The Yeast Pathway Kit - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

The MEC group developed a protocol for the in-vivo assembly of large metabolic pathways we call the Yeast Pathway Kit (YPK), see our publication in ACS Synthetic Biology: Pereira et. al 2015.

Quick links:

• Primer design for genes to be cloned in pYPKa (often necessary to express a new gene using the Yeast Pathway Kit)
• How to clone using the pYPKa vector (in silico assembly of pYPKa_A_ATF1)
• How to assemble a yeast expression vector (TU) (in silico assembly of pTA1_TDH3_ATF1_PGI1).
• Available Plasmid, Promoter & Terminator sequence files.

We use this protocol for the generation of expression cassettes (TU, transcriptional units) as well as large metabolic pathways that are yet relatively compact compared to pathways assembled with other protocols in Saccharomyces cerevisiae. YPK relies on natural intergenic sequences which might be positive for genetic stability.

The genetic building block DNA fragments (promoters, genes and terminators) are all cloned in an E. coli positive selection vector called pYPKa. The fragments are cloned one at a time, creating one plasmid per fragment.

These plasmids are used as template for PCR amplification and joined together by homologous recombination into single gene expression vectors (Transcriptional Units, TU) using a S. cerevisiae/E. coli shuttle vector such as pYPKpw or pTA1.

These TU vectors can be further assembled into large (at least 13 genes has been successfully assembled) metabolic pathways by homologous recombination between promoters and terminators of the transcriptional units.

The pYPKa vector is a derivative of the positive selection vector pCAPs. This vector is very efficient and permits direct cloning of PCR products directly from the PCR mix.

Promoters, genes and terminators are cloned in one of three unique restriction sites in pYPKa all producing blunt cuts (Table#1).

Table#1	Element	Cloning site
	Promoters	ZraI
	Gene	AjiI BtrI BmgBI
	Terminator	EcoRV Eco32I

These sites are located close together in pYPKa in the order given in Table#1. The figure below shows the ZraI and AjiI cut sites separated by 50 bp (red in the figure below) and AjiI and EcoRV separated by 31 bp (green).

NB only one DNA fragment (a promoter, gene or a terminator) is cloned per plasmid.

The resulting plasmids are named using an established nomenclature.

A pYPKa plasmids carrying the ABC1 fragment cloned in the ZraI site are named pYPKa_Z_ABC1, where "ABC1" is a short reference to the cloned DNA fragment. Optionally, a short prefix can be added indicating the strain or organism from which the gene was sourced.

We use the following prefixes: Sc for Saccharomyces cerevisiae, Ec for Escherichia coli and Yl for Yarrowia Lipolytica and At for Arabidopsis thaliana. For other cases, consider using the KEGG three letter abbreviation, but with an initial capital letter.

The insert designation must allow the plasmid name to be a file name, so only use ASCII letters (a -z A -Z 0-9), hence the following characters should be avoided: ! " # $ % & ' ( ) * + , - . / : ; < = > ? @ [ \ ] ^ _ ` { | } ~

Thus, vectors with DNA fragments cloned in ZraI, AjiI and EcoRV are designated according to Table#2 below:

Table#2	Element	Cloning site	Name
	Promoter	ZraI	pYPKa_Z_ABC1
	Gene	AjiI	pYPKa_A_ABC1
	Terminator	EcoRV	pYPKa_E_ABC1

One of the advantages of the system is the reuse promoters and terminators in pYPKa_Z and pYPKa_E vectors. This repository has over sixty S. cerevisiae intergenic sequences cloned in pYPKa.

Certain conventions should be followed for primer design for genes to be cloned in the pYPKa for gene expression, i.e. creating a new pYPKa_A_*** vector.

It is a good practice to create a new cloning project in-silico prior to starting the lab work. This example is provided for how to use the excellent DNA editor ApE in combination with PydnaWeb to manually assemble a pYPKa clone in-silico.

Look at the pYPKa cloning protocol for how to clone a PCR product using pYPKa in the lab.

The purpose of the pYPKa_* vectors described in the previous section is to provide building blocks for S. cerevisiae expression vectors, each expressing one gene.

Single genes are cloned between a promoter and a terminator by in-vivo homologous recombination between three PCR products obtained from pYPKa_* vectors and a linearized S. cerevisiae/E. coli shuttle vector.

Since promoters genes and terminators are cloned in essentially the same vector, DNA fragments sharing terminal homology are easily produced by choosing the right PCR primers.

Approximate location of six PCR primers used for this purpose are indicated by numbers in the figure below (577, 567), (468, 467) and (568, 578).

Promoters are amplified using primers 577, 567, genes using 468, 467 and terminators using 568, 578.

The three PCR products are mixed with a linearized shuttle vector (pYPKpw or similar). The linear vector is the red dashed line in the figure below. The vector carries regions of homology to the promoter and terminator PCR products, gray and pink boxes respectively.

See the specific protocol for how to construct a TU vector in the lab. A combination of web services, the software package pydna and and Google colab can be used to rapidly assemble the sequence (see here).

Metabolic pathways can later be built by linking single gene expression cassettes together in a second assembly step. This assembly has to be carefully planned already before the construction of the TU vectors. Transcriptional units are joined by recombination between mutually shared promoter and terminator sequences.

For this to be possible, promoters and terminators need to be identical DNA fragments in adjacent transcriptional units.

Primer locations around the ZraI, AjiI and EcoRV sites in pYPKa:

Primer locations around the ZraI, AjiI and EcoRV sites in pYPKpw and derived vectors, such as the pTAx series:

A short summary of the Yeast Pathway Kit:

PDF versions of the images above are available here.