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Metabolic Pathway Assembly

Metabolic Pathway Assembly Service

ORF cDNA Clones and Custom Clones

Metabolic Pathway Engineering is a powerful approach to optimize production of desired biomolecules, but assembling and validating the DNA constructs encoding re-engineered metabolic pathways can be laborious. Our Metabolic Pathway Assembly service allows you to focus on design while we perform the molecular biology. Going beyond our expertise in gene synthesis, we offer customizable services to assemble genetic components into full-length constructs or modular libraries.

Metabolic Pathway Assembly Service Specifications

Research Strategy Recommended Service Deliverable Format How to Order
Rational Design
Metabolic Pathway Assembly –
Constructs
Cat. No. SC1702
  • Individually sequence-verified plasmids, 4µg per construct
  • Price and turnaround depend upon sequence length, complexity, and number of constructs desired.
Library Screening
Metabolic Pathway Assembly –
Library
Cat. No. SC1707
  • 10 μg of pooled plasmids
  • Price and turnaround time depend upon sequence length, complexity, theoretical library size, and validation requests e.g. restriction analysis and sequencing to confirm accuracy and diversity of pooled library.

Workflow of Pathway Assembly for
Metabolic Engineering & Synthetic Pathway Optimization

Workflow of OLMA Pathway Assembly for 
Metabolic Engineering & Synthetic Pathway Optimization

Our proprietary Oligo Linker Mediated Assembly (OLMA) technology was developed by, and is exclusively licensed through, our academic research partners Dr. Chunbo Lou and Dr. Yong Tao of The Institute of Microbiology of the Chinese Academy of Sciences (IMCAS). OLMA offer several advantages:

  • one-step assembly of gene components to create modular libraries with high levels of accuracy, coverage, and diversity.
  • oligo linkers can be designed either as functional segments (e.g. encoding RBS variants to tune the translation initiation rate in prokaryotic hosts) or to enable seamless assembly between conserved regions.
  • flexibility to choose whether to preserve the order of gene components, link the order of only specific components, or shuffle the order of components across the entire construct.
  • flexibility to define a modular unit any way you like; you can define promoter-gene-terminator cassettes,  or vary domains within a coding region – any design you can imagine, our technology can accommodate!

Our in-house scientists have performed numerous case studies with diverse assembly design strategies. We will discuss all parameters of the assembly project design with you to customize each experiment for your needs.

Metabolic Engineering is a powerful approach to optimize genetic circuits, such as biosynthetic pathways that drive "cellular factories" for efficient, industrial-scale production of natural products, such as:

  • More efficient synthesis of valuable biomolecules (natural products). Natural products can often be obtained from plant extracts or from total chemical synthesis of the desired compound, but purification from natural sources can be problematic due to the need to resolve complex mixtures of closely related compounds, and our current best methods for chemical synthesis are limited by low yield and low specificity requiring additional painstaking purification. As an alternative approach, metabolic engineering allows biosynthetic pathways to be reconstructed in model organisms so that useful quantities of the desired product can be harvested. Flavanoid, Isoprenoids, polyphenols and other natural metabolites are often synthesized in microbiological hosts such as Escherichia coli, but other prokaryotes, yeast, plant, or other hosts can be used as well.

    In one example, Brazier-Hicks and Edwards developed a method for efficient production of C-glycosylated flavanoids for dietary studies by using gene synthesis to re-engineer a metabolic circuit in yeast. They designed synthetic variants of five genes that comprise the flavone-C-glycoside pathway in rice plants, which were subsequently codon-optimized for expression in yeast. These synthetic genes were used to construct a polyprotein cassette that expresses the entire metabolic circuit in a single step.


  • Protein Production Scale-Up: Metabolic pathway engineering can be used to enhance protein yield for any large-scale protein purification needs; when the expression and stability of your protein of interest depends upon multiple enzymatic steps, metabolic pathway feedback loops, or checks on protein modification or degradation, then your experiments require more than a simple expression cassette with a strong promoter driving your protein of interest. Even if you only need to express one protein, the same approach of modular assembly can be used to find the optimal combination of different promoters, ribosome binding sites, codon-optimized ORFs, and terminators that maximize the yield of soluble, properly-folded protein.

  • Sustainable alternatives to petroleum products. Most organic chemicals used today are derived from petroleum, but it's been estimated that up to 2/3 of those could be generated from renewable raw materials rather than oil, which could bring both economic and environmental benefits. The U.S. Department of Energy has provided grant funding for metabolic engineering projects undertaken in industrial as well as academic settings that could yield new bio-based production facilities for widely-used biomolecules. While most efforts to develop biofuels have focused on carbohydrates and related compounds, Dellomonaco et al. showed that fatty acids can serve as biomass for sustainably produced biofuels by using gene synthesis to engineer several native and heterologous fermentative pathways to function in E.coli under aerobic conditions. These synthetically engineered bacteria convert fatty acid-rich feedstocks into desirable biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate), with higher yield than more widely used lignocellulosic sugars.

