The enhancement of microorganisms for chemical modification

利用芳烃的细菌在环境生物修复和特定化学品生产方面越来越受到关注。本研究是与伦敦大学学院合作进行的。本研究的目的是增强微生物对特定化学品的降解和修饰。在联合研究中取得的成果如下.分析了假产碱假单胞菌（Pseudomonaspseudoalcaligenes）KF 707编码联苯转化为苯甲酸的bphABCXD基因的结构和功能。联苯被一种多组分酶氧化成二氢联苯，联苯双加氧酶由四个不同的亚基组成：末端双加氧酶的大小亚基（由bphA 1和bphA 2编码），铁氧还蛋白（bphA 3）和铁氧还蛋白还原酶（bphA 4）。然后通过脱氢酶（bphB）将二氢二醇二氢化为2，3-二羟基联苯基。2，3-二羟基联苯二氧合酶（bphc）是一种将2，3-二羟基联苯转化为 ...更多信息 环间位断裂化合物2-羟基-6-氧代-6-苯基己-2，4-二烯酸，其最终被水解酶（bphD）降解为苯甲酸。利用吉布森等人的方法对恶臭假单胞菌F1的甲苯代谢tod操纵子进行了分析。发现bph和tod操纵子非常相似，不仅在基因组织上，而且在相应基因组分的大小和氨基酸序列上。因此，bphA 1/todC 1、bphA 2/todC 2、bphA 3/todB和bphC/todE之间的氨基酸序列的同一性为53- 65%。利用KF 707的联苯不能利用甲苯。结果表明，将含有todC 1C 2BADE基因的质粒pDTG 351导入到KF 707中，使重组菌株能够在甲苯中生长。更重要的是，仅引入todC 1C 2支持KF 707在甲苯上的生长。使用转座子突变株KF 748（bphB：：Tn 5-B21）和KF 744（bphC：：Tn 5-B21），从各种联苯衍生物和联苯相关化合物产生二氢二醇和二羟基化合物，表明bph编码的酶具有非常宽松的底物特异性。少

Aromatic hydrocarbon utilizing bacteria have become an increased concern in the aspect of environmental bioremediation and production of specific chemicals. The present research has been conducted in a collaboration with University College London. The aim of this research is to enhance microorganisms for degradation and modification of specific chemicals. The results obtained in the joint research are as follows.1. The structure and function of bphABCXD genes of Pseudomonas pseudoalcaligenes KF707 coding for conversion of biphenyl to benzoic acid were analyzed. Biphenyl is oxidized to dihydrodio by a multi-component enzyme, biphenyl dioxygenase which is composed of four different subunits : large and small subunits of terminal dioxygenase (encoded by bphA1 and bphA2), ferredoxin (bphA3) and ferredoxin reductase (bphA4). The dihydrodiol is then dihydrogenated to 2,3-dihydroxybiphneyl by a dehydrogenase (bphB). 2,3-dihydroxybiphenyl dioxgenase (bphc) convert 2,3-dihydroxybiphenyl into th … More e ring-meta-cleavage compound, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, which is eventually degraded to benzoic acid by a hydrolase (bphD).2. A toluene catabolic tod operon of P. putida F1 was analyzed by Gibson's group. It was found that bph and tod operon are very similar, not only in the gene organization, but also the size and amino acid sequences of the corresponding gene components. Thus' identities of amino acid sequences between bphA1/todC1, bphA2/todC2, bphA3/todB, and bphC/todE are 53-65%. Biphenyl utilizing KF707 is not able to utilize toluene. It was demonstrated that introduction of plasmid pDTG351 containing todC1C2BADE genes into the KF707 allowed the recombinant strain to grow on toluene. More importantly, introduction of only todC1C2 supported the growth of the KF707 on toluene.3. Using transposon mutant strains, KF748 (bphB :: Tn5-B21) and KF744 (bphC :: Tn5-B21), dihydrodiol and dihydroxy compounds were produced from various biphenyl derivatives and biphenyl-related compounds, indicating bph-encoded enzymes possess very relaxed substrate specificities. Less