Tailoring the Oxidative Stress Tolerance of Clostridium tyrobutyricum

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Tailoring the oxidative stress tolerance of Clostridium tyrobutyricum CCTCC W428 by introducing trehalose biosynthetic capability Qian Wu, Liying Zhu, Qing Xu, He Huang, Ling Jiang, and Shang-Tian Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03172 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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Journal of Agricultural and Food Chemistry

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Tailoring the oxidative stress tolerance of Clostridium tyrobutyricum CCTCC

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W428 by introducing trehalose biosynthetic capability

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Qian Wu1,2#, Liying Zhu3,#, Qing Xu2, He Huang2, Ling Jiang1,*, Shang-Tian Yang4

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1

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China

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210009, PR China

College of Food Science and Light Industry, Nanjing Tech University, Nanjing 210009, PR

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing

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China

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Department of Chemical Engineering, The Ohio State University, Columbus, Ohio 43210, USA

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#

These authors contributed equally to this work.

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*

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Ling Jiang, [email protected]

College of Chemical and Molecular Engineering, Nanjing Tech University, Nanjing 210009, PR

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Corresponding author:

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ACS Paragon Plus Environment


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ABSTRACT: Fermentations employing anaerobes always suffer from the restriction of the

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stringent anaerobic conditions during the production of bulk and fine chemicals. This work aims

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to improve the oxidative stress tolerance of C. tyrobutyricum CCTCC W428, an ideal

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butyric-acid-producing anaerobe, via the introduction of trehalose biosynthesis capability.

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Compared with the wild type, a wider substrate spectrum, an improved metabolic profile and a

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significantly increased specific growth rate were shown by the engineered strain upon aeration

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and acid challenge. Molecular simulation experiments indicated that CoA transferase maintained

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its native folded state when protected by the trehalose system. Furthermore, qRT-PCR was

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combined assays for with acid-r31elated enzyme activities under various conditions to verify the

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effects of trehalose. These results demonstrate that introducing a trehalose biosynthetic pathway,

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which is redundant for the metabolism of C. tyrobutyricum, can increase the robustness of the

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host to achieve a better oxidative resistance.

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KEYWORDS: Clostridium tyrobutyricum, oxidative stress tolerance, trehalose synthase, omics

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analysis

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INTRODUCTION

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There has been a rise in global interest in the conversion of renewable biomass into chemical

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substances and valuable fuels by microorganisms in recent years, due to the anticipated peak of

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global fossil fuel production1. The utilization of microorganisms with unique metabolic

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properties is therefore of special interest, and these include the genus Clostridium2. Clostridium

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tyrobutyricum is an anaerobic, gram-positive, spore-forming bacterium, commonly found in soil,

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naturally fermented cheese and the intestinal tracts of animals and humans3. C. tyrobutyricum

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strains are capable of converting various carbon sources into precursors of biomaterials and

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biofuels such as butanol, H2, 1,3-propanediol and butyric acid4. Butyric acid, a typical short

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chain fatty acid (SCFA) with applications in a wide range of agricultural and food industries, is

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the major metabolite produced by C. tyrobutyricum5,6. SCFAs play an important role in

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maintaining animal fitness, including human body health, for example by acting as special

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nutrition and energy components of intestinal epithelial cells, protecting the intestinal mucosal

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barrier, lowering body inflammation levels, and strengthening the gastrointestinal motor

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function7. However, C. tyrobutyricum is sensitive to a number of different industrial process

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conditions. Since it is a typical obligatory anaerobe, for which continuous oxygen exposure is

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fatal, its robustness against multiple stress factors, such as air and acid exposure, needs to be

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improved. This is especially true concerning the synthesis of butyric acid.

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Trehalose is a naturally stable, nonreducing disaccharide with an ɑ,ɑ-(1,1)-glycosidic bond

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linking two glucose units, which has been isolated from a large number of prokaryotic and

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eukaryotic organisms including bacteria, yeasts, fungi, algae, insects, and invertebrates, as well 3



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as lower and higher plants, especially those living in harsh environments8. Initially, trehalose was

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thought to act solely as an energy reserve and carbon source in a manner similar to that of starch

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and glycogen, but a large body of research suggests that this disaccharide not only has extensive

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applications in the fields of foods and beverages, but also shows significant biological functions

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in cellular protection against diverse chemical and physical stressors, such as heat9, cold10,

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dehydration11, desiccation12, and oxygen radicals13. Our previous study demonstrated that

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trehalose improves the robustness of Propionibacterium acidipropionici, especially in response

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to acid stress14. Furthermore, an investigation of anhydrobiotic yeast cells revealed that

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intracellular trehalose diminishes their aggregation and denaturation and stabilizes enzymes in

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their native state15. Therefore, we investigated whether introducing trehalose biosynthetic

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capability into C. tyrobutyricum can improve its aero- and acid-tolerance. To test this hypothesis,

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we engineered C. tyrobutyricum for trehalose production by overexpressing the trehalose

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synthase (TreS, E. C. 5.4.99.16) gene, and looked into the effects of trehalose on cellular

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physiology.

