Effects of Simulated Mutational Biosynthetic Regulation on the Secondary Metabolites of Aspergillus terreus C23-3

doi:10.13560/j.cnki.biotech.bull.1985.2024-0110

Abstract

Abstract:

【Objective】Butyrolactone I is a small molecular natural product of Aspergillus terreus with versatile bioactivities, thus the exploration of its structural diversity extension is significant. A marine-derived A. terreus strain C23-3 featuring producing butyrolactone I was used as the base strain to study the effects of butyrolactone I precursor analogs and precursor synthase inhibitors on its secondary metabolism.【Method】The strain was statically fermented under chemically regulative conditions established by seawater potato liquid medium supplied with three precursor small molecules and three 4-hydroxyphenylpyruvate synthase inhibitors. The high-performance liquid chromatography（HPLC）, HPLC -ion trap mass spectrometry（MS）, and MS based molecular networking together with database mining were used to further analyze the yields and diversity of secondary metabolites.【Result】The most of the precursor analogs and synthase inhibitors suppressed the synthesis of butyrolactone I to different extents. Moreover, the precursor analog 3,4-dihydroxyphenylpyruvate at a high dose, the combination of the two 4-hydroxyphenylpyruvate synthase inhibitors and the combination of these three demonstrated remarkable inhibitory effects. In addition, the global synthesis for sorts of secondary metabolites including butenoids, territrems, lovastatins, etc., was downregulated when butyrolactone I was inhibited for production. However, the yield of a cyclopeptide was upregulated by more than ten folds under this situation, which was supposed to be a stress response to the synthetic suppression of butyrolactone I as a quorum sensing molecule and inducer of global transcriptive factor lae A. The detailed mechanism deserves further investigation.【Conclusion】The present study manifests that 4-hydroxyphenylpyruvate is the precursor of butyrolactone I and reveals that 3,4-dihydroxyphenylpyruvate and 4-hydroxyphenylpyruvate synthase inhibitors can be used as small molecule tools to suppress the production of butyrolactone I, which may be applied in further study on simulated mutational biosynthesis to generate diverse butyrolactone derivatives and induce the production of cyclopeptide.

Key words: marine fungus, chemical regulation, simulated mutational biosynthesis, butyrolactone, precursor analog, synthase inhibitor, LC-MS/MS, molecular network

MA Xiao-xiang, MA Ze-yuan, LIU Ya-yue, ZHOU Long-jian, HE Yi-fan, ZHANG Yi. Effects of Simulated Mutational Biosynthetic Regulation on the Secondary Metabolites of Aspergillus terreus C23-3[J]. Biotechnology Bulletin, 2024, 40(8): 275-287.

Figures/Tables 8

Fig. 1 Structures of the precursor-like small molecular inducers and enzyme inhibitors used in this study

Fig. 2 Extracellular, intracellular, and total contents of butyrolactone I of strain C23-3 at different culture time A: Standard curve used to calculate the content of butyrolactone I in the extracts. B-D: Extracellular, intracellular, and total contents of butyrolactone I, the incubation time is calculated from the time after inoculation. *, **, ***, **** refers to the difference between the other days and the first day, which are P values < 0.05, 0.01, 0.001, and 0.000 1, respectively. Ordinary one-way ANOVA multiple comparisons were used for difference analysis between groups

Fig. 3 Butyrolactone I contents（A）and total extract mass（B）of strain C23-3 under the effect of different enzyme inhibitors or inducers ADA: Enzyme inhibitor 1,3-adamantane diacetic acid; NTSP: enzyme inhibitor N-（p-toluenesulfonyl）-L-phenylalanine; PS: enzyme inhibitor phenelzine sulfate; HT: inducer 3-hydroxy-L-tyrosine; DHPPA: inducer 3,4-dihydroxy-p-phenylpyruvate; Far: inducer farnesol; DMSO: the DMSO control group; blank: the blank group. The number after the abbreviations indicate the concentrations, and the unit is mmol/L. *, **, *** refer to the differences between the other groups and blank, which were P values < 0.05, 0.01, and 0.001, respectively. Ordinary one-way ANOVA multiple comparisons were used for difference analysis between groups

Fig. 4 LC-MS/MS-PDA chromatogram of Aspergillus terreus C23-3 extracts under different cultural conditions A: BPC chromatogram; B: PDA chromatogram; C: UV chromatogram of each compound

