Development of a Combined Biological and Chemical Process for Production of Industrial Aromatics from Renewable Resources

141

156

ABSTRACTEscherichia coliwas metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) fromCupriavidus necatorwas introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) fromAzospirillum brasilenseand the phenylacetaldehyde dehydrogenase gene (feaB) fromE. coliwere introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereasipdCand the alcohol dehydrogenase gene (adhC) fromLactobacillus breviswere introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases inE. coliencoded by theyqhD,yjgB, andyahKgenes. Cointroduction and cooverexpression of each gene withipdCin the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of theyahKgene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion offeaB,pheA, and/ortyrAgenes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Section: Resultsmentioning

confidence: 97%

mentioning

confidence: 99%

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Koma

Yamanaka

Moriyoshi

et al. 2012

141

156

“…With advances in metabolic engineering and the discovery of novel biosynthetic pathways in plants, aromatic amino acids, which have been important commodities used as animal feeds, food additives, and supplements, can also serve as precursors to a variety of commercially valuable molecules and pharmaceutical drugs (13,42). Recently, several publications used L-tyrosineoverproducing strains of E. coli grown on glucose to produce biopolymer starting materials, such as p-hydroxycinnamic acid and p-hydroxystyrene (39), and drug precursors, such as reticuline, an important intermediate in the biosynthesis of benzylisoquinoline alkaloids (29,40). However, of the three aromatic amino acids derived from the shikimate (SHIK) pathway, the L-tyrosine yield is the lowest, ranging from 0.10 to 0.15 g per g glucose (Table 1).…”

mentioning

confidence: 99%

Modular Engineering of l -Tyrosine Production in Escherichia coli

Juminaga

Baidoo

Redding-Johanson

et al. 2012

247

203

Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/ shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.

“…Apart from its use as a dietary supplement, L-tyrosine also serves as a precursor for 3,4-dihydroxy-L-phenylalanine (L-DOPA, or levodopa), an important drug for the treatment of Parkinson's disease (5). Additionally, L-tyrosine is involved in the synthesis of p-hydroxycinnamic acid and p-hydroxystyrene, both of which serve as starting materials for a variety of novel polymers, adhesives and coatings, pharmaceuticals, biocosmetics, and health and nutrition products (20,23).Most prior work on the microbial production of aromatic amino acids has focused largely on two main goals: (i) alleviating the feedback regulation of the product-forming pathway and (ii) altering central carbon metabolism in order to increase the supply of the two main precursors, erythrose-4-phosphate and phosphoenolpyruvate (4,5,11,24). Although these approaches have certainly led to significant increases in aromatic amino acid production, further gains in yield and productivity may require the modulation of factors that are not directly involved in the biosynthetic pathway or the related precursorforming/utilization reactions.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Melanin-Based High-Throughput Screen for l -Tyrosine Production in Escherichia coli

Santos

Stephanopoulos

2008

Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.Traditional metabolic engineering has often focused on the rational design of metabolic pathways, relying on extensive a priori knowledge of cellular mechanisms in order to redirect metabolite flow, revise metabolic regulation, or introduce new pathways to achieve a particular phenotype (3). In recent years, however, considerable advances in molecular biology and the growing availability of annotated genome sequences have made combinatorial methods of metabolic engineering an increasingly attractive approach for strain improvement. With these search strategies, random, traceable genetic-level perturbations are introduced into a cell to yield a new population of strains with a diverse range of properties. A screen is then implemented in order to probe these mutant libraries for strains exhibiting enhancements in the trait of interest. Although the potential of the combinatorial approach has already been demonstrated for a number of genetic tools, including transposon mutagenesis, genomic complementation, and global transcription machinery engineering (1, 12), each of these examples has dealt with easily accessible phenotypes. In the case of lycopene production in Escherichia coli, desirable clones assumed a red pigmentation and could therefore be selected by visual inspection. For solvent (sodium dodecyl sulfate, ethanol) tolerance in E. coli and Saccharomyces cerevisiae, simple growth competition assays were used to heavily enrich a population with the best performers. For most other systems of interest, however, the widespread use of these combinatorial approaches is hampered by the absence of a high-throughput method for selecting strains with the desired cellular properties.Although L-tyrosine has received far less attention than the other aromatic amino acids, L-tryptophan and L-phenylalanine, it remains a valuable target compound for microbial production. Apart from its use as a dietary supplement, L-tyrosine also serves as a precursor for 3,4-dihydroxy-L-phenylalanine (L-DOPA, or levodopa), an important drug for the treatment of Parkinson's disease (5). Additionally, L-tyrosine is involved in the synthesis of p-hydroxycinnamic acid and p-hydroxystyrene, both of which serve as starting materials for a variety of novel polymers, adhesives and coatings, pharmaceuticals, biocosmetics, and health and nutrition products (20,23).Most prior work o...