Abstract
Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli). Twelve ldhA genes with sequentially shortened chromosomal upstream regions were cloned in an ldhA deletion, E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA). The varied ldhA upstream regions were further analyzed using program NNPP2.2 (Neural Network Promoter Prediction 2.2) to predict the possible promoter regions. Two-phase fermentations (aerobic growth and oxygen-limited production) of these strains showed that shortening the ldhA upstream sequence from 291 to 106 bp successively reduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase. Simultaneously, oxygen-limited lactate productivity was increased during the second phase. The putative promoter downstream of the −96 site of ldhA could function as a transcriptional promoter or regulator. B0013-080C/pTH-rrnB-ldhA8, with the 72-bp upstream segment of ldhA, could be grown at a high rate and achieve a high oxygen-limited lactate productivity of 1.09 g g−1 h−1. No transcriptional promoting region was apparent downstream of the −61 site of ldhA. We identified the latent transcription regions in the ldhA upstream sequence, which will help to understand regulation of ldhA expression.
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This work was partly funded by the Sino-South Africa Cooperation Program 2009DFA31300 and the National Natural Science Foundation of China no. 21006039.
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10295_2012_1116_MOESM2_ESM.tif
Representative experiments (conducted in duplicate) showing the production of lactate during the two phase fermentation by B0013-080C/pTH-rrnB-ldhA0 to B0013-080C/pTH-rrnB-ldhA11 as well as B0013-070 and B0013-080C. The dotted lines (marked by 0 to 13) indicate the time when the cultures of the strains were shifted from the aerobic growth to the oxygen-limited production phase (TIFF 1075 kb)
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Zhou, L., Shen, W., Niu, DD. et al. Fine tuning the transcription of ldhA for d-lactate production. J Ind Microbiol Biotechnol 39, 1209–1217 (2012). https://doi.org/10.1007/s10295-012-1116-y
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DOI: https://doi.org/10.1007/s10295-012-1116-y