the accumulation of TreS in recombinant E. coli

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induced by cheap raw whey was comparable to what was obtained using lactose as the

293

inducer (12033 ±730 vs. 12121 ± 680 U/mL, Table 3), which indicates the possibility

294

of a significant cost reduction with whey as a substitute for lactose in the large-scale

295

production of TreS.

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Repeated use of the whole-cell biocatalysts for the production of trehalose

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from maltose. It is well-known that the use of intracellular enzymes in the form of

298

whole-cell biocatalysts is an effective way to lower enzyme production costs, since

299

complex separation and purification in downstream processing can be simplified or

300

even discarded.38 Accordingly, an integrated bioprocess for trehalose production and

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separation from maltose with recombinant TreS in permeabilized cells was proposed

302

in our previous study.8 In the present study, we found that cells from auto-induction

303

fermentation can directly produce trehalose from maltose in a simple phosphate buffer

304

system even with no need for cell permeabilization (Figure S3). To explore the effect

305

of auto-induction fermentation on the recombinant E. coli cells, TEM was applied to

306

compare the morphology to that of cells cultured using the IPTG-induction

307

fermentation mode. As can be seen in Figure 5, the plasma membrane was destroyed 14


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in the auto-induction fermentation, appearing thin but not broken, whereas the

309

structure of the plasma membrane remained intact in the IPTG-induction fermentation.

310

Under this circumstance, TreS stays inside the cell, while the substrate (maltose) and

311

the product (trehalose and glucose) can freely enter and leave the cells.

312

In most cases, the increase of trehalose concentration was accompanied by a

313

synchronously increase in the concentration of glucose throughout the course of the

314

TreS catalytic reaction.11 TreS catalyzes two competing reactions: the formation of

315

trehalose proceeds via a rearrangement of maltose, which represents an intramolecular

316

transglycosylation. By contrast, the formation of glucose stems from a nucleophilic

317

attack by water molecules which hydrolyze the glycosyl enzyme intermediate on the

318

catalytic pockets of TreS, leading to the transformation of a maltose molecule into

319

two glucose molecules (Figure 1).5 The formation of glucose has been shown to be

320

irreversible and thus could lead to a decline in trehalose yield. Meanwhile, the

321

presence of glucose in the reaction mixture could further hamper trehalose synthesis

322

by acting as a competitive inhibitor.11 However, E. coli cells cultivated by

323

auto-induction can conduct the whole-cell catalysis in situ, which inspired us to

324

couple the expression of TreS and the subsequent TreS catalytic reaction process.

325

As shown in Figure 6, the repeated catalysis with the utilization of auto-induction

326

cells was operated at a large scale of 500-L fermenter system, which displayed high

327

trehalose productivity during a total reaction time of almost 200 h. The whole-cell

328

biocatalysts exhibited very good operational stability, with 83.2 ± 5.0% of the initial

329

trehalose productivity remained after 10 batches of reaction (134.5 ± 4.1 g/L of

330

trehalose in the first batch vs. 112.2 ± 3.7 g/L of trehalose in the 10th batch).

331

Intriguingly, we found that even after repeated utilization of the biocatalyst for 200 h,

332

the viability of the auto-induction cells remained above 80%. This value was even 15



333

higher than that of control cells that did not perform biocatalysis at all (Figure S4).

334

We proposed that the glucose produced as a by-product supplied nutrients for cell

335

growth in this process, as was confirmed by the low concentration of glucose, which

336

was less than a fourth of that found in the traditional catalytic process (1.8 ± 0.2 vs.

337

7.0 ± 0.6 g/L) at the end of the last 8 batch of biocatalysis.39 The reusage of glucose

338

not only reduced the need for carbon sources in the feeding process, but also

339

promoted the catalytic reaction of TreS. The conversion rate of maltose was therefore

340

increased to 90.5 ± 5.7%, which, to our best knowledge, is the highest value obtained

341

using recombinant TreS so far.1,17 Furthermore, this whole-cell catalytic process

342

significantly shortened the total trehalose production time, that is, from 24 h in a

343

previous report to 14 h in the present case.9 The high trehalose yield and low glucose

344

gain is preferable from the viewpoint of improved productivity and simplified

345

downstream processing in the mass production of trehalose.

