20.109(F20):M3D1

Introduction

In this module you will build upon the progress of former 109ers! In previous semesters, students engineered the the metabolism of E. coli in an attempt to increase the production of a commercially relevant product. Specifically, metabolic engineering was used to increase the ethanol yield via manipulating the fermentation pathway native to E. coli cells. Ethanol, or ethyl alcohol, is used as fuel – most commonly as a biofuel additive in gasoline. Current methods for producing ethanol involve the use of agricultural feedstocks and concern is mounting given the effect this may have on food prices and resources. Using E. coli as an ethanol-generating machine is promising; however, the ethanol output of a native cell is very low.

Metabolic engineering refers to the alteration of genetic and/or regulatory circuitry within organisms. The native circuitry involves a series of enzymes that perform biochemical reactions that function to convert raw substrates into products that are required for the organism’s survival. When this native circuitry is altered using metabolic engineering techniques, the goal is to use the host organisms as machines that produce valuable materials in large quantities and at low cost.

Example illustrating pathway to desired product.

Common strategies employed in metabolic engineering are:

• Increasing expression of a gene that encodes an enzyme responsible for a rate-limiting step.
• Inhibiting competing pathways that divert substrate away from the pathway of interest.
• Incorporating genes from other organisms into the host.
• Altering the structure of the enzyme such that yield is increased.

To increase production of a desired product, you can either target proteins that use the substrate to generate alternate products (see A in image on right) or you can target proteins that use the desired product to generate alternate products (see B in image on right). In A the substrate is siphoned away from the reaction that generates the desired product and in B the desired product is used as substrate in a subsequent reaction. By eliminating the proteins that catalyze the reactions that result in alternate products, you can potentially increase production of your desired product.

To manipulate the metabolism of E. coli, the CRISPRi system was used. Unlike more commonly used CRISPR-based technologies, CRISPRi is used to modulate expression from the genome rather than to modify the genome. This distinction is due to the use of an enzymatically-inactive dCas9 (or ‘dead’ Cas9) protein. Because dCas9 is enzymatically inactive, it is unable to cleave the DNA upon binding to a gRNA/DNA complex. The lack of DNA cleavage results in gene silencing through impeding RNA polymerase binding, transcription factor binding, and/or transcription elongation. The method of repression is largely due to the location of the genome that is targeted.

Schematic of CRISPRi preventing transcription by RNA polymerase. Image taken from Qi Lab Homepage.

Protocols

The focus for today is to understand the key concepts and methods that were used to generate the data you will critically evaluate in this module. First, it is important to consider how targets were selected for the metabolic engineering approach. Second, you will review the CRISPRi technology used for the metabolic engineering approach.

Part 1: Review E. coli ethanol metabolism

In E. coli ethanol is a product of the mixed-acid fermentation pathway. You will complete this task using the CRISPRi system, which targets genes such that transcription of the target gene is decreased, thus resulting in less of the protein encoded by the targeted gene.

With your laboratory partner, review Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products by Forster & Gescher. Using the information within this article and the genes that you identified above, select one gene that you will target in an attempt to increase the production of either ethanol or acetate. Be sure to include notes on your decision and thought process in your laboratory notebook.

Use the fermentation pathway from E. coli (included below) and the example above to answer the following questions with your partner.

1. Which gene(s) might you target to increase the availability of substrate for ethanol production? For acetate production?
2. Which gene(s) might you target to decrease the amount of substrate used to generate products in steps downstream of ethanol production? of acetate production?

Mixed-acid fermentation in E. coli. Image from Forster and Gescher, Front. in Bioeng. and Biotech., 2014.

Part 2: Use CRISPRi system to engineer E. coli metabolism

Reagents list

Navigation links

Next day: Align gRNA sequences with genomic targets

Previous day: Examine putative small molecule binders for common features ??