Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose

Jungseok Lee,Youngsoon Um,Han Min Woo,Jack N Saddler

doi:10.1186/s12934-016-0420-z

Abstract

BackgroundAn efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease.ResultsWe have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cellobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic analysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharification and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine.ConclusionsThe combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0420-z) contains supplementary material, which is available to authorized users.

Highlights

An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels
This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels
Utilization of cellobiose in C. glutamicum using metabolic engineering and adaptive evolution To test whether heterologous expressions of either CDT-1 transporter and bG or bG alone allow utilization of cellobiose in C. glutamicum, the N. crassa cdt-1 and gh1-1 gene were codon-optimized and introduced into a CoryneBrick vector [11], pBbEB1c (Table 1)

Summary

Introduction

An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. An industrial amino acid producer, Corynebacterium glutamicum [6] showed a broad range of sugar utilization. Since there are great potentials of C. glutamicum as a microbial cell factory to produce other commercially relevant chemicals and fuels [8,9,10] from renewable lignocellulosic biomass, efficient utilization of cellulosic sugars is an inevitable goal. The C. glutamicum R-CEL strain has been shown to utilize cellobiose, glucose and xylose simultaneously, but only possible under anaerobic conditions [16]

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