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CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase. [ABSTRACT FROM AUTHOR]

Alanine aminotransferase (AlaAT) catalyses the reversible transfer of an amino group from glutamate to pyruvate to form 2-oxoglutarate and alanine. The regulation of AlaAT in several plant species has been studied in response to low-oxygen stress, light and nitrogen application. In this study, induction of Arabidopsis AlaAT1 and AlaAT2 during hypoxia was observed at the transcriptional level, and an increase in enzyme activity was detected in hypoxically treated roots. In addition, the tissue-specific expression of AlaAT1 and AlaAT2 was analysed using promoter:GUS fusions. The GUS staining patterns indicated that both AlaAT genes are expressed predominantly in vascular tissues. We manipulated AlaAT expression to determine the relative importance of this enzyme in low-oxygen stress tolerance and nitrogen metabolism. This was done by analysing T-DNA mutants and over-expressing barley AlaAT in Arabidopsis. The AlaAT1 knockout mutant ( alaat1-1) showed a dramatic reduction in AlaAT activity, suggesting that AlaAT1 is the major AlaAT isozyme in Arabidopsis. Over-expression of barley AlaAT significantly increased the AlaAT activity in the transgenic plants. These plants were analysed for metabolic changes over a period of hypoxic stress and during their subsequent recovery. The results showed that alaat1-1 plants accumulate more alanine than wild-type plants during the early phase of hypoxia, and the decline in accumulated alanine was delayed in the alaat1-1 line during the post-hypoxia recovery period. When alanine was supplied as the nitrogen source, alaat1-1 plants utilized alanine less efficiently than wild-type plants did. These results indicate that the primary role of AlaAT1 is to break down alanine when it is in excess. Therefore, AlaAT appears to be crucial for the rapid conversion of alanine to pyruvate during recovery from low-oxygen stress. [ABSTRACT FROM AUTHOR]

Expression of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex (PDH) complex) and activity of the complex were investigated in cells grown under several conditions. Comparable amounts of PDA1 mRNA and E1α subunit were detected in cells from batch and chemostat cultures grown on various carbon sources, showing constitutive expression of PDA1 gene is only regulated to a small extent, if at all, by the GCN4 mechanism. Contrary to the constant levels of PDA1 mRNA and E1α subunit in both batch and chemostat cultures, the specific activity of the PDH complex varied with the culture conditions. The activity of the PDH complex in chemostat cultures was approximately two-threefold higher than in batch cultures grown on the same carbon sources. Overproduction of the E1α subunit in batch cultures resulted in a two-threefold increase in the activity of the PDH complex. Taken together, these results indicate that the activity of the PDH complex is mainly regulated by post-translational modification of the E1α subunit. Expression of PDA1 and acitivity of the PDH complex were also detected in cultures grown under conditions where no physiological significance of the PDH complex was expected, i.e. during anaerobic growth on glucose or acrobic growth on ethanol. Apparently, the switch from oxidative growth to fermentation occurs without much effect on the PDH complex. These observations suggest that the PDH complex has an alternative function besides sugar catabolism. [ABSTRACT FROM AUTHOR]

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 µkal/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC. the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxta and would be activated by small changes in pyruvate concentration. [ABSTRACT FROM AUTHOR]