Nieboer M, Vis A J, Witholt B
Institute of Biotechnology, ETH Hönggerberg (HPT), Zürich, Switzerland.
Eur J Biochem. 1996 Oct 15;241(2):691-6. doi: 10.1111/j.1432-1033.1996.00691.x.
When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10-15% of the total protein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110[pGEc47] affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction, Mol. Microbiol. 8, 1039-1051]. In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes. Using [35S]methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min after addition of inducer, when up to 15% of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorporation of 14C-labeled acetate and 33P-labeled phosphoric acid into phospholipids. Despite a negative effect of the inducer on the growth rate of W3110[pGEc47], net phospholipid synthesis was significantly enhanced as a result of the expression of alkB. Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB. Induction did not increase 33P incorporation into lipids in the control recombinant alk+ strain which lacked alkB. Simultaneous with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic acid (C16:1) into 9,10-methylene hexadecanoic acid (C17:ocyc) was reduced in the alk+ recombinant. Overall, these data show that the production of a foreign membrane protein in E. coli can engender a response of the phospholipid-synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more toxic to the cells. Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.
当由alkBFGHJKL和alkST基因组成的食油假单胞菌（Pseudomonas oleovorans）alk系统在大肠杆菌W3110中表达时，可观察到宿主磷脂代谢发生显著变化。细胞质膜蛋白烷烃羟化酶（AlkB）似乎起主要作用，在该菌株中，其合成量可达总蛋白的10% - 15%[Nieboer, M., Kingma, J. & Witholt, B. (1993)食油假单胞菌的烷烃氧化系统：大肠杆菌W3110[pGEc47]中alk基因的诱导影响膜生物合成，并导致烷烃羟化酶在不同细胞质膜亚组分中过表达，《分子微生物学》8, 1039 - 1051]。在本文中，我们通过研究这些过程的动力学，探讨了膜蛋白合成与磷脂和脂肪酸合成之间的联系。使用[35S]甲硫氨酸标记表明，添加诱导剂后30 - 60分钟内AlkB的诱导达到最大值，此时所有新合成蛋白中高达15%是AlkB。通过测量14C标记的乙酸盐和33P标记的磷酸掺入磷脂的情况来跟踪磷脂合成。尽管诱导剂对W3110[pGEc47]的生长速率有负面影响，但由于alkB的表达，净磷脂合成显著增强。alkB的诱导对所有三种主要磷脂的合成均有类似程度的刺激。在缺乏alkB的对照重组alk +菌株中，诱导并未增加33P掺入脂质。与AlkB合成同时，alk +重组体中不饱和9 - 十六碳烯酸（C16:1）向9,10 - 亚甲基十六烷酸（C17:ocyc）的转化减少。总体而言，这些数据表明，在大肠杆菌中产生外源膜蛋白可引发宿主磷脂合成系统的反应。在没有这种反应的情况下，alk系统的诱导对细胞的毒性会大得多。显然，增加的磷脂合成在使AlkB过量产生菌株能够生长方面起重要作用。
