Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Furubayashi, Maiko; Li, Ling; Katabami, Akinori; Saito, Kyoichi; Umeno, Daisuke

doi:10.1016/j.febslet.2014.07.028

Cited by 9 publications

(12 citation statements)

References 31 publications

(55 reference statements)

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“…Protein engineering is currently a powerful tool for improving enzyme activity, substrate specificity, and evolutionary fitness through the evolution of the enzymes of interest. Directed evolution of SQS genes was performed to determine the activity and evolutionary adaptability of the gene that could allow improved production of squalene (Furubayashi et al, 2014b). This study demonstrated that the direct evolution of different SQS genes from S. cerevisiae , humans, and Thermosynechococcus elongatus BP-1 could exhibit numerous beneficial mutations that could expand the activity of SQS (Furubayashi et al, 2014b).…”

Section: Engineering Of Microorganisms For Squalene Productionmentioning

confidence: 99%

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

Gohil

Bhattacharjee

Khambhati

et al. 2019

Front. Bioeng. Biotechnol.

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The triterpene squalene is a natural compound that has demonstrated an extraordinary diversity of uses in pharmaceutical, nutraceutical, and personal care industries. Emboldened by this range of uses, novel applications that can gain profit from the benefits of squalene as an additive or supplement are expanding, resulting in its increasing demand. Ever since its discovery, the primary source has been the deep-sea shark liver, although recent declines in their populations and justified animal conservation and protection regulations have encouraged researchers to identify a novel route for squalene biosynthesis. This renewed scientific interest has profited from immense developments in synthetic biology, which now allows fine-tuning of a wider range of plants, fungi, and microorganisms for improved squalene production. There are numerous naturally squalene producing species and strains; although they generally do not make commercially viable yields as primary shark liver sources can deliver. The recent advances made toward improving squalene output from natural and engineered species have inspired this review. Accordingly, it will cover in-depth knowledge offered by the studies of the natural sources, and various engineering-based strategies that have been used to drive the improvements in the pathways toward large-scale production. The wide uses of squalene are also discussed, including the notable developments in anti-cancer applications and in augmenting influenza vaccines for greater efficacy.

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Section: Engineering Of Microorganisms For Squalene Productionmentioning

confidence: 99%

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

Gohil

Bhattacharjee

Khambhati

et al. 2019

Front. Bioeng. Biotechnol.

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“…Two early, sequential committed steps in the conversion of farnesyl pyrophosphate (FPP) to phytoene in carotenoid biosynthesis are catalysed by geranylgeranyl diphosphate synthase and phytoene synthases [ 54 ]. Notably, the recent reports show that squalene synthase (SQS) is involved in carotenoid biosynthesis, and can convert to a dehydrosqualene synthase for production of the C 30 carotenoid backbone [ 55 , 56 ]. An early report showed the conversion of FPP to squalene by SQS in hot pepper [ 57 ].…”

Section: Discussionmentioning

confidence: 99%

Characterization of the hot pepper (Capsicum frutescens) fruit ripening regulated by ethylene and ABA

Hou

Han

et al. 2018

BMC Plant Biol

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BackgroundRipening of fleshy fruits has been classically defined as climacteric or non-climacteric. Both types of ripening are controlled by plant hormones, notably by ethylene in climacteric ripening and by abscisic acid (ABA) in non-climacteric ripening. In pepper (Capsicum), fruit ripening has been widely classified as non-climacteric, but the ripening of the hot pepper fruit appears to be climacteric. To date, how to regulate the hot pepper fruit ripening through ethylene and ABA remains unclear.ResultsHere, we examined ripening of the hot pepper (Capsicum frutescens) fruit during large green (LG), initial colouring (IC), brown (Br), and full red (FR) stages. We found a peak of ethylene emission at the IC stage, followed by a peak respiratory quotient at the Br stage. By contrast, ABA levels increased slowly before the Br stage, then increased sharply and reached a maximum level at the FR stage. Exogenous ethylene promoted colouration, but exogenous ABA did not. Unexpectedly, fluridone, an inhibitor of ABA biosynthesis, promoted colouration. RNA-sequencing data obtained from the four stages around ripening showed that ACO3 and NCED1/3 gene expression determined ethylene and ABA levels, respectively. Downregulation of ACO3 and NCED1/3 expression by virus-induced gene silencing (VIGS) inhibited and promoted colouration, respectively, as evidenced by changes in carotenoid, ABA, and ethylene levels, as well as carotenoid biosynthesis-related gene expression. Importantly, the retarded colouration in ACO3-VIGS fruits was rescued by exogenous ethylene.ConclusionsEthylene positively regulates the hot pepper fruit colouration, while inhibition of ABA biosynthesis promotes colouration, suggesting a role of ABA in de-greening. Our findings provide new insights into processes of fleshy fruit ripening regulated by ABA and ethylene, focusing on ethylene in carotenoid biosynthesis and ABA in chlorophyll degradation.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1377-3) contains supplementary material, which is available to authorized users.

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“…In previous studies on the production of squalene by introducing hSQS gene into E. coli (Furubayashi, Li et al 2014, Katabami, Li et al 2015, the nucleic acid sequence of hSQS was directly derived from human cDNA and was the truncated version at the N-terminal (Thompson, Danley et al 1998), while this study used the E. coli codon optimization of gene sequence. The experimental results showed that it also had the ability to catalyze and generate squalene in prokaryotic cells, but the yield was low, which might be the result of low supply of FPP.…”

Section: Discussionmentioning

confidence: 99%

Using Engineered Escherichia coli to Synthesize Squalene with Optimized Manipulation of Squalene Synthase and Mevalonate Pathway

Sun

Ding²,

Cheng³

et al. 2020

Preprint

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Squalene is the metabolic precursor of sterols and naturally synthesized in the deep-dea shark liver and human sebum. The utilization of squalene is wide such as food, cosmetical, and pharmaceutical industries. This experiment used engineered Escherichia coli to construct the gene circuit for the biosynthesis of squalene. Human squalene synthase (hSQS) efficiently catalyzes the synthesis of squalene. Also, mevalonate (MVA) pathway would increase the yield of the precursor of squalene, farnesyl diphosphate, which then increased the yield of squalene. Meanwhile, the regulation of MVA pathway via different inducer IPTG concentrations and creation of selection pressure by antibiotics were investigated.

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Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Cited by 9 publications

References 31 publications

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities

Characterization of the hot pepper (Capsicum frutescens) fruit ripening regulated by ethylene and ABA

Using Engineered Escherichia coli to Synthesize Squalene with Optimized Manipulation of Squalene Synthase and Mevalonate Pathway

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