Как выбрать гостиницу для кошек
14 декабря, 2021
Сегодня каждый, кто собирается в отпуск и не знает, с кем оставить своего котика или кошку, может во[...]
Ethanologenic Biocatalysts KO11 and LY01
Engineering Scheme
The development of ethanologenic E. coli has included a combination of directed engineering and metabolic evolution; the overall scheme is summarized in Fig. 3. The Z. mobilis homoethanol pathway (PET operon) was introduced
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Fig. 3 Ethanologenic E. coli design summary. Our design of ethanologenic KO11, LY01, and LY168 has featured a combination of directed engineering, as indicated on the left of each arrow, and metabolic evolution, as indicated in {}. For clarity, only major directed metabolic mutations are indicated. KO11 was constructed from E. coli W by the introduction of pdc and adhB from Z. mobilis and deletion of frd to prevent succinate production. The Z. mobilis genes, along with adhE and ackA, were removed during conversion of KO11 to SZ110. Lactic-acid producing SZ110 was re-engineered to ethanologenic LY168 by removal of ldhA, reinsertion of the Z. mobilis genes and restoration of the native pflB. Please see the text for complete details on each strain |
into E. coli in plasmids and these derivatives produced ethanol as the main fermentation product [13-16]. The PET operon was stably integrated into the chromosome at the pfl locus along with an antibiotic resistance marker; spontaneous mutants exhibiting high ADH activity and high antibiotic resistance were selected to ensure high PET activity. Side reactions that drain carbon away from ethanol were eliminated either by mutation (frd — succinate) or physiologically (differences in Km for pyruvate) (Fig. 2). The resulting strain KO11 produced ethanol at a yield of 95% in complex media [17]. While it was originally reported that KO11 was derived from E coli B, it has recently been discovered that E coli W is the parental strain (Jarboe and Ingram, unpublished).
While the rate of ethanol production by KO11 is as high as yeast, the ethanol tolerance is lower than the commercially employed yeast strains. In complex media, KO11 shows a complete lack of growth in the presence of 35 gL-1 ethanol and only 10% survival from 30 s of exposure to 100 gL-1 ethanol [18]. Using strain KO11 as a starting point, mutant strains with significantly increased ethanol tolerance were isolated. The 3-month metabolic evolution consisted of alternating periods of selection in liquid media for increased ethanol tolerance and selection on solid media for increased ethanol production. The final product of this evolution, strain LY01, was able to grow in the presence of 50 gL-1 ethanol and had greater than 80% survival from 30 s of exposure to 100 g L-1 ethanol. The method of metabolic evolution used to derive LY01 from KO11 has proved to be successful and has been applied to the design of other ethanologenic biocatalysts and to the production of other commodity products, as described in Sect. 5.