Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

Nicolaus A Herman; Jeffrey Li; Ripika Bedi; Barbara Turchi; Xiaoji Liu; Michael J Miller; Wenjun Zhang

doi:10.1128/AEM.02942-16

Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

Appl Environ Microbiol. 2016 Dec 30;83(2):e02942-16. doi: 10.1128/AEM.02942-16. Print 2017 Jan 15.

Authors

Nicolaus A Herman¹, Jeffrey Li¹, Ripika Bedi², Barbara Turchi³, Xiaoji Liu³, Michael J Miller³, Wenjun Zhang⁴

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of California-Berkeley, Berkeley, California, USA.
² Department of Chemistry, University of California-Berkeley, Berkeley, California, USA.
³ Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
⁴ Department of Chemical and Biomolecular Engineering, University of California-Berkeley, Berkeley, California, USA wjzhang@berkeley.edu.

Abstract

While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 10⁶ CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production.

Importance: This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.

Keywords: ABE fermentation; Clostridium; biofuel; genetics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biofuels / analysis*
Butanols / metabolism*
Clostridium / metabolism*
Fermentation
Metabolic Engineering / methods*

Substances

Biofuels
Butanols