Tien M, Myer S B
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Appl Environ Microbiol. 1990 Aug;56(8):2540-4. doi: 10.1128/aem.56.8.2540-2544.1990.
Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.
木材降解真菌黄孢原毛平革菌的木质素分解系统的合成是在二次代谢过程中由氮、碳或硫饥饿诱导产生的。我们在此描述一种筛选突变体的策略，这些突变体在营养丰富的条件下（一次代谢期间）具有木质素分解能力（木质素→二氧化碳）并过量产生木质素降解酶（木质素酶）。该策略基于使用赖氨酸与木质素模型化合物的加合物。这种加合物的木质素酶依赖性氧化会释放出游离赖氨酸，从而补充赖氨酸营养缺陷型对赖氨酸的需求。因此，通过紫外线照射使赖氨酸营养缺陷型发生诱变，并将存活菌株接种到含有该加合物和高氨氮的培养基上。通过该程序分离出了四个过量产生木质素酶同工酶的突变体。对其中一个突变体PSBL-1的进一步表征表明，产生的主要同工酶是H1（pI = 4.7）和H2（pI = 4.4）。通过藜芦醇氧化法测定，PSBL-1的木质素酶活性在第5天达到峰值，超过1000 U·升-1。突变体PSBL-1也能够将[14C]木质素降解为14CO2，这表明完整的木质素分解系统不受调控。