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MATERIALS AND METHODS

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Plasmids, strains and culture conditions. The plasmids and strains used in the present study

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are listed in Table 1. E. coli was cultivated in LB medium with 100 µg/mL ampicillin or 25

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µg/mL tetracycline added if necessary. C. tyrobutyricum CCTCC W428 was derived from C.

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tyrobutyricum ATCC 25755 via long-term immobilized-cell fermentation, which was described

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in detail previously16. C. tyrobutyricum CCTCC W428 was grown anaerobically at 37 °C in 4


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reinforced clostridial medium (RCM) containing (per liter): 10 g tryptone (OXOID, England), 10

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g beef extract (Hopebio, China), 5 g maltose (Aladdin, China), 5 g NaCl, 3 g yeast extract

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(OXOID, England), 3 g anhydrous sodium acetate, 1 g soluble starch (Aladdin, China), and 0.5 g

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L-cysteine (Sigma-Aldrich, USA). 20 g agar (Solaribio, China) was added to produce solid

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media, as needed. All the media were sterilized by autoclaving at 115 °C, 15 pounds per square

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inch gauge (psig) for 30 min.

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DNA extraction, manipulation, and transformation. Plasmids were extracted from E. coli

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using the Plasmid Extraction Kit (E.Z.N.A, China), while the genomic DNA of C. tyrobutyricum

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and E. coli were isolated using the Bacterial DNA Isolation Kit (E.Z.N.A, China). DNA cloning

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and restriction digestion were performed following standard procedures17. Electrotransformation

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of C. tyrobutyricum was performed as described previously18.

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Construction of the engineered C. tyrobutyricum strain. The plasmid pSY6, harboring the

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lac and ptb promoters, was used for general cloning procedures19. E. coli TOP10 (PAN2) was

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used for DNA methylation. Firstly, the new treS gene identified and preserved in our laboratory20

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was

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5’-CCGCTCGAGCAGCCAAAGCTTTCACTTCGC-3’ and treS-A: 5’-CGCTGTACATCATCA

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GATGACCGGGGACCC-3’ (the recognition sites of the restriction enzymes XhoI and BsrGI are

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underlined). The resulting PCR product was inserted into the pSY6 plasmid using the same

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restriction sites and ligated using T4 ligase, yielding the treS expression plasmid pSY6-treS. The

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resulting recombinant plasmid was methylated in E. coli Top10 (pAN2)21 and after verification

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by DNA sequencing (GENEWIZ, China), the recombinant plasmid was used for the

amplified

through

PCR

using

5


the

primers

treS-S:


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electrotransformation of C. tyrobutyricum CCTCC W428.

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Aerobic challenge experiments. The aerobic challenge experiments were conducted in two

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groups. In the first group, colonies of the wild-type strain were picked from RCM agar, seeded

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into AN (anaerobic) microplates (Table S1) and grown overnight under aerobic (79% N2 and

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21% O2) or anaerobic conditions (79% N2, 10% H2 and 11% CO2). In another group, both the

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wild-type and the engineered strain were cultivated under hypoxic conditions (79% N2, 11% CO2

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and 10% O2) overnight and the resulting cultures were transferred into AN microplates.

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Normoxic (21% O2) or hypoxic (10% O2) conditions were maintained using a H35 hypoxic

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workstation (HEPA, Don Whitley Co., UK).

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GC-MS for metabolomics analysis. Ten milliliter aliquots of culture broth from the overnight

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fermentations (the wild type and engineered strains cultivated under hypoxic conditions), and the

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wild type cultivated under anaerobic conditions, were used for a comparison. The standard

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chemicals (Sigma, USA) were injected into the GC-MS system for the identification of bioactive

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compounds. The GC-MS instrument (Thermo Electron Corporation, USA) was run in single ion

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monitoring (SIM) mode. Two-stage chemical derivatization was performed on the residue

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according to Ding22. The detailed GC-MS method has been described previously23.

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Setup of fermentation system. A 5 L stirred-tank bioreactor (B. Braun Biotech International,

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Germany) containing 2.0 L of culture medium was used for batch fermentations. Before use, the

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bioreactor was autoclaved for 30 min at 121 °C, held overnight, and then autoclaved again for

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another 30 min for complete sterilization. The temperature was kept at 37 °C, with 100 rpm

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agitation. The pH was adjusted to the required values (4.0-7.0) by the addition of 5 M H2SO4 or 6


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10 M NaOH. Details of the bioreactor setup have been given elsewhere14. Hypoxic or aerobic

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conditions were maintained by constant purging with a gas mixture comprising 79% N2+11%

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CO2+10% O2 or 79% N2+21% O2 at a gas flow rate of 10 mL/min. At the beginning of

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fermentation, 150 mL of cell suspension in a serum bottle was used to inoculate the fermentor.

