Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

doi:10.1038/srep32208

Cited by 41 publications

(36 citation statements)

References 35 publications

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“…All strains and plasmids used in this study are listed in Table 1 . The xllp01 strain was renamed in this experiment and the genome background of the strain was the same as the HD-1 strain in the publication [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

Genetic engineering of Escherichia coli to improve L-phenylalanine production

Liu

Ding

et al. 2018

BMC Biotechnol

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BackgroundL-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli.ResultsWe inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01.ConclusionThe metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0418-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Genetic engineering of Escherichia coli to improve L-phenylalanine production

Liu

Ding

et al. 2018

BMC Biotechnol

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“…The PAL enzyme is also a major contributor to the enzymatic synthesis of "industrially relevant biomolecules," namely pure L-phenylalanine (L-Phe), L-phenylalanine methyl ester (L-PM), and para-hydroxycinnamic acid (p-HCA) [28]. L-Phe, being an essential amino acid, is used in food formulations, feed for livestock, dietary supplements, and nutraceuticals [29]. L-PM is a precursor in the production of the sweetener aspartame [28], and p-HCA has utility in the cosmetic, health, and flavoring industries [30].…”

Section: Enzymesmentioning

confidence: 99%

The Oleaginous Red YeastRhodotorula/Rhodosporidium: A Factory for Industrial Bioproducts

Lyman¹,

Urbin²,

Strout³

et al. 2019

Yeasts in Biotechnology

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Rhodotorula genus, amended in 2015, is polyphyletic and contains Rhodotorula species that grow as single-cell yeast (monomorphic) and reproduce asexually via budding/fission (anamorphic); it also contains Rhodosporidium species that reproduce sexually (teleomorphic) and alternate between a yeast phase and dikaryotic filamentous phase (dimorphic). Several species of these "red yeast" produce industrial bioproducts, namely biofuel feedstocks, carotenoids, enzymes, and biosurfactants. This chapter highlights the biotechnology areas that Rhodotorula/ Rhodosporidium contributes to and the future market value of those industries. The primary yeast species to be discussed include Rhodosporidium toruloides, Rhodotorula glutinis, Rhodosporidium diobovatum, Rhodosporidium kratochvilovae, Rhodotorula graminis, Rhodotorula babjevae, and Rhodotorula taiwanensis.

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“…Extensive work has been done on the production of PHE, as it is a valuable amino acid with diverse applications in food- and pharma-industry. In the most notable recent work the optimum concentrations of six enzymes ( aroK, aroL, aroA, aroC, pheA , and tyrB ) along the shikimate pathway were adjusted in E. coli allowing an impressive final titer of 62.47 g/L to be reached (Ding et al, 2016 ), with a yield of roughly half of theoretical maximum and the third highest productivity of an aromatic product. This is even more impressive when considering how far downstream of the main pathway the compound is derived and underlines the importance for identification of bottlenecks in order to balance the pathway and tailor specific strain construction strategies.…”

Section: Results—strain Engineering Strategies For Production Of Arommentioning

confidence: 99%

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

Averesch

Krömer

2018

Front. Bioeng. Biotechnol.

163

125

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The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

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Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

Cited by 41 publications

References 35 publications

Genetic engineering of Escherichia coli to improve L-phenylalanine production

Genetic engineering of Escherichia coli to improve L-phenylalanine production

The Oleaginous Red YeastRhodotorula/Rhodosporidium: A Factory for Industrial Bioproducts

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

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