Team:TUST China/Results/G.xylinus

Team:TUST China 2017

Optimization of
metabolic pathway

Description :

  • Cellulose can be synthesized by many organisms, ranging from prokaryote to animal. In prokaryotic organisms, bacterial cellulose is synthesized mainly by Gluconacetobacter, Rhizobium, Agrobacterium, Escherichia, Enterobacter and salmonella. In nature, only bacteria can synthesize cellulose pellicle with water-holding texture, among which the most studied bacteria are the genus Gluconacetobacter. Several common strains in the genus Gluconacetobacter can be distinguished by different representations. Bacteria that can synthesize bacterial cellulose are mainly G. Europaeus, G. intermedius, G. Oboediens, G. Nataicola, G. Sacchari and G.xylinus. Researchers have been studying the biosynthesis of bacterial cellulose in recent decades. This G.xylinus has long been recognized as a model strain. Many classical cellulose synthesis pathways and regulatory models were obtained in G.xylinus. Therefore,we choose G.xylinus as the research object in this experiment.

    The biosynthesis pathway of bacterial cellulose in G.xylinus is complicated including the synthesis of cellulose precursor, polymerization of glucan monomers and assembly of glucan chains.

    The biosynthesis of bacterial cellulose begins with the conversion of substrates to cellulose precursors, which are usually carbon sources, such as the conversion of glucose to uridine diphosphate glucose (udp-glucose). Uridine diphosphate glucose is the substrate of cellulose synthase, and it is catalyzed by cellulose synthase to form β-1 and 4 glucoside bonds between glucose monomers. There are specific sites on the outer membranes of the bacterial cells, which are coupled with the cellulose synthase complexes and is the spot where the polymerization is conducted [9]. When the polymerization reaction is over, the newly generated cellulose is secreted to the extracellular through these sites. The following figure is a partial schematic of the production of cellulose by G. xylinus

    We hope to optimize the metabolic pathway of bacterial cellulose, redirecting more carbon sources into the production of bacterial cellulose; meanwhile expressing key genes in BC synthesis process. Through these two methods we hope to achieve high yield of BC . Therefore, we designed the following experiments:

Design & Engineering :

  • Phosphoglucomutase(PGM) is able to catalyze the conversion of glucose-1-phosphate and glucose-6-phosphate and plays an significant role in both glucose metabolism and BC metabolic network.

    Cyclic dimeric guanosine monophosphate (c-di-GMP) is a new type of second messenger molecule that is ubiquitous in bacteria. Bacteria utilize it to perceive cell surface signals and activate targets within cells, which involves amplification of the original signal and the subsequent expression of a series of specific genes in the cell. These genes affects the various physiological and biochemical processes in the cells. c-di-GMP control a wide range of process, and has a role in transcription, translation and post-translation levels. In addition, c-di-GMP participates in the regulation of multiple biological functions and affects biofilm formation. In a nutshell, it is a crucial elements to the formation of membrane-like bacterial cellulose and the improvement of BC production.

    This experiment attempts to improve BC production by over-expressing PGM and c-di-GMP, a small signal transfer molecule of bacteria.the goal is to implement both to realize the high yield of BC.

    The process of the experiment is divided into following steps, firstly, we transform the constructed plasmids into the E. coli DH5α. When expressing successfully, then we transform it into G.xylinus through electroporation and put it in comparison with the common G.xylinus. We try to explore the influence of each plasmids on the production of BC after fermentation.

    We use PCR to amplify gene PGM and c-di-GMP，and transform reconstructed plasmids J23119+PGM and J23119+c-di-GMP into G.xylinus. Then we put them in comparison with the control group PAsr+PGM and PAsr+c-di-GMP and measure its effect on the yield of BC respectively.

Results

  • At the beginning of the experiment, it was found that the yield of BC was higher than that of G.xylinus cultured individually when cultured with E.coli at pH=5 ，so we first construct the PPGM-PGM-331 plasmid and verify its effect on BC yield after overexpression.

    The results show that the overexpression of PGM has no significant effect on the increase of BC yield.

    1. We acquire genes of PAsr from K12 genome of E.coli via PCR

    2. We acquire genes of PGM and c-di-GMP from genome of G.xylinus.(With and without PGM)

      Construction of vector PPGM-PGM-pSEVA331. Transformation of plasmids into E.coli.

      Verification of homologous recombination via colony PCR

      Co-culture of E.coli and G.xylinus

  • Second, we found a strong promoter -J23119, linked it with the PGM, at the same time linked it with c-di-GMP which is a signal molecule could improve the yield of cellulose synthase.

    The results show that the yield of BC was not significantly increased, but the yield is increased compared to PPGM-PGM-331 plasmid, specially J23119-c-di-GMP.

    1. We acquire gene sequence J23119 and complete its linking with PGM and c-di-GMP
    2. We insert gene segments into plasmid pSEVA331 and transform it into chemically-competent E.coli.
    3. Verification of homologous recombination via colony PCR
    4. Co-culture of E.coli and G.xylinus

  • Third, due to G.xylinus is co-cultured with E.coli in the environment of pH=5 as we designed, we also try to linked PGM with the acid-controlled promoter PAsr, and linked c-di-GMP with PAsr. We explore and compare the effects of overexpressing PGM and c-di-GMP on the yield of BC in the environment of co-culture and pH=5. At the same time,the effect of it and pH-responsive promoter PAsr on BC production will be compared.

    The results showed that the yield of BC was further improved than that of the second step, especially when PAsr was connected with c-di-GMP, and the yield of BC was significantly improved under mixed culture.

    1. We acquire gene sequence of PAsr＋PGM via PCR
    2. We acquire gene sequence of PAsr＋c-di-GMP via PCR
    3. We insert gene sequences into plasmid pSEVA331and transform them into chemically-competent cell into E.coli.
    4. Verification of homologous recombination via colony PCR
    5. Co-culture of E.coli and G.xylinus

Reference

  1. Peter Ross， Raphael Mayer， and Moshe Benziman. Cellulose Biosynthesis and Function in Bacteria [J] . Microbiological Reviews，1991，55(1):35-58
  2. Peter Ross, Raphael Mayer, Haim Weinhouse, et al. The Cyclic Diguanylic Acid Regulatory System of Cellulose Synthesis in Acetobacter xylinum[J]. The J. of Biological Chemistry, 1990,265(31):18933-18943
  3. Alan L. Chang, Jason R. Tuckerman, Gonzalo Gonzalez, et al. Phosphodiesterase A1, a Regulator of Cellulose Synthesis in Acetobacter xylinum, Is a Heme-Based Sensor[J].Biochemistry, 2001,40:3420-3426