Creation and Exploratory Research of the Longest Bacterial
Polyynes with a Pentayne Structure
Kenji KAI
Graduate School of Agriculture, Osaka Metropolitan University
Research objective
Bacterial polyynes, which have a conjugated carbon-carbon triple bond (C≡C) startingfrom the terminal alkyne, are natural products with strong biological activities, such asantifungal, antioomycete, and nematocidal activities. Natural products with polyyne structures
are used as medicines, traditional medicines, and health-promoting nutritional agents.
Therefore, bacterial polyynes are an attractive group of compounds in chemistry and biology
but also for industrial applications. However, only five classes of bacterial polyynes have beenfound thus far, with protegenins and caryoynencins, which have a tetrayne structure, being thelongest polyynes (four C≡C repeats).1,2) Our efforts to discover novel bacterial polyynes andanalyze their biosynthesis have laid the groundwork for the creation of genetically engineeredbacterial polyynes. In this study, we tested whether enzymes (fatty acyl-ACP ligase andacetylenase) in the protegenin biosynthetic gene cluster could be artificially modified toconstruct pentyne structures or even longer polyyne structures.
Methods and results
1. Biosynthetic mechanism of the teterayne structure in protegninsProtegnins are bacterial polyynes biosynthesized by Pseudomonas protegens strain Cab57.2)
We found that protegnins are biosynthesized by a pro gene cluster. However, the function ofeach of these genes remains unknown. Therefore, we generated a strain deficient in each gene.
Deletion of proA (ligase), proB (desaturase), and proD (ACP) resulted in a loss of the ability
to produce protegenin A. Intermediate levels of accumulation were not observed. The deletionof proH slightly reduced the production of protegenin A. Deletion of proF, which encodes
rubredoxin, greatly reduced the production of protegenin A.
The pro gene clusters were then expressed in E. coli to test whether the enzymes/proteins
they encoded could also function in E. coli. The entire cluster was cloned by PCR and
expressed in E. coli BL21(DE3) using the pET-21a(+) vector, after which the bacteria acquired
the ability to produce protegenin A, confirming that the enzymes in the cluster can function
within E. coli. To examine in detail how polyyne formation occurs, we optimized the inductionof proD expression alone and then co-expressed proD and proA and examined the progressionof the acylation of ProD, an ACP, by SDS-PAGE, western blotting, and LCMS. Expression

and acylation of ProD were confirmed. We are currently expressing the remaining modified
enzymes and investigating the sequence of unsaturated fatty acids bound to ProD.
ACP
HOS

O

ACP
OS

ProB

HOO

ProD

ProC

ProE

ProA SH

ProE

ProG

LigaseDesaturase/Acetylenase

O

Thioesterase/Hydrolase

Octadecanoic AcidProtegenin AFig 1. Proposed biosynthetic pathway for protegein A

2. Introduction of fatty acy-ACP ligase using C20 fatty acids as substrates
Previous studies have shown that bacterial polyynes are biosynthesized from C16 fatty acids
with a triyne structure, and from C18 fatty acids with a tetrayne structure. Therefore, if thesubstrate specificity of fatty acyl-ACP ligase (also known as fatty acyl-AMP ligase) in thebiosynthetic gene cluster could be changed from C18 to C20, it may be possible to createbacterial polyynes with a pentagonal structure.
Although several reactions that use C20 fatty acids as substrates in secondary metabolism
in bacteria are known, we focused on a specific fatty acyl AMP ligase from a gram-negative
bacterium and examined its expression in the Cab57 strain. However, the expected conversion
was not observed, indicating that additional enzymes should be investigated.
3. Investigation of bacterial polyynes detection by induced Raman analysis
Because of their unique structure, polyyne compounds exhibit Raman absorption atpositions that are different from those of other biological components in Raman spectroscopy.
Utilizing this structure, we expected that bacterial polyynes in cells could be detected
selectively and with high sensitivity.
To verify this, we performed induced Raman microscopy analysis and found that the P.
protegens Cab57 strain produced relatively high levels of protegenin A. We constructed anexperimental system using these cells as model, but we were unable to achieve the expected
sensitivity and were only able to detect protegenin A using a combination of integration counts
and image processing. The reason for this is that, although protegenin A was produced at ahigher level than other bacterial polyynes, the amount was not sufficient to be detected byinduction Raman microscopy.
Conclusion
Knowledge of the protegenin A biosynthetic pathway has progressed; however, themechanism by which desaturases/acetylenases are involved in the formation of the polyyne
moiety remains unclear. The use of induced Raman spectroscopy to efficiently search forbacterial species producing bacterial polyynes was considered, but the sensitivity was not ashigh as expected; therefore, other methods need to be considered.
References1) Ross, C., Scherlach, K., Kloss, F. and Hertweck, C. (2014) The molecular basis of conjugatedpolyyne biosynthesis in phytopathogenic bacteria. Angew. Chem. Int. Ed. 53: 7794-7798.
2) Murata, K., Suenaga, M., and Kai, K. (2022) Genome mining discovery of protegenins A–D,
bacterial polyynes involved in the antioomycete and biocontrol activities of Pseudomonas
protegens. ACS Chem. Biol. 17: 3313-3320.
3) Kai, K., Sogame, M., Sakurai, F., Nasu, N., Fujita, M. (2018) Collimonins A–D, unstable
polyynes with antifungal or pigmentation activities from the fungus-feeding bacterium
Collimonas fungivorans Ter331. Org. Lett. 20: 3536-3540.