Recombinant strains

Western blotting of above mentioned recombinant strains shows a clear increase of rhamnogalacturonase production compared to the wild strain. 

To check the activity, fermentation broths of the recombinant strains were added to the isolated substrate resulting in a small increase of the extinction. 

Therefore this method can not be used to compare rhamnogalacturonase activity presoit in the wild strain and the recombinant strains. 

Dionex analysis of hydrolysis products of RG by these recombinant strains (see figure 4 ) shows the formation of various oligomers as published by Schols d al.(1992). 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Overhage, J., Steinbuechel, A. and Priefert, H. (2006) Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167. Journal of Biotechnology, 125, 369-376. 

Mayer, A.F., Hellmuth, K., Schlieker, H. et al. (1999) An expression system matures a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenulapolymorpha. Biotechnology and Bioengineering, 63 (3), 373-381. 

Type III-PHA synthase is represented by the enzyme of Chromatium vino-sum and is encoded by phaECCv. It consists of the two different subunits PhaCCv and PhaECv exhibiting molecular weights of 39,730 and 40,525 Da, respectively [23]. The native PHA synthase, as isolated from recombinant strains of E. coli, exhibited a molecular weight of approximately 390 and 400 + 20 kDa as revealed in our laboratory [54] or 360 + 50 kDa but also 520 + 50 kDa as revealed in another laboratory [55]. The lower molecular weights for the holoenzyme are... 

There are only very few PHA synthases which can incorporate 3HASCL as well as 3HAmcl into the accumulated PHAs. Examples are the PHA synthases of T. pfennigii and Aeromonas caviae, which synthesize, for example, copolyesters of 3HB, 3HHx plus 3HO [26], or of 3HB plus 3HHx [59], from fatty acids, respectively. Recently it was found that the type I-PHASCL synthase of R. eutropha also conferred the incorporation of 3HHx [60] or of 3HO plus 3HD [61] into PHAs in addition to 3HB to certain recombinant strains of enterobacteria. 

PHAs consisting of 4HB are other examples which can be synthesized from simple, unrelated carbon sources. Recombinant strains of E. coli expressing phaCRe and orfZ from Clostridium kluyveri synthesized poly(3HB-co-4HB) or even poly(4HB) homopolyester if 4-hydroxybutyrate was used as carbon source [90]. When additional succinate degradation genes from C. kluyveri were expressed in the recombinant E. coli, poly(3HB-co-4HB) with a low content of 4HB was synthesized from glucose [118]. 

It is worth mentioning that metabolic engineering of E. coli recently provided recombinant strains which synthesized PHAMCL from gluconate. For this, beside phaC2Po or phaClPa> the thioesterase I from E. coli (TesA) [128] or the acyl-ACP thioesterase from Umbellularia californica [129], respectively, were expressed in E. coli. However, the amounts of PHAMCL accumulated in the cells were rather low, and this artificial pathway was not very efficient. 

Much effort has been made to use recombinant strains of E. coli, which expressed the PHA biosynthesis genes from R. eutropha, or other combinations of a suitable host organism and foreign PHA biosynthesis genes for high cell density cultivation and production of bulk amounts of PHAs. The interested reader is referred to a different chapter of this book [13] and the references cited therein in which these systems are described in detail. 

The recombinant strain expressed the Staphylococcus nuclease directed to the periplasm without affecting PHA production. During downstream processing, the viscosity of the recombinant lysate was significantly reduced to facilitate the subsequent purification steps. 

Watanabe, K. Noda, K. Ohta, Y., and Maruhashi, K., Desulfurization of hght gas oil by a novel recombinant strain from Pseudomonas aeruginosa. Biotechnology Letters, 2002. 24(11) pp. 897-903. 

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