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)
1.Overview of our design
We plan to engineer facultative anaerobic Clostridium tyrobutyricum by introducing two gene fragments, the Dps (DNA-binding protein) gene and aceE gene.
pDps gene is isolated from the genome of Deinococcus wulumuqiensis R12. It can prevent H 2 O2 from reacting with divalent iron ions (Fenton reaction). Thus, it can prevent the production of hydroxyl radicals and protect DNA. aceE gene is a pyruvate dehydrogenase gene from E. coli MG1655. By introducing pyruvate dehydrogenase in the strain, Clostridium tyrobutyricum can convert pyruvate into acetyl coenzyme A after PFOR (pyruvate: ferredoxin oxidoreductase) is inactivated by oxygen, and complete the central metabolic pathway in the presence of oxygen.The coexpression of these two gene segments can make Clostridium tyrobutyricum more suitable for the growth in aerobic environment.
2.Dps gene for viability under aerobic conditions
On July 2022, Research group of Professor Jiang Ling of Nanjing University of Technology had isolated Deinococcus wulumuqiensis R12 from arid irradiated soil in Xinjiang province of China. Deinococcus wulumuqiensis R12 belongs to a genus that is well-known for its extreme resistance to ionizing radiation and oxidative stress. The team found that Dps play an important role in the metabolism, transportation and oxidation-reduction process of Deinococcus wulumuqiensis R12. Also, when implementing Dps gene to E. coli，through H 2 O2 treatment, the team discovered that the antioxidant capacity of the cell envelope was increased. Through this conclusion, we intend to apply this gene to the plasmid of Clostridium tyrobutyricum, hoping that Dps can enhance the viability of Clostridium tyrobutyricum in aerobic environment.[1]
3.aceE gene for aerobic respiration
In the energy metabolism pathway of the anaerobic bacteria Clostridium tyrobutyricum, PFOR is responsible for the catalysis of the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 [5]. However, when encountering oxygen, PFOR is inactivated, and the energy metabolism is disrupted, which affects the growth of Clostridium tyrobutyricum.
By comparing the pyruvate to acetyl-CoA reaction of the strict anaerobic Clostridium tyrobutyricum, the facultative anaerobic Escherichia coli and the facultative anaerobic Lactococcus lactis, we found that under aerobic conditions, facultative anaerobic bacteria can use pyruvate dehydrogenase (PDH) to catalyze pyruvate to acetyl-CoA and CO2[6]. To make a facultative anaerobic Clostridium tyrobutyricum strain, PDH can be considered as a good substitute for the inactivated PFOR to carry out the oxidative decarboxylation of pyruvate and maintain the energy metabolism under aerobic conditions.
aceE gene expresses pyruvate dehydrogenase E1 component in Escherichia coli (strain K12). Pyruvate dehydrogenase E1 component is a component of the PDH complex, that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2[7].
Therefore, our project introduced the aceE gene into Clostridium tyrobutyricum to make up for the functional vacancy in energy metabolism after PFOR inactivation.
4. Expression vector: pMTL82151 plasmid
We plan to use pMTL82151 plasmid as the expression vector for the Dps and aceE genes. According to a study, pMTL82151 with replicon pBP1 from C. botulinum is the best expression vector for Clostridium tyrobutyricum. It gives the highest transformation efficiency, segregational stability, enzymatic expression level, and butanol production from glucose and other carbon sources[2].
5. Promotors: Pthl and Plac
We plan to use a strong constitutive thiolase gene promoter Pthl to co-express the Dps and aceE genes to make sure the expression is robust[3]. Then we will replace the promoter with a lactose inducible promoter Plac to control the gene transcription. With the Plac promoter, the Dps and ace genes are only switched on and express proteins when lactose is present. This is because for the engineered Clostridium tyrobutyricum, the newly introduced gene expressions and other molecular pathways involved add burden to the strain itself. Therefore, when Clostridium tyrobutyricum are under anaerobic conditions such as in the digestive tract of animals, these genes are better switched off to relieve such burden. When the bacteria are in aerobic environment including feed preparation, transportation and fertilizer usage, lactose is added to induce gene expressions so as to ensure aerobic respiration and growth of Clostridium tyrobutyricum[4].
