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Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

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Microbial production host selection for converting second-generation feedstocks into bioproducts.

Karl Rumbold¹, Hugo J J van Buijsen, Karin M Overkamp

¹Team Microbial Production Processes, TNO Quality of Life, PO Box 360, 3700 AJ Zeist, The Netherlands. karl.rumbold@wits.ac.za

Microbial Cell Factories

|December 5, 2009

Summary

Microbial hosts vary in their ability to use lignocellulosic hydrolysates and resist inhibitors. Selecting hosts based on substrate utilization, not just product formation, simplifies metabolic engineering for industrial fermentation.

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Area of Science:

Biotechnology
Industrial Microbiology
Biorefining

Background:

Lignocellulosic biomass hydrolysates are increasingly used as feedstocks for industrial fermentations.
These hydrolysates contain sugars, inhibitors, and salts that impact microbial host performance.
Evaluating microbial host suitability for diverse lignocellulosic feedstocks is crucial for efficient bioprocessing.

Purpose of the Study:

To compare the performance of six industrially relevant microorganisms (bacteria, yeasts, fungi) on lignocellulosic hydrolysates.
To assess microbial utilization of monosaccharides, resistance to inhibitors, and growth on various biomass feedstocks.
To evaluate the ability of these hosts to utilize waste glycerol from biodiesel production.

Main Methods:

Six microbial hosts (Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia stipitis, Aspergillus niger, Trichoderma reesei) were tested.
Feedstock hydrolysates were generated using two methods: mild acid/enzymatic hydrolysis and concentrated sulfuric acid treatment.
Microbial performance was evaluated based on sugar utilization, inhibitor resistance, and growth on different lignocellulosic hydrolysates (corn stover, wheat straw, bagasse, willow wood).

Main Results:

Significant performance differences were observed among the six microbial hosts.
Carbon source versatility and inhibitor resistance were key factors differentiating host performance.
Pichia stipitis and Aspergillus niger demonstrated the best overall performance, while Corynebacterium glutamicum and Saccharomyces cerevisiae were least adapted.

Conclusions:

A substrate-oriented approach for microbial host selection is more effective than a product-oriented one for utilizing lignocellulosic hydrolysates.
This approach minimizes the need for extensive metabolic engineering, focusing instead on optimizing the product biosynthesis pathway.