346

Progress towards integrated trehalose production. Combined with the progress

347

made in trehalose separation and purification in our previous study,8 we proposed an

348

improved integrated strategy for the fermentation of recombinant E. coli expressing

349

TreS and concomitant trehalose production from maltose (Figure 7). In the upstream

350

fermentation procedure comprising auto-induction fermentation, TreS was produced

351

through three levels of fermentation with the application of the cheap industrial

352

by-products glycerol and whey, resulting in an auto-induction medium that costs only

353

58% as much as the IPTG-induction medium (Table S1 and S2). For the whole-cell

354

biocatalysis with auto-induction cells, the catalytic process was performed in the same

355

fermenter system, which eliminates centrifugation and filtration procedures for the

356

collection of E. coli cells, and also saves the costs of the construction of a special 16


Page 16 of 37

Page 17 of 37


357

reaction tank. In the downstream separation of trehalose only one SMB device is

358

needed to separate the trehalose from unreacted maltose in our improved integrated

359

system, since the residual glucose in the crude product mixture is less than 1% after

360

terminating the catalytic reaction and filtrating the auto-induction cells. The resulting

361

trehalose is further refined by simple crystallization as introduced previously.8

362

Furthermore, the single-cell protein derived from the waste cells and the maltose-rich

363

solution can be utilized as a high-value-added feed in agriculture and aquaculture,

364

which makes the entire technology more competitive on a larger scale.40,41

365

In this study, we thus proposed an integrated process for the auto-induction

366

expression of recombinant TreS and the in situ bioconversion of maltose into

367

trehalose. It is worth mentioning that the glucose produced as a by-product supplied

368

nutrients for cell growth and therefore helps maintain high cell viability even after

369

continuous catalysis for 200 h. At the same time, the low concentration of residual

370

glucose in the product stream will reduce the difficulty of the downstream purification

371

of trehalose. In this sense, large-scale fermentation of TreS and enzyme-catalyzed

372

trehalose synthesis from maltose were integrated, resulting in an overall process that

373

is characterized by low cost, high efficiency, and simple purification, which makes it

374

suitable for the industrial production of trehalose in the near future.

375

ACKNOWLEDGMENTS

376

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

377

(2017YFC1600404), the National Science Foundation for Young Scholars of China

378

(U1603112, 21506101), the Natural Science Foundation of Jiangsu Province 17



379

(BK20171461), and the Environmental Protection Project in Jiangsu Province

380

(2015053).

381

SUPPORTING INFORMATION

382

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

383

paper. SDS-PAGE analysis of proteins in whole-cells, Figure S1. Time courses of cell

384

growth and the TreS activity, Figure S2. Effects of buffer solution on the trehalose

385

conversion rate, Figure S3. Times courses of cell viability, Figure S4.

386

AUTHOR CONTRIBUTIONS

387

X.Y. and L.Z. performed the experiments, collected and analyzed the data, and

388

drafted the manuscript; L.J. and H.H. assisted in conceiving and designing the

389

experiments, and revised the manuscript; Y.Y. and Q.X. performed the molecular

390

dynamics simulation and analyzed the data; L.J. contributed reagents, materials and

391

analytical tools. All authors read and approved the final manuscript.

392

NOTES

393

The authors declare no competing financial interests.

394

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395

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396

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24


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521

FIGURE LEGENDS

522

Figure 1. An integrated strategy of trehalose production coupled auto-induction

523

expression of TreS with whole-cell biocatalysis.

524

Figure 2. Mapping model of visualization method.

525

Figure 3. Visual dimension-reducing mapping plane for the synthesis of TreS

526

(including optimal point).

527

Figure 4. Time courses of cell growth and the TreS activity in a 5-L fermenter system

528

using pH-stat culture mode at 37 °C for 24 h. The enzyme activity was analyzed by

529

using 150 mM maltose as substrate at 30 °C for 30 minutes. Error bars show SD for n

530

= 3.

531

Figure 5. Transmission electron micrographs of E. coli cells cultivated by

532

auto-induction (A) as compared with those of cultivated by IPTG-induction (B).