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Batch fermentation was carried out with 100 g/L glucose as the carbon source, and stopped when

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glucose in the fermentation broth was no longer consumed. Maltose was added at a concentration

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of 10 g/L as the substrate for TreS to produce trehalose. Samples were taken every 5 hours for

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the analysis of biomass, substrate, and product concentrations.

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Molecular dynamics (MD) simulations. Topology files for trehalose, with a flexible simple

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point charge (SPC) water model, were created based on data from the GlycoBioChem

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PRODRG2 server (http://davapc1.bioch.dundee.ac.uk/prodrg/). The original structure of the CoA

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transferase (CTF) with a resolution of 2.08 Å was downloaded from the Protein Data Bank (PDB

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code: 5H86)24. In order to simulate the molecules, a force field including the charge group

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assignment and the atomic charges was defined based on GROMOS96. The MD simulations

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were conducted on an Hp Z800 workstation by means of the GROMACS 4.5.4 package, uniting

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the atom protein force field via the GROMOS96 53a625. The Particle Mesh Ewald (PME)

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method was applied to reproduce the long-range electrostatic interactions with a cut-off of 10 Å.

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After energy minimization with steepest descent, the systems were equilibrated with position

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restraints on heavy atoms of the protein for a minimum of 30 ns. The MD simulations were done

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to relax the protein and simulate unrestrictedly with a time step of 2 fs at 313 K and pressure of 1

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atm. To determine the counteracting effect of trehalose on CTF, the MD simulations were carried 7



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out under three different conditions: in pure water; in 1 mol/L HCl and in 1 mol/L HCl with 0.5

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M trehalose.

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qRT-PCR. Quantitative real-time PCR (qRT-PCR) was carried out to verify the results of the

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molecular dynamics simulations of CTF, using an ABI STEPONE PLUS Real Time PCR

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System26. Primer Premier 6.0 was used to design the primers specific for the ctf gene, ctf-S:

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ATGAGTAATTATGGAAAACCTTTAG and ctf-A: TTATGCAATAGCCTCATCTT TTTTC.

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Cells were allowed to grow to an OD600~0.6 at pH 4.0 and total cellular RNA of both the

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engineered strain and the wild type were isolated using the RNeasy Mini Kit (Qiagen, Germany)

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according to the manufacturer’s protocol. The RNA was reverse-transcribed into single-stranded

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cDNA using a cDNA synthesis kit (New England Biolabs, USA), and detection of the PCR

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amplification was performed using the SYBR green fluorescence dye (TAKARA, China). The

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16S rRNA gene was used as calibration standard27,28.

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Determination of enzyme activities. The activity of trehalose synthase (TreS) was measured

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as previously reported20. The enzymatic activities of PTA, ACK and CTF29,30 under different

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conditions (pH 4.0-7.0 and different content of O2) were also determined using an Ultrospec

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3100 Pro spectrophotometer (Amersham Pharmacia Biotech, Cambridge, UK). PTA

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activity was assayed by detecting the liberation of CoA from acetyl-CoA. ACK

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activity was measured via the direction of acyl phosphate formation, while CTF

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activity was measured in the acetyl-CoA-forming direction using butyryl-CoA and

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acetate as the substrates. One unit of enzyme activity was defined as the amount of enzyme

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that catalyzes the conversion of 1 mL of substrate per minute. 8


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RESULTS

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Expression and enzyme purification from the recombinant strain. According to the NCBI

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database3, trehalose cannot be biosynthesized by C. tyrobutyricum CCTCC W428. In our

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previous study, we identified a novel treS gene from a metagenomic library of saline-alkaline

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soil20. A PCR product of approximately 1,820 bp was clearly visible on a 1% agarose gel (Figure

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S1-1). The recombinant expression vector pSY6-treS was constructed (Figure S1-2), and the

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TreS protein heterologously expressed in the strain CCTCC W428. After induction with IPTG

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for 6 and 8 h, the whole-cell lysates were analyzed by SDS-PAGE. A single band at

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approximately 69 kDa was identified on the SDS-PAGE gel (Figure S1-3), which was consistent

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with the size calculated based on the deduced amino acid sequence. The average activity of the

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recombinant TreS enzyme corresponded to 4,797.5 U/mL.

