Overview

We aim to make Clostridium tyrobutyricum facultative anaerobic and add it to animal feed. The engineered Clostridium tyrobutyricum can adapt to aerobic environment so it doesn’t require strictly anaerobic condition while fermenting. At the same time, when it is excluded from animals, the production of butyric acid by Clostridium tyrobutyricum can neutralize the alkaline soil. (If Clostridium tyrobutyricum is still strictly anaerobic, it cannot survive in soil) We engineered two facultative anaerobic Clostridium tyrobutyricum strains by introducing two genes, the dps (DNA-binding protein) gene and aceE gene, respectively. Ct(Plac-dps) strain is Clostridium tyrobutyricum transformed with pMTL-Plac-dps plasmid, and Ct(Plac-aceE) strain is Clostridium tyrobutyricum transformed with pMTL-Plac-aceE plasmid.

Therefore, we need to further verify ：

1.If the heterologous expression of dps protein in C. tyrobutyricum can improve its oxygen tolerance.

2.Whether heterologous expression of the AceE protein in Clostridium tyrobutyricum improves oxygen tolerance.

Proof 1: dps expression improves the oxygen tolerance of C. tyrobutyricum

We first examined whether Dps protein was successfully expressed in Clostridium tyrobutyricum by polyacrylamide gel electrophoresis (SDS-PAGE). Taking the strain with empty plasmid pMTL82151 as the control strain, it can be seen from Fig. 1 that a band appeared at 23.5 kDa of the recombinant strain, Ct (Plac-Dps), indicating that dps protein was successfully expressed in Clostridium tyrobutyricum.

In order to study whether the expression of dps protein affects the growth of the strain under anaerobic conditions, we studied the growth of the recombinant strain under anaerobic conditions. It can be seen from Fig. 2 that the maximum biomass of the recombinant strain (OD600) was 9.67 when the dps protein was overexpressed under anaerobic condition, while the maximum biomass of the control strain was 9.18. There was no significant difference in its maximum growth rate.

In order to study whether the expression of dps protein affects the growth of the strain under aerobic condition, we also studied the growth of the recombinant strain under aerobic conditions (100 rpm). It can be seen from Fig.3 that under aerobic conditions, the growth of the control strain was affected, and its delay period was long.

In addition, although the maximum biomass of the two strains had no significant difference, the growth rate of the recombinant strain was significantly higher than that of the control strain. Therefore, it can be found that overexpression of dps protein improved the growth performance of Clostridium tyrobutyricum under aerobic conditions. Overexpression of Dps protein can protect DNA in Clostridium tyrobutyricum from oxidative damage by binding Fe2+ and preventing Fe2+ and H2O2 from Fenton reaction.

Proof 2: aceE expression improves the oxygen tolerance of C. tyrobutyricum

We first examined whether aceE protein was successfully expressed in Clostridium tyrobutyricum by SDS-PAGE. Taking the strain with empty plasmid pMTL82151 as the control strain, it can be seen from Fig. 4 that 99.7 kDa bands appeared in the recombinant strain Ct (Plac-aceE), indicating that aceE protein was successfully expressed in Clostridium tyrobutyricum.

We studied the growth of the recombinant strain under anaerobic and aerobic conditions as well. It can be seen from Fig.5 that under anaerobic conditions, aceE protein was overexpressed, and the maximum biomass of the recombinant strain was lower than that of the control strain. It is speculated that under anaerobic conditions, the aerobic and anaerobic pathways from pyruvate to Acetyl-CoA in the recombinant strain may compete, which has a partial impact on the growth of the strain.

According to the research of the growth of recombinant strains under aerobic conditions (100 rpm), it can be seen from Fig.6 that under aerobic conditions, the growth of the control strain was affected, and its lag period was longer than that of the recombinant strain. In addition, the growth rate of the recombinant strain was significantly higher than that of the control strain.

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Therefore, it can be found that overexpression of aceE protein improved the growth performance of Clostridium tyrobutyricum under aerobic condition, and the overexpression of aceE protein under aerobic conditions enabled the reaction from pyruvate to Acetyl-CoA, a key step in the carbon metabolism pathway, to proceed normally.

Detailed experiments for the above proof results are shown below.

Experiments:

1.1Strain, plasmid and culture conditions

The strains used in the experiment included Clostridium tyrobutyricum transformed with empty plasmid pMTL82151 as control and Clostridium tyrobutyricum transformed with pMTL-Plac-dps and pMTL-Plac-aceE plasmids constructed by our team.

Clostridium tyrobutyricum used in the experiment is the strain L319 isolated by our laboratory, with the preservation number of GDMCC No. 62289. The Clostridium tyrobutyricum enriched medium (RCM) is used to cultivate Clostridium tyrobutyricum, which consists of 1% peptone, 1% beef extract, 0.5% sodium chloride, 0.5% glucose, 0.3% anhydrous sodium acetate, 0.3% yeast powder, 0.1% soluble starch, 0.05% cysteine hydrochloride, and pH 6.5. The fermentation medium TGY of Clostridium tyrobutyricum used is composed of 3% peptone, 2% glucose, 1% yeast powder, 0.13% cysteine hydrochloride and pH 6.8. Clostridium tyrobutyricum was cultured in anaerobic flask at 37 ℃.

1.2 Methods

1.2.1 Protein gel electrophoresis

Take the Clostridium tyrobutyricum bacterial solution freshly cultured to OD600~2.0, centrifugate it with 10000xg for 1 min to remove the supernatant, and then resuspend it with 1 mL PBS buffer solution with pH 7.0, centrifugate it with 10000xg for 1 min to remove the supernatant, and then add 0.5 mL PBS buffer solution for resuspension. The 150 W cells were broken by ultrasound for 10 min, and the broken transparent protein solution was centrifuged for 5 mi at 10000 xg to remove the residual cell fragments and insoluble substances. Then the supernatant was added to 160μl 4X SDS protein electrophoresis buffer and heated at 100 ℃ for 10 minutes. After cooling to room temperature, samples were taken for protein electrophoresis.

1.2.2 Cell culturing conditions for C. tyrobutyricum

Aerobic conditions: 100 mL anaerobic flask with rubber seal, half filled, rotation speed 100 rpm, at 37 °C, using Reinforced Clostridium Medium (RCM). Anaerobic conditions: 100 mL anaerobic flask with rubber seal, half filled, quiesced in an anaerobic incubator, at 37 °C, using Reinforced Clostridium Medium (RCM).

1.2.3 Growth performance assay

The OD600 data were determined by using UV-Vis spectrophotometer, sampling at different times and determining the light absorption values at 600nm.