Metabolic engineering of the shikimate pathway

#The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 416272009540SeqList.txt, date recorded: Aug. 30, 2012, size: 1 KB).

#The present disclosure relates to engineered microorganisms that produce amino acids and amino acid intermediates. In particular, the disclosure relates to recombinant nucleic acids encoding operons that increase production of aromatic amino acids and the aromatic amino acid intermediate shikimate; microorganisms with increased production of aromatic amino acids and the aromatic amino acid intermediate shikimate; and methods related to the production of aromatic amino acids, the aromatic amino acid intermediate shikimate, and commodity chemicals derived therefrom.

#The traditional approach for improving microbial production of natural products, such as amino acids, antibiotics, etc., includes altering key structural or regulatory genes of the biosynthetic pathway followed by measuring the amount of desired product that is produced. Each change then reveals the presence or absence of a bottleneck and, based on those results, the next gene is deleted or overexpressed, and the cycle repeats until product titers/yields can no longer be improved substantially. Although this step-wise approach can yield improvements in flux through these pathways, it is a tedious and time-consuming strategy, given that metabolic pathways tend to be well balanced and rarely does a single change increase flux dramatically. Indeed, some bottlenecks will not be revealed until others are relieved. This process typically leads to the identification of local yield maxima, but not the global optimal yield.

#The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

#FIG. 1A depicts the biosynthetic pathway of L-tyrosine (TYR) in E. coli from glucose. X5P corresponds to xylulose 5-phosphate; F6P corresponds to fructose 6-phosphate; PYR corresponds to pyruvate; PEP corresponds to phosphoenolpyruvate; E4P corresponds to erythrose 4-phosphate; G3P corresponds to glyceraldehyde 3-phosphate; DAHP corresponds to 3-deoxy-D-arobino-heptulosonate 7-phosphate; DHQ corresponds to 3-dehydroquinate; DHS corresponds to 3-dehydroshikimate; SHIK corresponds to shikimate; S3P corresponds to shikimate 3-phosphate; EPSP corresponds to 5-enolpyruvoylshikimate 3-phosphate; CHA corresponds to chorismate; PPA corresponds to prephenate; HPP corresponds to 4-hydroxyphenlypyruvate; L-Glu, glutamic acid; and α-KG, α-ketoglutarate. The enzymes (in bold) are PpsA, phosphoenolpyruvate synthase; TktA corresponds to transketolase A; AroG corresponds to DAHP synthase; AroB corresponds to DHQ synthase; AroD corresponds to DHQ dehydratase; YdiB corresponds to quinate/shikimate dehydrogenase; AroE corresponds to shikimate dehydrogenase; AroK/L corresponds to shikimate kinase I/II; AroA corresponds to EPSP synthase; AroC corresponds to chorismate synthase; TyrA corresponds to the chorismate mutase/prephenate dehydrogenase bifunctional enzyme; and TyrB corresponds to aromatic amino acid aminotransferase. QUIN and GA are side products quinate and gallic acid, respectively. QUIN is formed by YdiB from DHQ (18), while GA formed by AroE from DHS (18, 30). Dashed lines indicate where feedback inhibitions occur. Allosteric regulation of AroG and TyrA were removed by employing their respective feedback resistant mutants, AroG*[D146N] and TyrA*[M53I;A354V], respectively. FIG. 1B depicts the structure of the initial modules, S0 and Y0, for production of shikimate and L-tyrosine, respectively. Open blocks indicate the origin of replication; shaded block arrows indicate the genes; and the angled arrows indicate the promoters. For each operon, the genes are placed in the reverse order relative to the reaction pathway. FIG. 1C depicts SRM analysis of the protein production levels from S0 and Y0 in Strain A, when induced or uninduced with IPTG.

#FIG. 2A depicts stepwise improvements of the shikimate module by changing the origin of replication from pSC101 to pBBR1, and the promoter from PLtetO-1 to Plac-UV5 (S1), followed by codon optimization of aroB (S2), substitution of ydiB with aroE (S3), and insertion of a second promoter PLtetO-1 5′ of aroG* (S4). In S5, a combination of the rrnB terminator T1 (symbol T) and Ptrc was used to substitute PLtetO-1 5′ of aroG*, which resulted in significant reduction in protein and shikimate production. FIG. 2B depicts SRM results indicating the relative levels of TktA through YdiB/AroE as the consequence of the various modifications to the shikimate module.

#Definitions

#Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present disclosure, the following terms are defined.

#As used herein, “operon” refers to a configuration of separate genes that are transcribed in tandem as a single messenger RNA. For example, the genes encoding the enzymes necessary for a host cell to synthesize shikimate from phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) may be configured together so that they are transcribed together as a single messenger RNA. An“operon” of the present disclosure may include a group of two or more genes under the control of a single regulatory sequence, such as a promoter. Additionally, the transcribed genes of an“operon” of the present disclosure may be either translated together or each gene may be translated separately.