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Biosynthesis of trehalose in C. tyrobutyricum CCTCC W428 improves the oxygen

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tolerance of the host. Aerobic conditions are fatal to strictly anaerobic microorganisms,

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including C. tyrobutyricum CCTCC W428. Figure 1A illustrates that, under anaerobic

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conditions (79% N2+10% H2+11% CO2), the wild-type strain almost fully utilizes all substrates

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except for itaconic acid, which changed when it was exposed to normal atmospheric levels of

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oxygen overnight (79% N2+21% O2). As is clearly visible in Figure 1B, under these conditions

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little of the substrates were used by the bacteria, and the utilization rates thus significantly

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decreased compared to the results shown in Figure 1A. In details, it can be seen from Figure 1B

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that exposure to oxygen disturbed the utilization of a number of substrates, including 9



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monosaccharide, disaccharide, trisaccharide and some organic acids. On the other hand, after

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introducing the trehalose pathway into the host CCTCC W428, the fermentation performance

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was improved drastically. A comparison of Figure 1C with Figure 1D, which shows the results

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of incubation of the engineered strain and the wild type under hypoxic conditions (79% N2+11%

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CO2+10% O2), demonstrates that the capacity to utilize various substrates is restored by this

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intervention. In fact, the performance of the engineered strain under hypoxic conditions was

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comparable to that of the wild-type strain under the optimal, anaerobic conditions. This means

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that the engineered strain clearly grew better than the wild type under aerobic conditions. What’s

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more, by comparing Figure 1B with Figure 1D, we can conclude that increased oxygen

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concentration led to great difference in the utilization of substrates.

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Biosynthesis of trehalose in C. tyrobutyricum CCTCC W428 induces changes at the

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metabolic level, as observed by GC-MS analysis. To determine the physiological differences

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between the wild-type strain under anaerobic conditions and both the wild-type and engineered

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strain under hypoxic conditions at the metabolic level, GC-MS analysis was performed. Figure

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2A shows that numerous compounds were detected, and the color of each square represents the

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relative level of each corresponding metabolite. Red indicates an increase, while green means

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that the amount of the corresponding metabolite has decreased. Nearly 30 metabolites were

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common to both the wild type under anaerobic and hypoxic conditions and the engineered strain

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under hypoxic conditions (Table S2). These included organic acids (propionic, acetic, butyric,

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linolenic, malic, nonanoic, azelaic and pentanoic acid); phosphate compounds (phosphoric acid);

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saccharides (D-glucose, D-galactose, D-mannose, and fructose) as well as amino acids (L-proline, 10


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L-lysine, glycine and L-cysteine). These metabolites are mainly related to the central metabolic

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pathways: the glycolytic pathway (D-glucose), the tricarboxylic acid cycle (malic acid), the

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pentose phosphate pathway (phosphoric acid) and the diverse pathways of amino acid

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metabolism. It can be seen from Figure 2A that the levels of almost all of these analytes

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decreased when the cells were exposed to the aerobic atmosphere, while the engineered strain

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was able to improve these levels after intracellular trehalose accumulation.

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Biosynthesis of trehalose in C. tyrobutyricum CCTCC W428 improves the oxidative stress

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tolerance of the host. Low pH also has severe effects on the survival of C. tyrobutyricum. Batch

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fermentations of the wild-type and the engineered strain at different pH values from 4.0-7.0 and

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different oxygen concentrations were carried out to confirm the effects of trehalose on the

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cellular responses to oxidative stress. Figure 3 shows the benefits of the engineered trehalose

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production ability at the lower pH values (pH 4.0 in Figure 3A, and pH5.0 in Figure 3B), with

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more butyric acid accumulation when pH increased, while there was little or no difference

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between the wild-type and the engineered strain at the less acid or neutral pH (pH 6.0 in Figure

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3C, and pH 7.0 in Figure 3D). It can be concluded from Figure 3E and 3F that aerobic

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conditions are more severe to the host than low pH as nearly no cells were detected during

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fermentation and nearly half of the substrates were unavailable until 35 h after inoculation. Both

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the target product and byproducts decreased significantly compared to the anaerobic conditions

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at pH 7.0 (Figure 3D), which is the optimum conditions for fermentation. In addition, the

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specific growth rates were calculated from the growth data (see Figure S2) and it can be

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observed clearly that both the low pH and aerobic conditions were fatal to C. tyrobutyricum 11



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while introducing the trehalose biosynthesis pathways into the host indeed enhanced its tolerance

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to oxidative stress. Furthermore, we determined the concentrations of maltose and trehalose

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during batch fermentation under different conditions (Figure 4). Since the optimum for the TreS

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enzyme was pH 6.0-7.0 under anaerobic conditions, the conversion rate of maltose to trehalose

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reached as high as 51%. Under low pH and aerobic conditions, however, the activity of TreS

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decreased significantly, which reduced the conversion of trehalose to approximately 21% and

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12%, respectively. Even so, a small amount of trehalose (2.1 g/L in Figure 4A and 1.2 g/L in

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Figure 4B) is enough to play a role in protecting the proteins from oxidative stress according to

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previous studies14,15.

