We are combining techniques of metabolic engineering with of directed enzyme evolution for the production of useful novel compounds and materials in recombinant cells. To create these cell factories, we make use of the tremendous gene toolbox created in the course of the ongoing (and future) genome sequencing projects. We assemble genes from different organisms into pathways and create new catalytic functions by in vitro evolution. In this project, we apply this approach to the creation of new pathways for the production of a class of complex chemical compounds in E. coli - to the biosynthesis of unnatural porphyrin structures. Porphyrins are highly valuable chemicals with many applications in medicine, chemistry and material sciences. The complexity of many porphyrin structures makes their chemical synthesis often difficult or impossible and typically results in low yields. A biocatalytical approach using metabolically engineered cells, however, could produce specifically functionalized porphyrins in good yields. The biosynthetically produced functionalized porphyrins can then serve as scaffold for chemical modification and/or exhibit specific redox portentials useful for chemical catalysis or incorporation in heme-containing biocatalalysts, such as P450's.

Our first goal was the successful overproduction of porphyrins in E. coli. Presently, no efforts have been undertaken to engineer E. coli cells specifically for porphyrin overproduction. Next, methods for qualitative and quantitative analysis of porphyrin structures synthesized by recombinant E. coli cells needed to be established and methods suitable for high-throughput screening of E. coli libraries developed. To create new porphyrin structures in E. coli, we use combinatorial and in vitro evolution techniques to create in project I expanded porphyrins with more than four pyrrole rings and in project II different unnatural functionalized porpyrins.

During the past year, we have cloned 17 heme biosynthetic genes from different microorganisms. The genes were initially cloned into our constitutive expression vector pUCmod and subsequently several biosynthetic genes were cloned in a modular fashion on two plasmids pACmod and pBBR1, which can be stably propagated together with pUCmod in E. coli, for combinatorial assembly of different porphyrin pathways in E. coli. Because porphyrin overproduction in an engineered microbial host has not yet been described, toxicity of the porphyrins synthesized in E. coli was a major concern. However, after transforming E. coli with our modular heme gene expression plasmids we found that E. coli is capable of synthesizing very large quantities of porphyrins. In fact, the obtained production levels are so high that the produced porphyrins are excreted into the culture medium where they precipitate. HPLC, HPLC-MS and TLC methods for the detection, identification and quantification of porphyrin compounds have also been established. To produce novel porphyrin structures, we have begun to create libraries of hemF and hemH and transform those libraries into recombinant E. coli that synthesize different porphyrin precursor structures.

Because a first library of randomly mutated hemC did not yield a variant pathway for the synthesis of expanded porphyrins, we changed our strategy towards a semi-rational approach. Reasoning that increased flexibility of the protein structure would be necessary to accommodate an expanded polypyrrole chain, we created random insertions of 2-3 amino acids into loop regions. Surprisingly, the majority of variants of the loop insertion libraries retained their wild type activity. Based on these findings, we physically dissected hemC at these positions such that we express an a and ? fragment of the protein in order to investigate whether these fragments are able to complement each other functionally. In a second series of plasmids we expressed only the a-fragment. To test for functional hemC activity we created a hemC, hemD deletion strain of E. coli into which we transformed these constructs. E. coli cells lacking the ability to produce heme grow only very slow, if at all, under aerobic conditions. Growth is restored in this strain upon complementation with functional genes for hemC and hemD. We also integrated the gene for the heme uptake receptor hasR from Serratia marcescens (kindly provided by C. Wandersmann, Paris) into the E. coli chromosome and thus obtained an E. coli strain without a wild type porphyrin background and capable of aerobic growth when supplemented with hemin in the culture medium. Both properties are desirable when screening libraries for novel porphyrin structures, in particular if the new compounds would inhibit wild type heme biosynthesis. With most of the hemC dissections and surprisingly with most of the hemC truncations as well, heme biosynthesis is restored in E. coli. From these results it appears that domain 1 alone, containing the catalytically active arginine residue, apparently can provide a catalytically active scaffold for the synthesis of sufficient amounts of linear tetrapyrroles to enable growth. We are conducting additional studies (in vitro and in vivo) to further confirm that a minimal hemC protein with only one domain left exhibits catalytic activity and investigate the porphyrin products. If we find this to be the case, we will generate libraries of domain 1 and/or domain 2 to screen for new catalytically active scaffolds that would then allow the synthesis of expanded polypyrrols.

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