摘要
研究鉴定了All0769为鱼腥藻PCC 7120中乙酰辅酶A合成酶,通过CRISPR/Cpf1系统敲除鱼腥藻PCC 7120中的乙酰辅酶A合成酶(由all0769编码),探究了乙酰辅酶A合成酶在异形胞分化中的调控机制。结果所示:All0769能在体外反应中催化乙酰辅酶A的生成。在供氮环境下,敲除all0769会影响藻细胞生长速率。而无论环境中是否存在化合氮,Δall0769突变株的乙酰辅酶A和α-酮戊二酸含量均显著减少。在供氮环境下,Δall0769突变株中检测到(26.17±1.55)nmol/mg protein的乙酰辅酶A,而在野生型中检测出(43.04±1.09)nmol/mg的乙酰辅酶A。Δall0769突变株的α-酮戊二酸[(1.41±0.24)nmol/mg protein]低于野生型的α-酮戊二酸[(2.13±0.05)nmol/mg protein]。在缺氮环境下,Δall0769突变株中检测到(10.00±2.81)nmol/mg protein的乙酰辅酶A,而在野生型中检测出(29.82±4.04)nmol/mg protein的乙酰辅酶A。Δall0769突变株的α-酮戊二酸含量[(1.48±0.35)nmol/mg protein]低于野生型的α-酮戊二酸[(2.74±0.33)nmol/mg protein]。此外,Δall0769突变株异形胞分化频率(7.12%)显著低于野生型异形胞分化频率(9.22%)。综上所述:文章鉴定了All0769是鱼腥藻PCC 7120中的乙酰辅酶A合成酶。在鱼腥藻PCC 7120中,缺失乙酰辅酶A合成酶(All0769)会减少乙酰辅酶A的含量,进而下调α-酮戊二酸含量使异形胞频率降低。
In this study,we identified All0769 as acetyl coenzyme A(acetyl-CoA)synthetase in Anabaena sp.PCC 7120,and investigated the molecular mechanism of acetyl coenzyme A syntheses(encoded by all0769)in the regulation of heterocyst differentiation in Anabaena sp.PCC 7120 by disrupting all0769 with a CRISPR/Cpf1 system.Our results demonstrated that All0769 could catalyze the formation of acetyl-CoA in vitro and loss of the all0769 was found to affect the growth of Anabaena sp.PCC 7120 cells under combined nitrogen.The content of acetyl-CoA andα-ketoglutaric acid were significantly decreased inΔall0769 strain compared to that of WT under combined nitrogen and nitrogen deficiency conditions.We detected(26.17±1.55)nmol/mg protein acetyl-CoA inΔall0769 strain,whereas(43.04±1.09)nmol/mg protein acetyl-CoA was obtained in wild-type strain under combined nitrogen.For the content ofα-ketoglutaric acid,Δall0769 strain exhibited a decreaseα-ketoglutaric acid[(1.41±0.24)nmol/mg protein]relative to that of WT[(2.13±0.05)nmol/mg protein]under combined nitrogen.Upon deprivation of combined nitrogen,we measured(10.00±2.81)nmol/mg protein acetyl-CoA inΔall0769 strain,while(29.82±4.04)nmol/mg protein acetyl-CoA was received in wild-type strain.Additionally,the content ofα-ketoglutaric acid decreased in theΔall0769 strain[(1.48±0.35)nmol/mg protein],compared with the wild-type strain[(2.74±0.33)nmol/mg protein]after the removal of combined nitrogen.Furthermore,the heterocyst formation was measured andΔall0769 strain shows a significant difference in heterocyst frequency(7.12%)compared with the wild-type(9.22%)at 24h.In conclusion,we identified All0769 as acetyl coenzyme A(acetyl-CoA)synthetase in Anabaena sp.PCC 7120.And All0769 deletion led to impaired growth,content of acetyl-CoA,α-ketoglutaric acid and heterocyst frequency in Anabaena sp.PCC 7120.
作者
陈柏楠
余胜超
袁丽
杨明坤
葛峰
CHEN Bai-Nan;YU Sheng-Chao;YUAN Li;YANG Ming-Kun;GE Feng(College of Fisheries and Life Science,Dalian Ocean University,Dalian 116000,China;State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;University of Chinese Academy of Sciences,Beijing 100049,China)
出处
《水生生物学报》
CAS
CSCD
北大核心
2023年第7期1025-1035,共11页
Acta Hydrobiologica Sinica
基金
国家重点研发计划(2020YFA0907400)资助。