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Molecular dynamics simulation of the conformational properties of CoA transferase

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(CTF) under acidic conditions. Encouraged by these results, we decided to investigate whether

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trehalose can protect the butyric acid biosynthesis pathway under acidic conditions. We thus

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searched the NCBI database to confirm that the key enzyme of butyric acid formation in our

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strain is CoA transferase (CTF), and subsequently made molecular dynamics simulations of this

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enzyme31. In order to assess the trehalose-mediated protein stability enhancement quantitatively,

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two structure parameters, the root-mean-square deviation (RMSD) of the Cα atoms’ initial native

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state and the root mean squared fluctuate (RMSF) per residue of CTF protein, were applied to

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represent the CTF protein’s conformational changes under acidic conditions. Both RMSD and

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RMSF were displayed in the wild type, under normal and acidic conditions, as well as under

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acidic conditions with trehalose added. The theoretical models inferred that acid stress induces

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changes in the structural conformation of CTF, which is consistent with our fermentation results. 12


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Figure 5 shows that the Cα-RMSD values under acidic conditions increased up to 4.5 Å after 9

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ns, while they remained around 2.5 Å with the addition of trehalose. The lower the RMSD values,

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the more stable the CTF in this situation. The RMSF parameter, on behalf of the overall

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flexibility of CTF, was significantly higher under acidic conditions than in pure water, while the

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overall flexibility of CTF decreased with the addition of trehalose (Table 2). These results

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confirmed that trehalose has a remarkable capacity to offset the damage to CTF caused by acid

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stress.

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qRT-PCR analysis of butyric acid biosynthesis markers. To further verify the conclusion of

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the molecular dynamics simulation. The ctf gene was selected for qRT-PCR analysis based on its

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relevance to acidic conditions. As can be seen in Figure 6, the ctf gene had a 10.05-fold

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enhanced expression in the engineered strain compared to the wild-type under acidic conditions

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(pH 4.0). This was further verified by HPLC analysis, which showed that the engineered strain

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accumulated 8.9-fold more butyric acid than the wild type under acidic conditions (1.21 ± 0.01

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mg per 1011 cells vs. 0.14 ± 0.01 mg per 1011 cells) (Table S3). For the determination of enzyme

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activities under different conditions, both the low pH and aerobic conditions did not affect the

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activity of acetate kinase (ACK), while had a great difference on the phosphate acetyltransferase

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(PTA) and CoA transferase (CTF). Moreover, the differences appeared to be more significant at

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lower pH as well as with more oxygen (Figure 7).

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DISCUSSION Anaerobic microorganisms have drawn great attention due to their potential to produce 13



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valuable chemicals and due to their beneficial health effects. However, the complexity and

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expense of achieving strictly anaerobic, rigid production conditions, have limited their broad

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application at the industrial level. Experiments have shown that a 5 log decline in cell numbers

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happens within the first 3 h of aeration, while after 5 h of aeration, no viable cells were

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observed32. This phenomenon is so called lethal oxidative stress. Interestingly, there is an overlap

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in physiological reactions between the stress induced by low pH values and oxygen-induced

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stress25. In fact, the stress caused by acid has similarities with the stress caused by oxygen in

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nature, and both the acid stress and the air-induced stress can be summarized as oxidative stress25.

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Thus, an improvement of the oxidative stress resistance of anaerobic microorganisms is of great

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significance for industrial applications.

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Trehalose is a stress-induced metabolite that is widespread in microbial cells. It has the ability

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to protect the cells as a whole, as well as the cell membranes, proteins and other biological

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macromolecules under various adverse environmental conditions such as drought, dehydration,

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radiation and high permeability. So far, five different enzymatic pathways that are related to the

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biosynthesis of trehalose, have been uncovered and identified in naturally occurring

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microorganisms (Figure S3)33. However, no trehalose was detected in wild-type C.

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tyrobutyricum, suggesting that no gene homologous to any of these five pathways is present in

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the genome of C. tyrobutyricum. This was also consistent with the result of GC-MS, which

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showed that only the transgenic strain produced trehalose. Therefore, our research team has

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introduced a trehalose biosynthesis pathway by utilizing a single enzyme, trehalose synthase, to

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catalyze the conversion of maltose to trehalose via an intramolecular rearrangement34. Maltose is 14


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comparatively inexpensive, and both cost and time can be saved in process scale-up due to the

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simplicity of this single step trehalose production pathway. Thus, this simple genetic

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modification may be enough for obtaining an engineered strain with combined oxygen- and

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acid-resistance properties, which was consistent with our previous results obtained from the

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study of P. acidipropionici14.

