Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Recruitment of heterologous proteins enables extension of existing pathways to obtain new chemical products, alter posttranslational protein processing, and degrade recalcitrant wastes. Although some of the experimental and mathematical tools required for rational metabolic engineering are available, complex cellular responses to genetic perturbations can complicate predictive design.

https://science.sciencemag.org/content/sci/252/5013/1668.full.pdf

Toward a science of metabolic engineering

http://compbio.korea.ac.kr/wiki/images/8/83/Mmbr.1996.expression.pdf

Strategies for achieving high-level expression of genes in Escherichia coli.

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Methods and compositions for cellular and metabolic engineering

http://dbkgroup.org/Papers/davey_kell_micrev96.pdf

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

New method for generating deletions and gene replacements in Escherichia coli.

We desired the development of a commercializable process for the production of the amino acid L-phenylalanine, which has uses in the manufacture of aspartame and in parenteral nutrition. In order to approach the problem, we set performance targets for our envisioned process based upon the technical and scientific constraints on phenylalanine biosynthesis and upon the commercial constraints on the value of the product. One mole of phenylalanine is synthesized from two moles of glucose and one mole of ammonia in common bacterial systems. This establishes minimum raw material costs. We wished to develop an organism that could produce high concentrations of phenylalanine with relatively few by-products (including cell mass) by efficiently using raw materials and capital equipment (fermentors and downstream processing equipment). In order to do this, we pursued a systematic and thorough strategy covering organism selection, optimization of biosynthetic capacity, and development of fermentation and recovery processes.

Genetic Engineering of Metabolic Pathways Applied to the Production of Phenylalanine

An Escherichia coli chromosomal DNA segment is engineered for controllable excision and loss from the E. coli cell population. The DNA segment has a gene determining a function that is lost in the absence of that chromosomal segment. Specifically, the segment includes a pair of lambdoid phage att sites positioned at opposite ends thereof, and a gene encoding the function. The E. coli cell includes DNA encoding lambdoid phage int and xis, positioned for transcription under control of an external stimulus. By excising the chromosomal DNA segment once a desired cell population is achieved, cell growth and diversion of intermediates from a desired fermentation product are diminished or avoided.

Controlled gene excision

A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

Controllable alteration of cell genotype in bacterial cultures using an excision vector.

Integrating a gene in a bacterial chromosome and controlling excision of the gene from the chromosome in response to an external stimulus, uses DNA comprising:
 a) a vector that is incapable of replicating autonomously in the host and that includes the gene lor a site for the insertion of the gene) and an attachment site corresponding to an attachment site on the host chromosome; b) DNA capable of causing the integration of vector a) in the bacterial chromosome and DNA capable of causing the excision of that vector therefrom; and c) DNA capable of effecting control of excision of vector a) from the bacterial chromosome in response to an externally controlled stimulus.

Ramaswamy Balakrishnan

Papers

Genetic Engineering of Metabolic Pathways Applied to the Production of Phenylalanine

Controlled gene excision

A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

Controllable alteration of cell genotype in bacterial cultures using an excision vector.

Integration of a gene into a chromosome and controllable excision therefrom