Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance

Sun, Qiumeng; Gao, Song; Yu, Shiqin; Zheng, Pu; Zhou, Jingwen

doi:10.1016/j.synbio.2022.07.004

Cited by 14 publications

(13 citation statements)

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“…Given that F4′OMTs are SAM-dependent flavonoid methyltransferases, SAM might be a limiting factor for the biosynthesis of methyl flavonoids. , In this study, we detected minor amounts of (2 S )-eriodictyol and (2 S )-isosakuranetin at the end of fermentation in strain HE03 (Figure ), supporting the idea that SAM may be limited in our strains. When metK was overexpressed in strain HE04, it reduced (2 S )-isosakuranetin accumulation, but it also reduced the level of (2 S )-hesperetin, thus warranting further investigation.…”

Section: Discussionsupporting

confidence: 78%

Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli

Liu,

Xiao,

Zhang

et al. 2023

J. Agric. Food Chem.

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Enzyme promiscuity is evolutionarily advantageous to plants for gaining new enzyme functions when adapting to environmental challenges. However, this promiscuity can negatively affect the expression of genes encoding for plant enzymes in microorganisms. Here, we show that refining the promiscuity of flavonoid 3′-hydroxylase (F3′H) and 4′-O-methyltransferase (F4′OMT) improves (2S)-hesperetin production in Escherichia coli. First, we employed inverse molecular docking to screen a highly substrate-specific ThF3′H from Tricyrtis hirta, which could selectively convert 100 mg L–1 (2S)-naringenin to (2S)-eriodictyol but not (2S)-isosakuranetin, with a cytochrome P450 reductase from Arabidopsis thaliana. Second, we employed a directed evolution approach to restrict the promiscuity of MpOMT from Mentha × piperita. The strain harboring the MpOMTS142V mutant presented a remarkably increased preference for (2S)-eriodictyol. Finally, 27.5 mg L–1 (2S)-hesperetin was produced, while only minor amounts of (2S)-eriodictyol and (2S)-isosakuranetin accumulated as byproducts. This value represents a 14-fold increase in (2S)-hesperetin compared to the parental strain, along with a dramatic reduction in side products. Our work highlights the benefit of alleviating the promiscuity of plant enzymes when engineering production of natural products by microbial cell factories.

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Section: Discussionsupporting

confidence: 78%

Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli

Liu,

Xiao,

Zhang

et al. 2023

J. Agric. Food Chem.

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“…This is despite the fact that MetJ, a tried and tested tool as a SAM biosensor unit, 38,39 is derived from E. coli, and that E. coli is a common host in the production of methylated compounds. [23][24][25][26][27][28][29]32,34,40 This may be partially because a MetJ-based biosensor inevitably undergoes crosstalk with the endogenous MetJ regulon, which is the master regulator of SAM homeostasis in E. coli. Recently, riboswitch-based systems 41−43 have been shown to respond to L-methionine (L-Met) supplemented in the medium, indicating that they could be useful indicators of the intracellular SAM concentration in E. coli.…”

Section: ■ Introductionmentioning

confidence: 99%

“…23−26 To improve the production level of methylated compounds, metabolic engineering efforts have been conducted. These approaches encompass eliminating SAM metabolic regulators, 27,28 enhancing the known route for SAM biosynthesis, [23][24][25]29,30 pruning of the endogenous SAM consumption pathways, 31−33 and reinforcing SAM-regeneration pathways. 30,34 These approaches yielded, for instance, a 1. acid.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In the bioproduction of these methylated compounds, SAM availability has been suspected to be the major bottleneck. − To improve the production level of methylated compounds, metabolic engineering efforts have been conducted. These approaches encompass eliminating SAM metabolic regulators, , enhancing the known route for SAM biosynthesis, − ,, pruning of the endogenous SAM consumption pathways, − and reinforcing SAM-regeneration pathways. , These approaches yielded, for instance, a 1.5–12.6 times improvement in the production of a representative methylated compound ferulic acid. ,, Although impressive, the highest current titer for ferulic acid is 5.09 g/L, an order of magnitude lower than that (60 g/L) achieved for its precursor, l -phenylalanine.…”

Section: Introductionmentioning

confidence: 99%

“…coli, and that E. coli is a common host in the production of methylated compounds. − ,,, This may be partially because a MetJ-based biosensor inevitably undergoes crosstalk with the endogenous MetJ regulon, which is the master regulator of SAM homeostasis in E. coli.…”

Section: Introductionmentioning

confidence: 99%

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MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors

Watanabe,

Kimura,

Umeno

2024

ACS Synth. Biol.

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SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batchto-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.

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Biosynthesis of pterostilbene in Escherichia coli from resveratrol on macroporous adsorption resin using a two‐step substrate addition strategy

Yingling,

Xueqin,

Xiaolong

2024

Biotech & Bioengineering

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Pterostilbene (PST), a 3’,5’‐O‐methylated derivative of resveratrol (RSV), is a potent natural antioxidant produced by some plants in trace amounts as defense compound. It exhibits various health‐promoting activities, such as anticancer, antiviral, and antimicrobial effects. Large‐scale biosynthesis of PST is crucial due to the challenges associated with extracting it from plants. This study aims to develop an efficient method for PST production using an engineered Escherichia coli strain by feeding RSV as a precursor. We introduced a two‐step substrate addition strategy combined with immobilized RSV (IMRSV) on macroporous adsorption resin (MAR) to enhance PST production. Five MARs were selected for RSV immobilization, and the substrate addition strategy and fermentation parameters for PST synthesis were optimized. A maximum PST concentration of 403 ± 9 mg/L was achieved, representing a 239% increase over the control, which in a one‐step addition of free RSV. The PST titer reached 395 ± 24 mg/L in a 3‐L bioreactor. In conclusion, the combination of a two‐step substrate addition system and IMRSV is a promising approach for the economical and industrial‐scale production of PST.

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Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance

Cited by 14 publications

References 55 publications

Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli

Restricting Promiscuity of Plant Flavonoid 3′-Hydroxylase and 4′-O-Methyltransferase Improves the Biosynthesis of (2S)-Hesperetin in E. coli

MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors

Biosynthesis of pterostilbene in Escherichia coli from resveratrol on macroporous adsorption resin using a two‐step substrate addition strategy

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