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Module 1: Microbial Factories for Biofuels and Amino acids

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Microbial Factories for Biofuels and Amino acids - Lesson Summary

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Applications of Metabolic Engineering Summary
Module 2
The key points from this module are as follows:
In the last two decades, metabolic engineering has been exploited to:
- Improve traditional microbial fermentation processes
- Produce chemicals that are currently used as fuels, material, and pharmaceutical ingredients.

Strategies in metabolic engineering:
Genome editing and evolution
Tolerance engineering
Rewiring of metabolic fluxes
Growth-based selection and adaptive laboratory evolution
Subcellular compartmentalization and trafficking in eukaryotic organisms

The chemicals produced by microbial cell factories can be classified into:
(1) Biofuel (2) Commodity Chemicals and (3) Natural products
Challenges towards economical biofuel production:
(1) Carbon fluxes from the substrate dissipate into a complex metabolic network. Besides the desired products, microbial hosts direct carbon flux to synthesize biomass, overflow metabolites and heterologous enzymes.
(2) Microbial hosts need to oxidize a large portion of the substrate to generate both ATP and NADPH to power biofuel synthesis.
(3) Large transfer limitations in large bioreactors create heterogeneous growth conditions and micro-environmental fluctuations that induce metabolic stresses and instability.
Microbial factories for biofuel production – Extensive research have been performed on the microbial production of biofuels using renewable feedstock.
Metabolic engineering of ethanol production by microbes:
For lignocellulose to be amenable to fermentation, it needs to undergo treatments that release its monomeric sugars, which thane can be converted by microorganisms. The efficient utilization of the hemicellulosic components of raw materials e.g. hardwood, xylose, etc., offers an opportunity to reduce the costs of ethanol production by 25%. Two main steps are involved, and these steps are:
1. A pretreatment that releases hexoses and pentoses from hemicellulose (physical or chemical procedures).
2. An enzymatic treatment
The biological process of ethanol production by utilizing lignocellulose as substrate requires three steps:
Delignification: To liberate cellulose and hemicellulose from their complex with lignin.
Deplolymerization: To produce free sugars from the carbohydrate polymers (cellulose and hemicellulose).
Fermentation: To produce the ethanol biofuel from the mixed hexose and pentose sugars.
The raw materials for ethanol production are:
1. Crops e.g. sugar cane, corn, etc.
2. Lignocellulose – Consists ~45% cellulose, ~30% hemicellulose, and ~25% lignin.
The conversion of lignocellulosic biomass to ethanol involves five main steps:
1. Collection and delivery of feedstock to the plant
2. Pretreatment of the feedstock
3. Enzymatic saccharification
4. Fermentation
5. Product formulation
The choice of microorganisms used for biofuel production includes:
• Ethanologens
• Enteric bacteria
The essential traits improved through metabolic engineering include:
• Broad substrate-utilization range for S. cerevisiae and Z. mobilis.
• High ethanol yield and productivity in E. coli and other enteric bacteria.
• Minimal byproduct formation in yeasts and E. coli.
• Increased tolerance to ethanol by microorganisms
• Increased tolerance to inhibitors by microorganisms
• Tolerance to process hardiness
The desired traits improved via metabolic engineering include:
1. The capability for the simultaneous utilization of various sugars.
2. Cellulose/hemicellulose degradation by microorganisms
3. Other desirable traits, e.g. growth at low pH and high temperature, minimal nutrient requirements, etc.
Yeast metabolic engineering for hemicellulosic ethanol production
To enable xylose fermentation by yeasts, three main strategies are adopted, which are:
1. The insertion of bacterial xylose isomerase gene
2. The insertion of pentose utilizing gene
3. Improvement of xylose consumption
S. cerevisiae for efficient xylose metabolism:
S. cerevisiae has been engineered to express xylose isomerae (XI) or xylose reductase-xylitol dehydrogenase (XR/XDH) that converts xylose to D-xylulose. The D-xylulose can enter the pentose phosphate pathway via endogenous xylulokinase (XK) and be further metabolized through the central carbon metabolism.
The initial attempts to improve xylose fermentation by engineered yeasts include:
1. Optimization of xylose metabolic pathways
2. Introduction of heterologous xylose transporters
3. Deletion of endogenous metabolic pathways that siphon intermediates
4. Adaptive laboratory evolution with genome-wide analytical techniques.
Metabolic engineering strategies in E. coli towards ethanol biofuel production
E. coli is able to use both pentose and hexose sugars, and it’s highly amenable to metabolic engineering for fuel production. It is the preferred host for ethanol production from lignocellulose (also known as cellulosic biomass) because:
1. It is the most studied microorganism in terms of gene regulation and expression.
2. It can grow efficiently on a wide range of carbon substrates.
3. It can grow on inexpensive mineral media under aerobic and anaerobic conditions.
4. It sustains high glycolytic fluxes.
5. It is expresses reasonable ethanol tolerance.
6. Availability of detailed genetic info and diverse genetic tools for gene manipulation.
Application of metabolic engineering in amino acids production
The recent trends in microbial strain improvements towards amino acids production include:
1. System metabolic engineering
2. Single cell analysis
3. Synthetic biology
4. Evolutionary engineering
The common strategies for the design of amino acids producing strains are:
1. The amplification of biosynthesis pathway enzymes for the target amino acids.
2. Reduction of byproducts formation.
3. The release of feedback regulation for key enzymes by the target amino acid.
4. Increased supply of reducing equivalents such as NADPH.
5. Reduction of metabolic fluxes to the tricarboxylic acid TCA cycle.
The systems metabolic engineering strategies for amino acids high-producing strains are categorized as:
1. Pathway based approach.
2. Systems biology based approach
3. Evolutionary approach