6.Three stages of our design
In this study, facultative anaerobe Clostridium tyrobutyricum is constructed in three stages:
(1)A strain with lactose inducible expression of the Dps gene.
(2)A strain with lactose inducible expression of the aceE gene.
(3)A strain with lactose inducible co-expression of the Dps and aceE gene.
Stage 1: A strain with lactose inducible expression of the Dps gene.
A recombinant expression plasmid pMTL-Pthl-dps is constructed based on the pMTL82151 vector, containing the Dps gene from Deinococcus wulumuqiensis R12 and a Pthl promoter. The recombinant plasmid is transformed into E. coli CA434. After sequence validation, E. coli CA434 is conjugated to Clostridium tyrobutyricum. Dps protein expression and oxygen tolerance are verified. Then, lactose inducible Plac promoter is used to replace the Pthl promoter, and the Clostridium tyrobutyricum with lactose inducible expression of the Dps gene is constructed according the above steps.
dps gene was constructed as a part BBa_K4407001 . BBa_K4407010 and BBa_K4407012 were constructed as the parts for expressing dps under the control of Pthl and Plac promoters, respectively. Click here（BBa_K4407001 BBa_K4407010 BBa_K4407012） [SS1] for more details. The genetic circuit of the dps expression under the Plac promoter is shown in Figure 6.
Stage 2: A strain with lactose inducible expression of the aceE gene.
This strain is constructed by the same method as stage 1, using the aceE gene instead of Dps gene. The aceE gene is obtained by PCR amplification using E. coli DH 5a and L. lactis subsp. cremoris NZ9000.
aceE gene was constructed as a part BBa_K4407002. BBa_K4407011 and BBa_K4407013 were constructed as the parts for expressing aceE under the control of Pthl and Plac promoters, respectively. Click here（ BBa_K4407002 BBa_K4407011 BBa_K4407013 ） [SS1] for more details. The genetic circuit of the aceE expression under the Plac promoter is shown in Figure 7.
Stage 3: A strain with lactose inducible co-expression of the Dps and aceE gene.
Same method is followed as in stage 1. Plasmids that express lactose inducible Dps and aceE genes are respectively constructed and transformed into E.coli CA434 and subsequently conjugated to Clostridium tyrobutyricum to develop a strain with co-expression of the two genes.
BBa_K4407015 and BBa_K4407014 were constructed as the parts for co-expressing dps and aceE under the control of Pthl and Plac promoters, respectively. Click here（ BBa_K4407015 BBa_K4407014 ） [SS1] for more details. The genetic circuit of the dps and aceE co-expression under the Plac promoter is shown in Figure 8.
The simultaneous expression of the two genes can enhance the viability of Clostridium tyrobutyricum under aerobic conditions. In this way, feed preparation with Clostridium tyrobutyricum as probiotic additives can be easier and transportation will require no anaerobic devices. More importantly, livestock that eat the feeds with the engineered Clostridium tyrobutyricum as probiotics excrete feces with viable Clostridium tyrobutyricum. These bacteria can function well under aerobic conditions and produce butyric acid in the feces. Such feces can be used to help neutralize saline and alkaline land and change the current problems of uncultivated poor lands in many areas of China.
Reference:
[1]Chen, Y., Yang, Z., Zhou, X. et al. Sequence, structure, and function of the Dps DNA-binding protein from Deinococcus wulumuqiensis R12. Microb Cell Fact 21, 132 (2022). https://doi.org/10.1186/s12934-022-01857-7
[2]YU M, DU Y, JIANG W, et al. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum [J]. Applied Microbiology and Biotechnology, 2012, 93(2): 881-9.
[3]贾军皓, 曹丁, 陈勉华,等. 菌丝霉素在酪丁酸梭菌中的表达研究[J]. 食品工业科技, 2020, 41(18):6.
[4]刘本举, 李新梅. 乳糖操纵子[J]. 中学生物学, 2006, 22(12):3.
[5]Lee J, Jang YS, Han MJ, Kim JY, Lee SY. Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses. mBio. 2016 Jun 14;7(3):e00743-16. doi: 10.1128/mBio.00743-16. PMID: 27302759; PMCID: PMC4916380.
[6] https://www.genome.jp
[7]https://www.uniprot.org/uniprotkb/P0AFG8/entry
