De Novo Metabolic Engineering and the Promise of Synthetic DNA

Abstract 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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MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

Bioinformatics ◽

10.1093/bioinformatics/btaa140 ◽

2020 ◽

Vol 36 (11) ◽

pp. 3322-3326

Author(s):

Michael Schwarz ◽

Marius Welzel ◽

Tolganay Kabdullayeva ◽

Anke Becker ◽

Bernd Freisleben ◽

...

Keyword(s):

Dna Synthesis ◽

Web Application ◽

De Novo ◽

Supplementary Information ◽

Dna Fragments ◽

Synthetic Dna ◽

Custom Made ◽

Dna Fragment ◽

Polymerase Chain ◽

Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

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Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of l-DOPA from d-glucose

Microbial Cell Factories ◽

10.1186/s12934-019-1122-0 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 13

Author(s):

Eric Fordjour ◽

Frederick Komla Adipah ◽

Shenghu Zhou ◽

Guocheng Du ◽

Jingwen Zhou

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo ◽

Escherichia Coli Bl21

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Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

Genome Announcements ◽

10.1128/genomea.01291-16 ◽

2016 ◽

Vol 4 (6) ◽

Cited By ~ 5

Author(s):

Elena Geiser ◽

Florian Ludwig ◽

Thiemo Zambanini ◽

Nick Wierckx ◽

Lars M. Blank

Keyword(s):

Metabolic Engineering ◽

Genome Sequencing ◽

De Novo ◽

Draft Genome ◽

Essential Genes ◽

Smut Fungi ◽

Genome Sequences ◽

The Family

Some smut fungi of the family Ustilaginaceae produce itaconate from glucose. De novo genome sequencing of nine itaconate-producing Ustilaginaceae revealed genome sizes between 19 and 25 Mbp. Comparison to the itaconate cluster of U. maydis MB215 revealed all essential genes for itaconate production contributing to metabolic engineering for improving itaconate production.

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Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.624838 ◽

2021 ◽

Vol 9 ◽

Author(s):

Beom Gi Park ◽

Junyeob Kim ◽

Eun-Jung Kim ◽

Yechan Kim ◽

Joonwon Kim ◽

...

Keyword(s):

Fatty Acids ◽

Metabolic Engineering ◽

Palmitic Acid ◽

Yarrowia Lipolytica ◽

Cell Sorting ◽

De Novo ◽

Random Mutagenesis ◽

Oleaginous Yeasts ◽

Synthetic Promoters ◽

Base Strain

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

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Metabolic engineering strategies of de novo pathway for enhancing 2′‐fucosyllactose synthesis in Escherichia coli

Microbial Biotechnology ◽

10.1111/1751-7915.13977 ◽

2021 ◽

Author(s):

Mengli Li ◽

Chenchen Li ◽

Miaomiao Hu ◽

Tao Zhang

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo

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Metabolic engineering of Klebsiella pneumoniae for the de novo production of 2-butanol as a potential biofuel

Bioresource Technology ◽

10.1016/j.biortech.2015.08.086 ◽

2015 ◽

Vol 197 ◽

pp. 260-265 ◽

Cited By ~ 24

Author(s):

Zhen Chen ◽

Yao Wu ◽

Jinhai Huang ◽

Dehua Liu

Keyword(s):

Metabolic Engineering ◽

Klebsiella Pneumoniae ◽

De Novo

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Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties

Metabolic Engineering ◽

10.1016/j.ymben.2016.10.019 ◽

2017 ◽

Vol 39 ◽

pp. 80-89 ◽

Cited By ~ 48

Author(s):

Michael Eichenberger ◽

Beata Joanna Lehka ◽

Christophe Folly ◽

David Fischer ◽

Stefan Martens ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Engineering ◽

De Novo

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Draft Genome Sequence of Ustilago trichophora RK089, a Promising Malic Acid Producer

Genome Announcements ◽

10.1128/genomea.00749-16 ◽

2016 ◽

Vol 4 (4) ◽

Cited By ~ 5

Author(s):

Thiemo Zambanini ◽

Joerg M. Buescher ◽

Guido Meurer ◽

Nick Wierckx ◽

Lars M. Blank

Keyword(s):

Metabolic Engineering ◽

Genome Sequencing ◽

Genome Sequence ◽

Malic Acid ◽

De Novo ◽

Draft Genome ◽

Ustilago Maydis ◽

Essential Genes ◽

Smut Fungus ◽

Draft Genome Sequence

The basidiomycetous smut fungus Ustilago trichophora RK089 produces malate from glycerol. De novo genome sequencing revealed a 20.7-Mbp genome (301 gap-closed contigs, 246 scaffolds). A comparison to the genome of Ustilago maydis 521 revealed all essential genes for malate production from glycerol contributing to metabolic engineering for improving malate production.

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