The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies o...The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of thepMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes fromtwo individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recoveredtransgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5%respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicatingthat most transgenes were faithfully inherited during the four generations of reproduction. The other five classes weredifferent from the original configuration in both molecular weight and restriction map, indicating that a few transgeneshad undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types ofaberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carpβ-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermalkeratin 14, respectively.展开更多
L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engine...L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.展开更多
基金Supported by the National Natural Science Foundation of China(30970262,31000778)the 863 Program(2011AA100904)the Major S&T Projects on the Cultivation of New Varieties of Genetically Modified Organisms(Grant 2009ZX08009-120B)~~
基金supported bythe Major State Basic Research Development Program ofChina (No. 2004CB117406 and G2000016109)the National Natural Science Foundation of China (No.90208024 and 39823003).
文摘The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgeniccommon carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of thepMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes fromtwo individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recoveredtransgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5%respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicatingthat most transgenes were faithfully inherited during the four generations of reproduction. The other five classes weredifferent from the original configuration in both molecular weight and restriction map, indicating that a few transgeneshad undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types ofaberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carpβ-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermalkeratin 14, respectively.
基金supported by grants from Ministry of Science and Technology of China (Grant Nos.2008ZX09401-05 and 2010ZX09401-403)the National Natural Science Foundation of China (Grant No. 31100074)Chinese Academy of Sciences (Grant No. XBXA-2011-009)
文摘L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.
