Biosynthesis of odd-chain fatty alcohols in Escherichia coli

Cao, Yingxiu; Xiao, Wenhai; Liu, Duo; Zhang, Jinlai; Ding, Ming-Zhu; Yuan, Ying‐Jin

doi:10.1016/j.ymben.2015.03.005

Cited by 69 publications

(54 citation statements)

References 47 publications

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“…Cao et al [14] once improved odd-chain fatty alcohols production in Escherichia coli through balancing the expression level of TesA, αDOX, AHRs and the genes involved in fatty acids metabolism pathway. Meanwhile, through combinatorially screening the carotenogenic enzymes (CrtE, CrtB and CrtI) from diverse organisms and fine-tuning the expression level of CrtI, an optimal enzymes combination with the highest lycopene yield was obtained in Saccharomyces cerevisiae [15].…”

Section: Introductionmentioning

confidence: 99%

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

et al. 2017

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BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.Conclusions Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0665-1) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

et al. 2017

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“…These genes were delivered as pUC57-simple serious plasmids (Additional file 1: Table S2). All the Adx-CPY11B1-AdR expression plasmids were constructed based on vector pET-21a-YX, which was derived from vector pET-21a (Novagen, Germany) by Cao et al [53] through adding a SpeI site between the BamHI and BglI site of the initial plasmid. Therefore, genes Cyp11B1 , Adx 4 – 108 and AdR could be assembled into pET-21a-YX via BioBrick™ strategy [54].…”

Section: Methodsmentioning

confidence: 99%

Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation

et al. 2017

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Background21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.ResultsIn this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.ConclusionsHeterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0720-y) contains supplementary material, which is available to authorized users.

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“…Odd carbon-numbered PAs often yield materials with superior properties (Horn et al, 1963), but successful commercialization of those heavily depends on the availability of cheap precursors with an odd carbon count, for example, from ricinolic acid after oxidative cleavage (Teomim et al, 1999). Naturally, fatty acid-derived molecules with odd carbon number are scarce, although engineered microbes that overproduce them have been described (Cao et al, 2015;Wu and San, 2014). Monomers coming from cost-intensive multistep syntheses, or with high bulk material costs, limit the use of these polymers on broad scale (Finch, 2001).…”

Section: Introductionmentioning

confidence: 99%

Non‐hazardous biocatalytic oxidation in Nylon‐9 monomer synthesis on a 40 g scale with efficient downstream processing

Milker

Fink

Rudroff

et al. 2017

Biotech & Bioengineering

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This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670-1678. © 2017 Wiley Periodicals, Inc.

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Biosynthesis of odd-chain fatty alcohols in Escherichia coli

Cited by 69 publications

References 47 publications

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation

Non‐hazardous biocatalytic oxidation in Nylon‐9 monomer synthesis on a 40 g scale with efficient downstream processing

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