Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing
            <i>Escherichia coli</i>
            Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

Koma, Daisuke; Kishida, Takahiro; Yoshida, Eisuke; Ohashi, Hiroyuki; Yamanaka, Hayato; Moriyoshi, Kunihiko; Nagamori, Eiji; Ohmoto, Takashi

doi:10.1128/aem.00525-20

Cited by 21 publications

(25 citation statements)

References 64 publications

(98 reference statements)

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“…PEP is also consumed and converted to pyruvate in glycolysis [ 53 ]. In order to conserve PEP, we deleted the pykA and pykF genes encoding pyruvate kinases, and the triple deletion strain BTR16 produced 535.87 mg/L of l -phenylalanine, a 36.90% increase than strain BTR13, in consistent with previous reports [ 54 , 55 ]. l -Phenylalanine biosynthesis is negatively regulated by the repressor TyrR targeting the transcriptional expression of the aroG and aroF genes encoding 3-deoxy-arabino-heptulonate 7-phosphate (DAHP) synthase isoenzymes and the aroL gene encoding shikimate kinase [ 56 ].…”

Section: Resultssupporting

confidence: 88%

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

et al. 2021

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Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

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Section: Resultssupporting

confidence: 88%

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

et al. 2021

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“…Also, the chorismate mutase is affected by a feedback inhibition by Phe or Tyr and, accordingly, tyrA fbr and pheA fbr mutated proteins have been created ( Turnbull and Morrison, 1990 ; Vincent et al, 2002 ; Lütke-Eversloh and Stephanopoulos, 2005 ; Zhou et al, 2010 ; Cao et al, 2016 ). Nowadays, the co-overexpression of both feedback-inhibition resistant E. coli enzymes is common to gain aromatics production, mostly in E. coli and yeast ( Báez-Viveros et al, 2004 ; Zhang et al, 2014a ; Rodriguez et al, 2014 , 2015 ; Wang et al, 2019 ; Koma et al, 2020 ), as well as in cyanobacteria ( Ni et al, 2018 ; Brey et al, 2020 ; Deshpande, Vue and Morgan, 2020 ). Additionally, in the literature another reaction has been reported as high flux controlling step ( Luttik et al, 2008 ; Takai A, Nishi R, Joe Y, 2009; Darmawi et al, 2012 ; Rodriguez et al, 2015 ; Xu et al, 2019 ), i.e.…”

Section: Discussionmentioning

confidence: 99%

Combining metabolite doping and metabolic engineering to improve 2-phenylethanol production by engineered cyanobacteria

Usai

Cordara

et al. 2022

Front. Bioeng. Biotechnol.

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2-Phenylethanol (2-PE) is a rose-scented aromatic compound, with broad application in cosmetic, pharmaceutical, food and beverage industries. Many plants naturally synthesize 2-PE via Shikimate Pathway, but its extraction is expensive and low-yielding. Consequently, most 2-PE derives from chemical synthesis, which employs petroleum as feedstock and generates unwanted by products and health issues. The need for “green” processes and the increasing public demand for natural products are pushing biotechnological production systems as promising alternatives. So far, several microorganisms have been investigated and engineered for 2-PE biosynthesis, but a few studies have focused on autotrophic microorganisms. Among them, the prokaryotic cyanobacteria can represent ideal microbial factories thanks to their ability to photosynthetically convert CO2 into valuable compounds, their minimal nutritional requirements, high photosynthetic rate and the availability of genetic and bioinformatics tools. An engineered strain of Synechococcus elongatus PCC 7942 for 2-PE production, i.e., p120, was previously published elsewhere. The strain p120 expresses four heterologous genes for the complete 2-PE synthesis pathway. Here, we developed a combined approach of metabolite doping and metabolic engineering to improve the 2-PE production kinetics of the Synechococcus elongatus PCC 7942 p120 strain. Firstly, the growth and 2-PE productivity performances of the p120 recombinant strain were analyzed to highlight potential metabolic constraints. By implementing a BG11 medium doped with L-phenylalanine, we covered the metabolic burden to which the p120 strain is strongly subjected, when the 2-PE pathway expression is induced. Additionally, we further boosted the carbon flow into the Shikimate Pathway by overexpressing the native Shikimate Kinase in the Synechococcus elongatus PCC 7942 p120 strain (i.e., 2PE_aroK). The combination of these different approaches led to a 2-PE yield of 300 mg/gDW and a maximum 2-PE titer of 285 mg/L, 2.4-fold higher than that reported in literature for the p120 recombinant strain and, to our knowledge, the highest recorded for photosynthetic microorganisms, in photoautotrophic growth condition. Finally, this work provides the basis for further optimization of the process aimed at increasing 2-PE productivity and concentration, and could offer new insights about the use of cyanobacteria as appealing microbial cell factories for the synthesis of aromatic compounds.

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“…In S. cerevisiae , E4P and PEP were not abundant and were pathway bottlenecks based on the results of carbon tracing and 13 C metabolic flux analysis [ 31 ] . Many studies have revealed that the overexpression of transketolase (TktA) and phosphoenolpyruvate synthase ( PpsA) effectively raised the intracellular pool of E4P and PEP [ 32 , 33 ]. In yeast, the 3-deoxyD-arabino-heptulosonate-7-phosphate (DAHP) synthase ( ARO4 / ARO3 ) catalyzes the first step to condense PEP and E4P to form DAHP.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Saccharomyces cerevisiae for high-level production of gastrodin from glucose

Yin

Zhuang

et al. 2020

Microb Cell Fact

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Background The natural phenolic glycoside gastrodin is the major bioactive ingredient in the well-known Chinese herb Tianma and is widely used as a neuroprotective medicine in the clinic. Microbial production from sustainable resources is a promising method to replace plant extraction and chemical synthesis which were currently used in industrial gastrodin production. Saccharomyces cerevisiae is considered as an attractive host to produce natural plant products used in the food and pharmaceutical fields. In this work, we intended to explore the potential of S. cerevisiae as the host for high-level production of gastrodin from glucose. Results Here, we first identified the plant-derived glucosyltransferase AsUGT to convert 4-hydroxybenzyl alcohol to gastrodin with high catalytic efficiency in yeast. Then, we engineered de novo production of gastrodin by overexpressing codon-optimized AsUGTsyn, the carboxylic acid reductase gene CARsyn from Nocardia species, the phosphopantetheinyl transferase gene PPTcg-1syn from Corynebacterium glutamicum, the chorismate pyruvate-lyase gene UbiCsyn from Escherichia coli, and the mutant ARO4K229L. Finally, we achieved an improved product titer by a chromosomal multiple-copy integration strategy and enhancement of metabolic flux toward the aglycon 4-hydroxybenzyl alcohol. The best optimized strain produced 2.1 g/L gastrodin in mineral medium with glucose as the sole carbon source by flask fermentation, which was 175 times higher than that of the original gastrodin-producing strain. Conclusions The de novo high-level production of gastrodin was first achieved. Instead of chemical synthesis or plants extraction, our work provides an alternative strategy for the industrial production of gastrodin by microbial fermentation from a sustainable resource.

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Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

Cited by 21 publications

References 64 publications

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Combining metabolite doping and metabolic engineering to improve 2-phenylethanol production by engineered cyanobacteria

Metabolic engineering of Saccharomyces cerevisiae for high-level production of gastrodin from glucose

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