Fig. 5 Molecular network and its partial enlarged diagram of the metabolites of strain C23-3 under different cultural conditions based on MS2 relationships A-C：Enlarged images of metabolites clusters of butyrolactone I, territrem B and lovastatin congeners. Compounds 1, 3, 6, 7: Butyrolactone I，territrem B，lovastatin，and lovastatin hydroxy acid. Compounds 2, 4, 5, 8, 9：Possible derivatives of known compounds，i.e.,[M（butyrolactone I isomer）+ H]+,[M（butyrolactone I-2H）+ H]+,[M（butyrolactone I isomer + O）+ H]+,[M（butyrolactone I - H2O）+ H]+,[M（territrem B-O）+ H]+,[M（territrem B - CH2）+ H]+,[M（lovastatin - H2O）+ H]+,[M（lovastatin - O）+ H]+，and[M（lovastatin + 2H）+ H]+，respectively. The nodes in the molecular network are marked in 7 colors, , sequentially representing the 7 sample sources of compounds: blank group, control group with solvent DMSO; groups with the addition of 3,4-dihydroxyphenylpyruvate（0.05, 10 mmol/L）, N-（p-toluenesulfonyl）-L-phenylalanine（2.1 mmol/L）+ phenelzine sulfate（0.15 mmol/L）, 3,4-dihydroxyphenylpyruvate（0.05 mmol/L）+ N-（p-toluenesulfonyl）-L-phenylalanine（2.1 mmol/L）+ phenelzine sulfate（0.15 mmol/L）, 3,4-dihydroxyphenylpyruvate（10 mmol/L）+N-（p-toluenesulfonyl）-L-phenylalanine（2.1 mmol/L）+ phenelzine sulfate（0.15 mmol/L）respectively

Fig. 6 MS1 and MS2 spectra of compound 20

Fig. 7 Butyrolactone I content variation of strain C23-3 with the addition of enzyme inhibitors and inducers Blank: Blank group; DMSO: the solvent DMSO control group; NTSP: N-（p-toluenesulfonyl）-L-phenylalanine, PS: phenelzine sulfate. # P<0.05, DMSO control group vs blank group; * P<0.05, experimental group vs DMSO Control group; ** P<0.01, the sample group vs the control group, *** P<0.001, the sample group vs the control group

Table 1 Variation of compounds 1-20's contents（integrated peak areas）under different cultural conditions compared to CK

化合物序号Compound No.	化合物注释 Compound note	不同培养条件下的产物产量变化幅度 Changes in product yield under different cultivation conditions/%
化合物序号Compound No.	化合物注释 Compound note	DMSO group	0.05 mmol/L 3,4-DHPPA group	10 mmol/L 3,4-DHPPA group	2.1 mmol/L NTSP + 0.15 mmol/L PS group	2.1 mmol/L NTSP + 0.15 mM PS + 0.05 mmol/L 3,4-DHPPA group	2.1 mmol/L NTSP + 0.15 mM PS + 10 mmol/L 3,4-DHPPA group
1	丁内酯I Butyrolactone I	↓62.50	↓32.71	↓90.06	↓74.04	↓68.76	↓92.19
2	丁内酯I脱水产物 Butyrolactone I dehydrated product	↓71.53	↓34.28	↓78.36	↓100.00	↓67.42	↓100.00
3	土震素B Territrem B	↑4.40	↓26.45	↑9.58	↓73.15	↓78.94	↓57.47
4	土震素B脱氧产物 Territrem B deoxylation products	↑65.52	↓100.00	↑38.07	↓80.67	↓84.58	↓100.00
5	土震素B降碳产物 Territrem B carbon reduction products	↑50.57	↓56.52	↓33.36	↓100.00	↓100.00	↓86.79
土震素B类化合物总产量 Total production of territrem B compound		↑5.12	↓27.10	↑9.36	↓73.42	↓79.14	↓57.95
6	洛伐他汀Lovastatin	↑6.60	↓28.74	↓29.12	↑86.14	↑46.16	↓100.00
7	洛伐他汀羟酸 Lovastatin hydroxy acid	↓91.26	↓85.64	↓88.04	↓62.17	↓83.70	↓85.34
8	洛伐他汀脱水产物 Lovastatin dehydrated products	↓34.19	↑10.05	↓57.14	↑50.70	↑15.97	↓98.61
9	洛伐他脱氧产物 Lovastatin deoxygenation products	↓64.96	↓100.00	↓100.00	↑95.58	↑12.68	↓100.00
洛伐他汀类化合物总产量 Total production of lovastatin compounds		↓43.48	↓48.02	↓60.20	↑16.07	↓15.59	↓93.41
10	N/A	↓25.36	↓12.73	↓16.03	↓6.82	↓17.87	↓34.47
11	Aspernolide C	↑30.46	↓20.96	↑24.90	↓79.36	↓80.96	↓55.49
12	Isochromophilone VI脱氢产物 Isochromophilone VI dehydrogenation product	↓1.59	↓29.31	↓6.46	↓71.79	↓75.73	↓58.11
13	Epi-aszonaleni A	↑11.30	↓24.58	↓9.34	↓66.76	↓70.52	↓40.02
14	N/A	↓28.33	↑0.59	↓54.44	↓24.12	↓53.74	↓90.22
15	Asterriquinone CT3	↓21.98	↓31.05	↑34.49	↓81.50	↓84.16	↓11.36
16	Perinadine A	↓43.49	↑38.06	↓66.44	↓15.69	↓46.11	↓88.34
17	12a-dehydroxyisoterreulactone A or Terreulactone C	↓67.24	↓26.58	↓85.57	↑6.36	↓22.89	↓100.00
18	Teraspiridole B	↑8.11	↑14.79	↑148.27	↓13.45	↓38.63	↑94.77
19	N/A	↓26.37	↑1.29	↑13.45	↑50.55	↓3.21	↑19.44
20	N/A	↑111.26	↓81.63	↑1208.42	↑570.07	↑181.73	↑1399.99