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Phenotype analysis was utilized to investigate the effects of trehalose on the metabolic status

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of C. tyrobutyricum when exposed to different oxygen concentrations (Figure 1). The transgenic,

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trehalose-producing strain showed the best utilization of various substrates when challenged with

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oxygen, which implies that introducing a trehalose biosynthesis pathway into the host by

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expressing the treS gene can be applied, yielding even better engineered strains. Further

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experiments have also verified that the engineered strain obtained here shows a distinct

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advantage over the wild type when exposed to the low pH and oxygen in batch fermentations

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(Figure 3). Consistent with such a role, the presence of exogenous trehalose in toxin-producer C.

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perfringens may likewise have the same protective function. In the minimal medium, trehalose

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had an osmoprotective effect and enabled the bacteria to grow at NaCl concentrations of up to

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2%

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cells under stress conditions is thought to contribute to the maintenance of cell integrity and

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protein stability35. Further investigation on the omics level will be needed to elucidate the exact

306

mechanism of this improved resistance to oxidative stress.

33

. Previous research has revealed that accumulation of constitutive trehalose in microbial

307

Based on the metabolomics analysis (Figure 2A), we can conclude that oxygen indeed has a

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strong influence on the host, since almost all metabolites were down-regulated compared to the 15



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wild type grown under anaerobic conditions. On the other hand, things have improved

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significantly when the treS gene was introduced into the host, and the content of the measured

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metabolites in the transgenic strain grown under oxygen challenge was almost the same as that of

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the wild-type cultured in the optimal, anaerobic atmosphere. As shown in Figure 2B, enrichment

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analysis revealed that when CCTCC W428 was exposed to hypoxic condition, there were

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differences of metabolites in galactose metabolism, protein biosynthesis, alpha linolenic acid and

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linoleic acid metabolism, glutathione metabolism, fructose and mannose degradation, and biotin

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metabolism. Furthermore, butyrate metabolism was also significantly changed under hypoxic

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conditions. These results suggest that oxidative stress mainly affects protein biosynthesis and the

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amino acid metabolism of CCTCC W428, which was consistent with the results of pathway

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analysis shown in Figure 2C. It just so happens that there is a similar case, an integrated stress

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response also regulates amino acid metabolism and resistance to oxidative stress of E. coli36.

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These results confirm that introducing the trehalose pathway into the host confers on the ability

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to adapt to oxidative stress with a distinct phenotype. Only those cells with a strong tolerance to

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acid and oxygen were able to survive and thus represent good candidates for industrial processes.

324

Previous studies have shown that trehalose protects central metabolic enzymes, such as

325

pyruvate dehydrogenase (PDH)37 and up-regulates the synthesis of glycolytic enzymes in the

326

yeast Saccharomyces cerevisiae38. We assumed that trehalose might protect the central

327

metabolism of C. tyrobutyricum as well. The capacity of trehalose to counteract acid-induced

328

denaturation and stabilize proteins, was further verified as theoretically sound using MD

329

simulations. The overall topology remained stable in the presence of trehalose, whereas it 16


Page 17 of 39


330

changed markedly in the presence of acid without trehalose in the simulations (Figure 5). The

331

results thus illustrated that two parameters of CoA transferase (CTF), the Cα-RMSD and RMSF

332

values, displayed similar trend pattern in 1 mol/L HCl to those in pure water, while when the

333

trehalose was present in 1 mol/L HCl, the protein was the most stable. Thus, both MD

334

simulations and experiments confirmed the protective role of trehalose against the acid-induced

335

denaturation of the CTF protein.

336

So far, the exact mechanism of trehalose protection is unclear, but most explanations conform

337

to one of three hypotheses: "water replacement", "vitresence", and "preferential exclusion"

338

(Figure S4)39. It has been reported that trehalose is needed as protectant for growing bacteria

339

and/or enzymes directly submitted to oxidative treatment40. To further investigate the preferential

340

interactions of protection of CTF in acidic solutions, the distribution of solvent molecules at the

341

protein surface was calculated using RDFs (radial distribution functions) between the center of

342

mass (COM) of CTF and different atoms (water oxygen, acid carbon and trehalose hydroxyl

343

oxygen). As shown in Fig. 8, the water density from the COM of the protein was less than that of

344

HCl within a distance of about 0.2 nm (inset of Figure 8), but was significantly decreased with

345

the addition of trehalose. That is, HCl molecules, originally distributed around the CTF protein

346

and expelling water from the CTF surface, are excluded from the surface by trehalose.

347

Furthermore, the trehalose molecules were only distributed in the approximate range of more

348

than 0.2 nm from the COM of CTF protein, indicating that trehalose is preferentially excluded

349

from the domain of CTF, which has been confirmed to be the primary reason for protein

350

stabilization by trehalose. This result implied that the CTF protein is preferentially hydrated, that 17



Page 18 of 39

351

is, acid is expelled from the protein. This intriguing confirmation via MD illustrated that the

352

preferential exclusion of HCl by trehalose is the reason of its offsetting effects on the

353

acid-induced CTF denaturation. It is also possible that trehalose is able to reorient the water

354

structure around CTF, reduce its solvation radius, and weaken its mobility, such that the CTF

355

protein is stabilized. This conclusion is consistent with the protective role of trehalose in the

356

binary N,N-dimethylformamide (DMF) solution, where enzymes often suffer significant loss of

357

activity41.

