0. Abbreviation

CDP: cyclodipeptide
LC-MS: Liquid Chromatography-Mass spectrometry
. LB: LB medium
M9: M9 minimal culture medium
PBS: Phosphate buffered saline

1. Overexpression of cWA synthase in E. coli

Design 1

Our first step is to recombine E. coli to introduce the cWA synthase and show that E. coli can synthesize cWA.

Build 1

First, Streptomyces varsoviensis NRRL B-3589 and Streptomyces monomycini NRRL B-24309 were transferred from RIKEN BRC and cultured in PDA medium. The target genes were then cut out by colony PCR.
The pET28b vector was then cut with restriction enzymes and introduced by Gibson Assembly together with the insert. To confirm that the vector was formed, it was cut again with restriction enzymes and electrophoresed.

Test 1

The vector was then introduced into E. coli DE3 for expression, culture and expression were performed, and the culture medium was collected for LC-MS analysis.

Learn 1

We found that CDP was synthesized at approximately the same rate as in the original paper.

2. Separation of main culture and substance production, change of medium for substance production

Design 2

LB medium is the best medium for growth and expression of E. coli, but because it is rich in amino acids in both type and quantity, we wondered if E. coli would take up amino acids other than alanine and tryptophan to produce CDP other than cWA. On the other hand, if we narrowed down the types of amino acids in the main culture stage, growth and production of cWA synthase might be inhibited. Therefore, we decided to separate the main culture and material synthesis stages and use E. coli as a biocatalyst.

Build 2

To separate the main culture and substance production stages and to investigate the effect of changing the composition of the medium for substance production on the amount of synthesis, the following medium compositions, times, and temperatures were used.

LB main culture 3 hours

LB main culture -> Amino acid excess LB for substance synthesis 3 hours
LB main culture -> Amino acid excess LB for substance synthesis 6 hours
LB main culture -> Amino acid excess M9 for substance synthesis 6 hours
LB main culture -> Amino acid excess PBS for substance synthesis 12 hours

Test 2

The reaction solution was subjected to LC-MS. None of the values were higher than those of Test1. In addition, the synthesis was higher in the order of LB, M9, and PBS for the medium used during substance synthesis.

Learn 2

Since none of the media for material synthesis exceeded the Test 1 values, it was inferred that the yield decreased due to the separation of the main culture and material synthesis phases. The highest synthesis was observed in the order of LB, M9, and PBS, suggesting that the survival of E. coli played a major role in the synthesis of substances.

Design 3

Since we found that changing the composition of amino acids in the medium did not change the synthesis ratio of CDP, we wondered if overexpression of native alanine synthase in E. coli and increasing the amount of alanine in the body would increase the amount of synthesis.

Build 3

The following parts are incorporated into the pCDF vector in different combinations and expressed together with cWA synthase. Currently the vectors are not assembled and we are trying to do so by trial and error.
BBa_K4871010: avtA
BBa_K4871011: iscS
BBa_K4871012: alaD

Future Prospect

・Use more efficient enzyme (BBa_K4871002)
・Construct a cell-free system (Synthesis of aminoacyl-tRNA synthetase and RNA polymerase)