Table 1 Variation of compounds 1-20's contents（integrated peak areas）under different cultural conditions compared to CK

化合物序号Compound No.	化合物注释 Compound note	不同培养条件下的产物产量变化幅度 Changes in product yield under different cultivation conditions/%
化合物序号Compound No.	化合物注释 Compound note	DMSO group	0.05 mmol/L 3,4-DHPPA group	10 mmol/L 3,4-DHPPA group	2.1 mmol/L NTSP + 0.15 mmol/L PS group	2.1 mmol/L NTSP + 0.15 mM PS + 0.05 mmol/L 3,4-DHPPA group	2.1 mmol/L NTSP + 0.15 mM PS + 10 mmol/L 3,4-DHPPA group
1	丁内酯I Butyrolactone I	↓62.50	↓32.71	↓90.06	↓74.04	↓68.76	↓92.19
2	丁内酯I脱水产物 Butyrolactone I dehydrated product	↓71.53	↓34.28	↓78.36	↓100.00	↓67.42	↓100.00
3	土震素B Territrem B	↑4.40	↓26.45	↑9.58	↓73.15	↓78.94	↓57.47
4	土震素B脱氧产物 Territrem B deoxylation products	↑65.52	↓100.00	↑38.07	↓80.67	↓84.58	↓100.00
5	土震素B降碳产物 Territrem B carbon reduction products	↑50.57	↓56.52	↓33.36	↓100.00	↓100.00	↓86.79
土震素B类化合物总产量 Total production of territrem B compound		↑5.12	↓27.10	↑9.36	↓73.42	↓79.14	↓57.95
6	洛伐他汀Lovastatin	↑6.60	↓28.74	↓29.12	↑86.14	↑46.16	↓100.00
7	洛伐他汀羟酸 Lovastatin hydroxy acid	↓91.26	↓85.64	↓88.04	↓62.17	↓83.70	↓85.34
8	洛伐他汀脱水产物 Lovastatin dehydrated products	↓34.19	↑10.05	↓57.14	↑50.70	↑15.97	↓98.61
9	洛伐他脱氧产物 Lovastatin deoxygenation products	↓64.96	↓100.00	↓100.00	↑95.58	↑12.68	↓100.00
洛伐他汀类化合物总产量 Total production of lovastatin compounds		↓43.48	↓48.02	↓60.20	↑16.07	↓15.59	↓93.41
10	N/A	↓25.36	↓12.73	↓16.03	↓6.82	↓17.87	↓34.47
11	Aspernolide C	↑30.46	↓20.96	↑24.90	↓79.36	↓80.96	↓55.49
12	Isochromophilone VI脱氢产物 Isochromophilone VI dehydrogenation product	↓1.59	↓29.31	↓6.46	↓71.79	↓75.73	↓58.11
13	Epi-aszonaleni A	↑11.30	↓24.58	↓9.34	↓66.76	↓70.52	↓40.02
14	N/A	↓28.33	↑0.59	↓54.44	↓24.12	↓53.74	↓90.22
15	Asterriquinone CT3	↓21.98	↓31.05	↑34.49	↓81.50	↓84.16	↓11.36
16	Perinadine A	↓43.49	↑38.06	↓66.44	↓15.69	↓46.11	↓88.34
17	12a-dehydroxyisoterreulactone A or Terreulactone C	↓67.24	↓26.58	↓85.57	↑6.36	↓22.89	↓100.00
18	Teraspiridole B	↑8.11	↑14.79	↑148.27	↓13.45	↓38.63	↑94.77
19	N/A	↓26.37	↑1.29	↑13.45	↑50.55	↓3.21	↑19.44
20	N/A	↑111.26	↓81.63	↑1208.42	↑570.07	↑181.73	↑1399.99

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