Kraak M N, Smits T H, Kessler B, Witholt B
Institute of Biotechnology, ETH Hönggerberg, Zürich, Switzerland.
J Bacteriol. 1997 Aug;179(16):4985-91. doi: 10.1128/jb.179.16.4985-4991.1997.
A functional antibody highly specific for polymerase C1 of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P(alk) promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% (wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h.
制备了一种对食油假单胞菌GPo1的聚合酶C1具有高度特异性的功能性抗体，并将其用于体内实验中测定聚合酶C1的水平。聚合酶C1抗体与食油假单胞菌的聚合酶C2没有交叉反应。在野生型食油假单胞菌GPo1和恶臭假单胞菌KT2442中，相对于总蛋白，分别发现聚合酶含量为0.075%和0.06%。食油假单胞菌GPo1(pGEc405)在其天然启动子的控制下含有额外的聚合酶C1编码基因拷贝，相对于总蛋白，其聚合酶C1含量为0.5%。当通过食油假单胞菌GPo1(pET702, pGEc74)中的P(alk)启动子过表达聚合酶C1编码基因时，聚合酶C1达到细胞总蛋白的10%。当在食油假单胞菌中表达额外的聚合酶C1时，在非氮限制条件下聚(R - 3 - 羟基链烷酸酯)(PHA)的含量显著增加。在这些条件下，食油假单胞菌产生34%(重量/重量)的PHA，而诱导后食油假单胞菌GPo1(pGEc405)的PHA水平可达64%(重量/重量)，食油假单胞菌GPo1(pET702, pGEc74)的PHA水平可达52%(重量/重量)，而未诱导对照的PHA水平为13%。所有含有额外聚合酶C1的重组假单胞菌菌株的PHA组成都有微小变化。与野生型相比，发现3 - 羟基己酸单体含量增加，3 - 羟基辛酸和3 - 羟基癸酸含量减少。开发了两种不同的方法来量化新单体掺入预先存在的PHA颗粒中的速率。在氮限制条件下生长的食油假单胞菌GPo1细胞显示出与生长阶段相关的掺入速率。在稳定期中期发现最高的PHA合成速率为9.5 nmol C8/C6单体/毫克细胞干重(CDW)/分钟，这相当于每千克CDW每小时生产80克PHA的速率。