533

Figure 6. Reusability of auto-induction cells in a 500-L fermenter for the production

534

of trehalose from in situ supplied maltose. Error bars show SD for n = 3.

535

Figure 7. Simplified flow sheet for integrated process of trehalose production and

536

separation from maltose. The entire technological scheme integrates auto-induction

537

fermentation, whole-cell biocatalysis, separation, purification, crystallization, etc.

538

SMB, simulated moving bed chromatography.

25



Page 26 of 37

Figure 1 Lactose

Extracellular Intracellular

Lactose permease

Inducer

Repressor

Induction LacI

Escherichia coli

LacP

LacO

Z

plasmid

Glucose

supply for cell growth Glu-334

Leu-331

Maltose

Trehalose Tyr-221

Intracellular

Y

Asn-261

Trehalose synthase

Cell membrane Extracellular Trehalose

Maltose

26


Page 27 of 37


Figure 2

27



Figure 3

28


Page 28 of 37

Page 29 of 37


Figure 4

11 10 9 8

13 12

10000

35

11 30

8000

25

7

20

6000

6 15

5

4000

4 10

3

10 9 8 7 6 5 4 3

2000

2

TreS activity (U/mL)

12

14

40

OD

13

12000

5

2 1

1 0

0

0

2

4

6

8

10

12

14

16

18

20

Time(h)

29


22

24

0

0

Acetic acid concentration (g/L)

Glycerol in pH-stat Glycerol in constant-feeding OD in pH-stat OD in constant-feeding TreS activity in pH-stat TreS activity in constant-eeding Acetic acid in pH-stat Acetic acid in constant-feeding

14

Glycerol concentration (g/L)

15

45

15


Figure 5

30


Page 30 of 37

Page 31 of 37


Figure 6

160

Maltose

150

Trehalose

Glucose

140 130

Sugar concentration(g/L)

120 110 100 90 80 70 60 50 40 30 20 10 0

0

14

28

42

56

70

84

98

112

126

140

Time(h)

31


154

168

182

196


Figure 7

32


Page 32 of 37

Page 33 of 37


Table 1. Factors and Levels of Orthogonal Experimental Design L9 (34) Temperature

Concentration of glucose

(°C)

(g/L)

A

B

C

D

1

27

0

5.5

5

2

32

10

6.0

10

3

37

20

7.0

15

levels

pH

Concentration of glycerol (g/L)

33



Page 34 of 37

Table 2. Design of the orthogonal experiment and results factor Enzyme No. of

Temperature Concentration of

pH

Concentration of activity

experiments

(°C)

glycerol (g/L)

glycerol (g/L) (U/mL)

R

A

B

C

D

1

27

0

5.5

5

4811

2

27

10

6.0

10

4113

3

27

20

7.0

15

3321

4

32

0

7.0

10

5114

5

32

10

5.5

15

4321

6

32

20

6.0

5

3841

7

37

0

6.0

15

3814

8

37

10

7.0

5

6113

9

37

20

5.5

10

5821

K1

122

137

149

119

K2

132

145

117

150

K3

157

129

143

114

k1

40.667

45.667

49.667

39.667

k2

44.000

48.333

39.000

50.000

k3

52.333

43.000

47.667

38.000

11.666

5.333

10.667

12.000

K: the sum of yields at each level; k: the average of K at each level; R: range, the 34


Page 35 of 37


difference between the maximal k and minimal k. The value is expressed as the mean (n = 3) and the S.D. is less than 5%.

35



Page 36 of 37

Table 3. Performance of recombinant TreS production with fed-batch fermentation using pH-stat and constant-feeding strategies Feeding strategy

Inducer Maximum

cell Concentration

concentration

of Maximum TreS

acetic acid (g/L)

activity (U/mL)

(OD600) pH-stat

lactose

31.2 ± 2.3

2.6 ± 0.4

12121 ±680

whey

25.7 ± 1.8

2.8 ± 0.5

12033 ±730

25.1 ± 2.2

4.5 ± 0.9

9775 ± 530

Constant-feeding lactose

36


Page 37 of 37


P


In situ Biocatalytic Production of Trehalose with Auto-Induction

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