Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

Shen, Yuping; Niu, Fu-Xing; Yan, Zhibo; Fong, Lai San; Huang, Yijia; Liu, Jianzhong

doi:10.3389/fbioe.2020.00407

Cited by 43 publications

(30 citation statements)

References 167 publications

(232 reference statements)

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“…In our previous paper [ 13 ], applying a combinatorial strategy of the directed evolution of pathway enzymes and quorum-sensing-based dynamic regulation of the pathway further improved the production of 4HPAA, which reached 17.39 g/L in 2-L bioreactor fed-batch fermentation. However, the titer of 4HPAA in engineered microorganisms is much lower than that of other aromatic compounds [ 14 ]. Thus, further work is required to increase the production of 4HPAA.…”

Section: Introductionmentioning

confidence: 99%

ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

Shen

Liao

et al. 2021

Biotechnol Biofuels

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Background 4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis. Results We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date. Conclusion This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

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Section: Introductionmentioning

confidence: 99%

ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

Shen

Liao

et al. 2021

Biotechnol Biofuels

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“…Even if all belong to the recombinant protein of Rg PAL/TAL, the K m values for substrates l -Phe and l -Tyr were quite different. Obviously, the K m for TAL observed in this study indicated its high affinity to l -Tyr substrate, which would be valuable to produce a wide range of aromatic compounds that using l -Tyr or its derivative as a precursor (Shen et al, 2020 ).…”

Section: Resultsmentioning

confidence: 73%

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Xue

Liu

et al. 2021

Front. Bioeng. Biotechnol.

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PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

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“…The data represent means of three replicates and error bars represent standard deviations. Shen et al, 2020). Aguilar et al (2012Aguilar et al ( , 2015 reported that deletion of the 2-acyl-glycerophosphoethanolamine cycle allows fast growth in glucose and improves the production of aromatic compounds in E. coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS).…”

Section: Host Strain Engineering Increasing the Availability Of L-tyrosinementioning

confidence: 99%

Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

et al. 2021

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Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

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Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

Cited by 43 publications

References 167 publications

ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

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