358

Overall, the present data suggest that simply introducing a single trehalose synthase gene into

359

C. tyrobutyricum is enough to induce the production of trehalose. Moreover, the produced

360

trehalose was found to play physiological roles and that the strategy of introducing trehalose

361

biosynthesis into C. tyrobutyricum indeed improves its robustness. To the best of our knowledge,

362

this is the first time that metabolic engineering has been used in C. tyrobutyricum specifically to

363

promote its robustness under process conditions. Based on the genotypic and phenotypic

364

differences between the engineered and the wild-type strain, we can conclude that trehalose

365

biosynthesis improved the tolerance to acid and oxygen stresses, which are tough issues

366

encountered by the cells during industrial processes. Trehalose, a compound that is not found in

367

wild-type C. tyrobutyricum strains, was therefore demonstrated to confer protection on the

368

bacterium against the damage that results from exposure to oxygen and acids. Moreover, this

369

study reveals a theoretic basis and is a proof-of-concept for the evolution of more robust C.

370

tyrobutyricum strains that can survive, grow and perform well in industrial-scale working

371

environments. 18


Page 19 of 39


372 373

ACKNOWLEDGEMENTS

374

We would like to thank Prof. Sheng Yang from Institute of Plant Physiology & Ecology,

375

SIBS, CAS to give the pSY6 plasmid. This work was supported by the National Science

376

Foundation for Young Scholars of China (21506101, U1603112), the State Key Laboratory of

377

Bio-organic and Natural Products Chemistry, CAS (SKLBNPC15429), the Six Talent Peaks

378

Project in Jiangsu Province (2015-JY-009), and the Environmental Protection Project in Jiangsu

379

Province (2015053).

380 381

SUPPORTING INFORMATION

382

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

383

The PCR results of treS gene amplification, Figure S1-1. Map of the recombinant plasmid

384

harboring the treS gene, Figure S1-2. SDS-PAGE analysis, Figure S1-3. Specific growth rates of

385

the wild type and the engineered strain in batch fermentations under different content of O2 (A)

386

and different pH values (B), Figure S2. Five trehalose biosynthesis pathways found in different

387

microorganisms, Figure S3. Three hypotheses on how trehalose protects proteins, Figure S4.

388 389

AUTHOR CONTRIBUTIONS

390

Q.W. and L.Z. performed the experiments, collected and analyzed the data, and drafted the

391

manuscript; L.J. and H.H. assisted in conceiving and designing the experiments, and revised the

392

manuscript; Q.X. performed the molecular dynamics simulation and analyzed the data; S.Y. 19



393

contributed reagents, materials and analytical tools. All authors read and approved the final

394

manuscript.

Page 20 of 39

395 396 397

NOTES The authors declare no competing financial interests.

398 399

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[34] Park M, Mitchell WJ, Rafii F. 2016. Effect of trehalose and trehalose transport on the

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fluoroquinolone-resistant mutant. Int J Microb 2016:1-9.

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[38] Bonini BM, Dijck PV, Thevelein JM. 2013. Trehalose metabolism: enzymatic pathways and physiological functions. Biochem Mol Biol 3:291-332.

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[39] Jain NK, Roy I. 2009. Effect of trehalose on protein structure. Protein Sci 18:24-36.

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[40] Alvarez-Peral FJ, Zaragoza O, Pedreno Y, Argüelles JC. 2002. Protective role of trehalose

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during severe oxidative stress caused by hydrogen peroxide and the adaptive oxidative stress

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[41] Yang X, Jiang L, Jia Y, Hu Y, Xu Q, Xu X, Huang H. 2016. Counteraction of trehalose on N,

505

N-dimethylformamide-induced Candida rugosa lipase denaturation: spectroscopic insight

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507

25



Page 26 of 39

508

FIGURE LEGENDS

509

Fig. 1 The growth differences visualized by phenotype microarrays. A: the wild type under

510

anerobic conditions (79% N2+10% H2+11% CO2, overnight); B: the wild type under aerobic

511

conditions (79% N2+21% O2, overnight); C: the engineered strain under hypoxic conditions

512

(79% N2+11% CO2+10% O2, overnight); D: the wild type under hypoxic conditions (79%

513

N2+11% CO2+10% O2, overnight). The normoxic (21% O2) or hypoxic (10% O2) conditions

514

were generated inside a H35 hypoxic workstation.

