Optimizing Production and Evaluating Biosynthesis in Situ of an Herbicidal Compound

Optimizing Production and Evaluating Biosynthesis in situ of an Herbicidal Compound, Mevalocidin, from Coniolariella sp.

Vincent P. Sica,†,*Mario Figueroa,†,§,*Huzefa A. Raja,†Tamam El-Elimat,†Blaise A. Darveaux,‡Cedric J. Pearce,‡ and Nicholas H. Oberlies†*

† Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, P.O. Box 26170, Greensboro, North Carolina 27402, United States

§ Facultad de Química, Universidad Nacional Autónoma de México, Mexico, DF 04510, Mexico

‡Mycosynthetix, Inc., 505 Meadowlands Dr. #103, Hillsborough, NC 27278, United States

Figure S2. Phylogram of the most likely tree (-lnL = 1461.94) from a PHYML analysis of 21 sequences based on a portion of the D1/D2 divergent domains of the 28SrDNA (396 bp). Numbers refer to PHYML bootstrap support values based on 1000 replicates. MSX56446 nested within the Coniolariella clade with the type species, C. gamsii, highlighted in gray.

Figure S3. General procedure for the extraction, fractionation, and initial chromatography utilized for the isolation of mevalocidin and/or methylidene mevalonolactone. The percentages refer to the approximate amount of mevalocidin in each fraction as monitored by 1H NMR. The dashed route is an addition to the isolation procedure to convert mevalocidin to methylidene mevalonolactone.

Figure S4. 1H NMR spectrum of mevalocidin (1; top) [400 MHz, D2O]. 1H NMR spectrum of methylidene mevalonolactone (2; bottom) [400 MHZ, CDCl3].

Figure S5. The base peak chromatograms (top) of both mevalocidin (1) and methylidene mevalonolactone (2) for their accurate (±5 ppm) molecular ion peaks of 161.0808 and 143.0703, respectively. The accurate mass spectra (bottom) for both compounds with labeled ions including [M+H]+, [M+Na]+, [M-H2O+H]+, and [M-2H2O+H]+ where appropriate.

Figure S6. (A) The extracted ion chromatogram (XIC) of m/z 161.0808 (±5 ppm) for the mevalocidin (1) standard. (B) The XIC of m/z 143.0703 (±5 ppm) for the methylidene mevalonolactone (2) standard. The XIC for (C) m/z 161.0808 (±5 ppm) and (D) m/z 143.0703 (±5 ppm) of the direct fungal culture extraction via the droplet–LMJ–SSP. The peak at 3.54 min in chromatogram D is a result of the loss of water on mevalocidin due to in source fragmentation.

Figure S1. Phylogram of the most likely tree (-lnL = 1766.4) from a PHYML analysis of 18 sequences based on complete ITS rDNA (467 bp). Numbers refer to PHYML bootstrap support values based on 1000 replicates. MSX strains used in the present study are nested within the Coniolariella clade with the type species, C. gamsii. The Coniolariella clade is highlighted in gray. Bar indicates nucleotide substitutions per site.
Figure S2. Phylogram of the most likely tree (-lnL = 1461.94) from a PHYML analysis of 21 sequences based on a portion of the D1/D2 divergent domains of the 28SrDNA (396 bp). Numbers refer to PHYML bootstrap support values based on 1000 replicates. MSX56446 nested within the Coniolariella clade with the type species, C. gamsii, highlighted in gray.
Note:Gerwick et al. [13] sequenced DA092917_KF698738 and DA056446_KF698737 for D2 region of the 28S, which is a small portion about 300 bp. We compared, MSX56446, which we sequenced for the complete ITS region and partial D1/D2 regions as a single contig using primers ITS4-LR3. We then compared the D2 region of MSX56446 with both DA092917 and DA056446. MSX56446 was <1% different compared to the D2 region of DA092917, but was 16% different compared to D2 region of DA056446. Additionally, the ITS region of both MSX92917 and MSX56446 was identical.
Figure S3. General procedure for the extraction, fractionation, and initial chromatography utilized for the isolation of mevalocidin and/or methylidene mevalonolactone. The percentages refer to the approximate amount of mevalocidin in each fraction as monitored by 1H NMR. The dashed route is an addition to the isolation procedure to convert mevalocidin to methylidene mevalonolactone.
Mevalocidin
Methylidene mevalonolactone
Figure S4.1H NMR spectrum of mevalocidin (1; top) [400 MHz, D2O].1H NMR spectrum of methylidene mevalonolactone (2; bottom) [400 MHZ, CDCl3].
Mevalocidin / Mevalonolactone
Figure S5.The base peak chromatograms (top) of both mevalocidin (1) and methylidene mevalonolactone (2) for their accurate (±5 ppm) molecular ion peaks of 161.0808 and 143.0703, respectively. The accurate mass spectra (bottom) for both compounds with labeled ions including [M+H]+, [M+Na]+, [M-H2O+H]+, and [M-2H2O+H]+ where appropriate.
Figure S6. (A) The extracted ion chromatogram (XIC) of m/z 161.0808 (±5 ppm) for the mevalocidin (1) standard. (B) The XIC of m/z 143.0703 (±5 ppm) for the methylidene mevalonolactone (2) standard. The XIC for (C) m/z 161.0808 (±5 ppm) and (D) m/z 143.0703 (±5 ppm) of the direct fungal culture extraction via the droplet–LMJ–SSP. The peak at 3.54 min in chromatogram D is a result of the loss of water on mevalocidin due to in source fragmentation.