Optimization of culture conditions for the sophoridine-producing new fungal species Achaetomium sophor strain HY17 isolated from seeds of Sophora alopecuroides

doi:10.11686/cyxb2024444

Abstract

Abstract:

The aim of this study was to screen for endophytic fungal strains of Achaetomium sophora infecting Sophora alopecuroides that produce sophoridine alkaloid， classify and identify them， and then identify in-vitro culture conditions that optimize the alkaloid production of the endophyte fungus. The overall goal was to improve alkaloid yield and obtain excellent strains for culture. Fifty strains of A. sophora isolated from healthy S. alopecuroides seeds were screened using alkaloid precipitation， acid dye colorimetry， and high-performance liquid chromatography analyses. The strains were identified through morphological and molecular analyses. Subsequently， a series of methods including the single factor test， Plackett-Burman （PB） test， steepest climb test， and response surface test （Box-Behnken test） were used to investigate the effects of medium composition （medium type， carbon and nitrogen sources）， culture conditions （incubation time and pH）， and precursors and inducers on the alkali yield of selected fungal strains. From the results of these analyses， the optimal alkali-producing medium， culture conditions， and precursors for sophoridine alkaloid production were determined. The fungal strain HY17 producing sophoridine at high levels was identified as a new species， Achaetomium sophora. The optimal culture conditions for A. sophora HY17 were determined to be SDY liquid medium with an initial pH of 6 and a culture period of 8 days， with maize flour as the carbon source and casein as the nitrogen source. Tyrosine， L-lysine， L-piperidinic acid， and phenylalanine had significant effects on sophoridine production by strain HY17 during culture. The addition of L-lysine （1.044 g·L^-1）， L-piperidinic acid （0.081 g·L^-1）， and phenylalanine （1.995 g·L^-1） resulted in a maximum alkaloid yield of 1.369 mg·g^-1 mycelium under these optimized conditions， representing a remarkable increase of 61.95% compared with that obtained under the original culture conditions. A. sophora HY17 produced sophoridine at a high and stable rate. Optimization of the culture conditions resulted in a notable increase in alkaloid production. The results of this study offer a novel approach for sophoridine production through in-vitro culture of the fungal endophyte， A. sophora strain HY17 isolated from S. alopecuroides.

Key words: Sophora alopecuroides HY17, screening and identification, sophoridine, optimization of fermentation conditions, response surface methodology （RSM）

Zhi-yun TANG, Wen-kai WANG, Guan-lan LIU, Pei-wen GU. Optimization of culture conditions for the sophoridine-producing new fungal species Achaetomium sophor strain HY17 isolated from seeds of Sophora alopecuroides[J]. Acta Prataculturae Sinica, 2025, 34(10): 151-163.

Figures/Tables 13

References 41

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目标基因Target gene	引物名称Primer	引物序列Primer sequences （5′-3′）	退火温度Annealing temperature （℃）	参考文献Reference
ITS	ITS4	TCCTCCGCTTATTGATATGC	55	［23］
ITS	ITS5	GGAAGTAAAAGTCGTAACAAGG	55	［23］
LSU	LSU1Fd	GRATCAGGTAGGRATACCCG	55	［24］
LSU	LR5	TCCTGAGGGAAACTTCG	55	［24］
TUB2	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	58	［25］
TUB2	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	58	［25］

目标基因Target gene	引物名称Primer	引物序列Primer sequences （5′-3′）	退火温度Annealing temperature （℃）	参考文献Reference
ITS	ITS4	TCCTCCGCTTATTGATATGC	55	［23］
ITS	ITS5	GGAAGTAAAAGTCGTAACAAGG	55	［23］
LSU	LSU1Fd	GRATCAGGTAGGRATACCCG	55	［24］
LSU	LR5	TCCTGAGGGAAACTTCG	55	［24］
TUB2	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	58	［25］
TUB2	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	58	［25］

变量 Codes	因素 Factors	水平Levels
变量 Codes	因素 Factors	-1	1
X₁	L-赖氨酸 L-lysine	1.00	2.00
X₂	L-哌啶酸 L-piperidinic acids	0.08	0.16
X₃	α-酮戊二酸 α-ketoglutaric acid	0.01	0.02
X₄	苯丙氨酸 Phenylalanine	2.00	5.00
X₅	丙酮酸 Pyruvate	1.00	4.00
X₆	水杨酸 Salicylate	1.00	2.00
X₇	茉莉酸甲酯Methyl jasmonate	0.10	0.50
X₈	种子提取物 Seed extract	2.00	5.00

变量 Codes	因素 Factors	水平Levels
变量 Codes	因素 Factors	-1	1
X₁	L-赖氨酸 L-lysine	1.00	2.00
X₂	L-哌啶酸 L-piperidinic acids	0.08	0.16
X₃	α-酮戊二酸 α-ketoglutaric acid	0.01	0.02
X₄	苯丙氨酸 Phenylalanine	2.00	5.00
X₅	丙酮酸 Pyruvate	1.00	4.00
X₆	水杨酸 Salicylate	1.00	2.00
X₇	茉莉酸甲酯Methyl jasmonate	0.10	0.50
X₈	种子提取物 Seed extract	2.00	5.00

项目 Item	因子 Factors	水平 Levels
项目 Item	因子 Factors	-1	0	1
X₁	L-赖氨酸 L-lysine	0.50	1.00	1.50
X₂	L-哌啶酸 L-piperidinic acids	0.04	0.08	0.12
X₄	苯丙氨酸 Phenylalanine	0.50	2.00	3.50