Metabolic Pathway Optimization approaches that can be used alone or in combination include:

  • Altering the relative expression levels of multiple enzymes in a circuit, which can often be achieved by mixing and matching gene regulatory elements such as ribosome binding sites (RBS) in prokaryotic hosts or eukaryotic promoters or terminators.
  • Mining naturally-occurring genetic diversity by using homologs, orthologs, or infologs with subtle differences in enzymatic activity to manipulate metabolite flow through a biosynthetic pathway.
  • Codon optimization (or “de-optimization”), which introduces synonymous mutations that can alter translation rate and other steps in protein expression.
  • Kallio et al. An engineered pathway for the biosynthesis of renewable propane. Nat Commun. 2014 Sep 2;5:4731. Free Full Text
  • Xu et al. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. Proc Natl Acad Sci U S A. 2014 Aug 5;111(31):11299-304. Free Full Text
  • Xue et al. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol. 2013 Aug;31(8):734-40. Free Full Text
  • Nguyen et al. Redirection of metabolic flux for high levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds. Plant Biotechnol J. 2014 Jul 26. doi: 10.1111/pbi.12233. Free Full Text

Search or browse peer-reviewed publications on metabolic engineering that cite GenScript services & products.

The International Genetically Engineered Machine (iGEM) Competition showcases synthetic biology and metabolic engineering innovations that create new tools for research, healthcare, energy, or environmental applications. In the 2014 Jamboree, GenScript-sponsored teams won accolades for their work to develop novel genetic circuits that turned their genetically engineered bacteria into “cellular factories” to purify water of heavy metals or methane, to keep burn wound sites free of infection, to produce biodegradable elastin polymers that could replace plastics, and to detect pathogenic bacteria. Read more about the projects of GenScript-sponsored iGEM teams from 2009-2014.

To download a printable PDF of this case study, click here.

Lycopene biosynthetic pathway to be optimized through metabolic engineering Introduction: Lycopene, a carotenoid phytochemical best known for its bright red color and anti-oxidant properties, has various biological functions and is widely used in pharmaceutical, food and cosmetic industries. Structurally, it consists of six isopentenyl diphosphate (IPP) and two dimethylallyl diphosphate (DMAPP) molecules. The precursors IPP and DMAPP can be converted to lycopene by co-expressing four exogenous genes in E.coli cells: isopentenyl-diphosphaste delta-isomerase (idi); geranylgeranyl disphosphate (GGPP) synthase (crtE); phytoene synthase (crtB); and phytoene desaturase (crtI).

In this case study, we applied a new technology to perform an all-in-one reaction to assemble multiple variants of each part of the lycopene biosynthetic pathway in many unique combinations. The resulting high-diversity pooled library was then transformed into E. coli hosts and colonies were screened to identify transformants with enhanced lycopene yield. The recombinant genetic circuits that gave rise to the best yield improvements could then be identified through restriction analysis and/or sequencing.

Experimental Design: Four homologs of crtE, crtB, and crtI from Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, and Rhodobacter sphaeroides were cloned into a module plasmid with the same overhang for each gene. idi from E.coli K12 was cloned into the backbone plasmid, and fixed on the last position of the gene circuit. For each gene, 20 ribosome binding sites (RBS), including 10 reverse designed and 10 forward designed RBS, were applied to balance the expression of crtE, crtB, and crtI genes. Each RBS for crtE, crtB, and crtI was synthesized as an oligo linker containing one of three versions of overhangs, in order to enable assembly of the first three genes in any order. An all in-one-reaction was performed to generate a modular metabolic pathway assembly construct library.


Metabolic Pathway Assembly using oligo linker mediated assembly technique

lycopene yield optimized through metabolic pathway engineering

Results: The plasmids mixture was transformed into E.coli PXIDF and the transformations were cultured for lycopene production. Lycopene was quantified by measuring OD472 absorption after extracting by ethanol-acetone (V4:1). 1080 red colonies were randomly picked for lycopene production measurement. A wide range of lycopene yield was observed. Simultaneous optimization of RBS, gene order, and homologs using oligo linker mediated assembly enabled the rapid identification of genetic circuits that drive vastly enhanced lycopene production in E. coli.

ORF cDNA Clones and Custom Clones

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