Genetic and metabolic engineering

Recent advances in molecular biology techniques, analytical methods and mathematical tools have led to a growing interest in using metabolic engineering to redirect metabolic fluxes for industrial and medical purposes. Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology (Stephanopoulos, 1999). This multidisciplinary field draws principles from chemical engineering, biochemistry, molecular and cell biology, and computational sciences. The aim of this article is to give an overview of the various strategies and tools available for metabolic engineers and to review some of the recent work that has been conducted in our laboratories in the metabolic engineering area

Metabolic engineering and flux analysis of Escherichia coli

The advances in molecular biology, genome sequencing and mathematical tools have led to growing interest in metabolic engineering, the goal of which is to redirect metabolic fluxes for industrial and medical purposes. Escherichia coli was chosen as the model system for several reasons: the ease of genetic manipulation, the wealth of genetic information, its capacity for rapid growth, and the availability of inexpensive and standardized cultivation protocols. The effects of precise perturbation in the central metabolic pathway of E. coli with an emphasis on the competition at the pyruvate node was investigated using recombinant DNA technology to create the changes and metabolic flux analysis to quantify the results. First, the effects of competition at the pyruvate node was studied by two modifications of central metabolism, the heterologous expression of the Bacillus subtilis acetolactate synthase (ALS) and the removal of the major acetate production pathway. This study was primarily a continuation of the application of metabolic engineering strategies to increase recombinant protein production through acetate reduction. The next study involved the effect of an additional mutation at the nuo gene encoding an NADH dehydrogenase recently mapped near the genes (ack4-pta ) controlling the acetate production pathway. In the earlier studies involving strains deficient in ackA-pta gene, lactate instead of acetate or ethanol was the major fermentation product. A natural extension of this project was to examine the effects of deficiency and overexpression of the lactate dehydrogenase (LDH) enzyme responsible for lactate production pathway. Next, the role of intermediate pool levels, specifically pyruvate, on redistribution of carbon flux and partitioning at the pyruvate node was examined. Sensitivity or flux control coefficients were calculated to gain further insight into dynamics of the overall reaction network. Finally, the effects of different enzyme properties on metabolic patterns were explored through the integration and expression of ALS from Klebsiella pneumoniae and a comparison with the B. subtilis ALS

Genetic and metabolic engineering

Recent advances in molecular biology techniques, analytical methods and mathematical tools have led to a growing interest in using metabolic engineering to redirect metabolic fluxes for industrial and medical purposes. Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology (Stephanopoulos, 1999). This multidisciplinary field draws principles from chemical engineering, biochemistry, molecular and cell biology, and computational sciences. The aim of this article is to give an overview of the various strategies and tools available for metabolic engineers and to review some of the recent work that has been conducted in our laboratories in the metabolic engineering area

Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells · h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae