Wiebe M G, Robson G D, Shuster J, Trinci A P
School of Biological Sciences, 1.800 Stopford Bldg., University of Manchester, Manchester, M13 9PT, UK.
Biotechnol Bioeng. 2001 Apr 20;73(2):146-56. doi: 10.1002/bit.1046.
Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates.
剧毒镰刀菌JeRS 325是A3/5菌株的转化体，该菌株在尖孢镰刀菌类胰蛋白酶启动子的控制下产生黑曲霉葡糖淀粉酶（GAM）。在以NaNO₃或(NH₄)₂SO₄作为氮源的葡萄糖限制恒化器培养物中研究了JeRS 325的进化情况。从NaNO₃恒化器中分离出13个分支更多的突变体和4个比亲本菌株分支更稀疏的突变体。在该恒化器中检测到的高度分支突变体并未取代分支稀疏的群体。从NaNO₃恒化器中分离出的突变体与先前从以(NH₄)₂SO₄为氮源的剧毒镰刀菌A3/5葡萄糖限制恒化器培养物中分离出的代表性菌株互补，但它们之间几乎没有互补作用。相比之下，从(NH₄)₂SO₄恒化器培养物中分离出的一个高度分支突变体取代了分支稀疏的菌丝体群体。从NaNO₃或(NH₄)₂SO₄恒化器中分离出的突变体均未产生与JeRS 325一样多的GAM。Southern印迹分析表明，除一个突变体外，所有突变体均丢失了葡糖淀粉酶和乙酰胺酶（选择标记）基因的拷贝。然而，特定GAM的产生不一定与观察到的glaA基因丢失程度相关。此外，10个突变体失去了以乙酰胺作为唯一氮源生长的能力，尽管它们保留了amdS基因的拷贝。在竞争研究中，不能利用乙酰胺的突变体取代了能利用乙酰胺的突变体。JeRS 325中存在外源DNA导致比生长速率降低（与A3/5相比），但外源DNA的存在并未阻止该菌株的进化或阻止分离出生长速率提高的突变体。