515

Fig. 2 Metabolomic GC-MS analysis of C. tyrobutyricum CCTCC W428. A: Clustering of

516

metabolites of the wild type under anaerobic conditions, the wild type under hypoxic conditions

517

(79% N2+11% CO2+10% O2) and the engineered strain under hypoxic conditions (79% N2+11%

518

CO2+10% O2); B: Enrichment analysis of the metabolites in the CCTCC W428 that were

519

exposed to hypoxic conditions (79% N2+11% CO2+10% O2); C: Pathway analysis of the

520

metabolites in the CCTCC W428 that were exposed to hypoxic conditions (79% N2+11%

521

CO2+10% O2).

522

Fig. 3 Batch fermentations of the wild type and the engineered strain at different pH values

523

(4.0-7.0) and different content of oxygen (anaerobic, hypoxic and areobic). Symbols: squares

524

represent the glucose concentration; circles represent the OD600; triangles represent the butyric

525

acid concentration; rhombus represent the acetic acid concentration. Solid symbols denote the

526

engineered strain; hollow symbols denote the wild type. Each error bar indicates the average

527

value obtained in the three experiments.

528

Fig. 4 The concentrations of maltose and trehalose during the batch fermentation of engineered 26


Page 27 of 39


529

strain under different pH values (4.0-7.0) and different content of oxygen (anaerobic, hypoxic

530

and areobic). Symbols: squares represent pH 4.0; circles represent pH 5.0; triangles represent pH

531

6.0; pentagrams represent pH 7.0; rhombuses represent hypoxic; pentagons represent aerobic,

532

respectively. Each error bar indicates the average value obtained in the three experiments.

533

Fig. 5 RMSD from the initial native structure for Cα atoms under three different conditions. Blue

534

represents the CTF in pure water; Red represents the CTF in 1 mol/L HCl and black represents

535

the CTF in 1 mol/L HCl with the trehalose presented.

536

Fig. 6 qRT-PCR of the ctf gene in the wild type under normal conditions, the wild type at pH 4.0

537

and the engineered strain at pH 4.0. Each error bar indicates the average value obtained in the

538

three experiments.

539

Fig. 7 The determination of enzyme activities under different conditions. A, B and C: the

540

activities of phosphate acetyltransferase (PTA), acetate kinase (ACK) and CoA transferase (CTF)

541

under different pH (4.0-7.0); D, E and F: the activities of phosphate acetyltransferase (PTA),

542

acetate kinase (ACK) and CoA transferase (CTF) under different content of oxygen (anaerobic:

543

79% N2+11% CO2+10% H2, hypoxic: 79%. Each error bar indicates the average value obtained

544

in the three experiments.

545

Fig. 8 g (r) as a function of time for the simulations of CoA transferase (CTF) under three

546

different conditions. Blue represents the CTF in pure water; Red represents the CTF in 1 mol/L

547

HCl and black represents the CTF in 1 mol/L HCl with the trehalose presented.

27



Fig. 1

28


Page 28 of 39

Page 29 of 39


Fig. 2 A

29



B

C

30


Page 30 of 39

Page 31 of 39


Fig. 3

31



Page 32 of 39

Fig. 4 A 12

12

10

8

8

6

6

4

4

2

2

0

Trehalose (g/L)

Maltose (g/L)

pH 10

0 0

5

10

15

20

25

30

35

Time (h)

B 12

12

10

10

8

8

6

6

4

4

2

2

0

0 0

5

10

15

20

25

30

Time (h)

32


35

Trehalose (g/L)

Maltose (g/L)

O2

Page 33 of 39


Fig. 5

33



Fig. 6

ctf 25

mRNA level

20

15

10

5

0 Wild type

Wild type (acid) Engineered strain (acid)

34


Page 34 of 39

Page 35 of 39


Fig. 7

35



Fig. 8

36


Page 36 of 39

Page 37 of 39


Table 1. Strains and plasmids used in this study Strains and Plasmids

characteristic

Source or reference

Clostridium tyrobutyricum

wild type

CCTCC

E. coli DH5α

competent cells

Vazyme

E. coli TOP10

carries the pAN2 vector

Invitrogen

pAN2

methylated vector, Φ3TI, pl5A

[34], [35]

CCTCC W428

ori, TetR pSY6

general vector for Clostridium sp.

[36]

pSY6-treS

recombinant plasmid

This study

Mutant strain

Clostridium strain with

This study

introduced treS gene

37



Page 38 of 39

Table 2. Total RMSF (sum of RMSF per residue) of CoA transferase (CTF) averaged over the last 6 ns of each simulation under three conditions. Conditions

Total RMSF (nm) of CTF

No trehalose (CTF in pure water)

51.23

No trehalose + acid (CTF in 1 mol/L HCl)

54.11

Trehalose + acid (CTF in 1 mol/L HCl with trehalose

43.69

presented)

38


Page 39 of 39


TOC graphic

39


Tailoring the Oxidative Stress Tolerance of Clostridium tyrobutyricum

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