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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
5
210009, China
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2
7
Nanjing 210009, China
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3
9
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
11
China
College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009,
12 13 14 15 16 17 18 19 20
#
21
*
22
Ling Jiang,
[email protected] 23
These authors contributed equally to this work.
Corresponding author:
1
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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.
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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
231
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
234
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
245
method was applied and obtained with the contour of the objective function to predict
246
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
253
reference, and using a step size of 1.1, a predicted point indicated by a red asterisk
254
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
265
cultures and the utilization of highly concentrated substrate, which can reduce
266
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
269
proteins.35 Especially, cell growth and product formation is usually limited by
270
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
273
recombinant TreS in the case of using glycerol as the carbon source. The profiles of
274
cell growth, as well as the production of TreS and acetic acid were compared with
275
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
277
the pH-stat culture than those in the constant-feeding culture, with a higher OD600
278
value (31.2 ± 2.3 vs. 25.7 ± 1.8 in the constant-feeding fed-batch culture), and less
279
acetic acid after 24 h of fermentation (2.6 ± 0.4 vs. 4.5 ± 0.9 g/L in the
280
constant-feeding fed-batch culture). The maximum activity of TreS also increased
281
1.24-fold in the pH-stat fed-batch culture over the constant-feeding fed-batch mode
282
(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
286
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
288
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
292
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.
296
Repeated use of the whole-cell biocatalysts for the production of trehalose
297
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
301
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
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structure of the plasma membrane remained intact in the IPTG-induction fermentation.
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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
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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
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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
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(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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FIGURE LEGENDS
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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
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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
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Figure 2
27
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Figure 3
28
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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
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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
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Figure 5
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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
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168
182
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Figure 7
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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
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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
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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%.
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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
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