Elsevier

Bioresource Technology

Volume 249, February 2018, Pages 953-961
Bioresource Technology

The myo-inositol/proton symporter IolT1 contributes to d-xylose uptake in Corynebacterium glutamicum

https://doi.org/10.1016/j.biortech.2017.10.098Get rights and content

Highlights

  • Deletion of iolR improves growth of C. glutamicum on d-xylose containing media.

  • Permease IolT1 contributes to d-xylose uptake in C. glutamicum.

  • Engineering of the iolT1 promoter renders iolR-deletion unnecessary.

  • Minimally engineered C. glutamicum WMB2 PO6 iolT1 allows for high growth rates on d-xylose.

Abstract

Corynebacterium glutamicum has been engineered to utilize d-xylose as sole carbon and energy source. Recently, a C. glutamicum strain has been optimized for growth on defined medium containing d-xylose by laboratory evolution, but the mutation(s) attributing to the improved-growth phenotype could not be reliably identified. This study shows that loss of the transcriptional repressor IolR is responsible for the increased growth performance on defined d-xylose medium in one of the isolated mutants. Underlying reason is derepression of the gene for the glucose/myo-inositol permease IolT1 in the absence of IolR, which could be shown to also contribute to d-xylose uptake in C. glutamicum. IolR-regulation of iolT1 could be successfully repealed by rational engineering of an IolR-binding site in the iolT1-promoter. This minimally engineered C. glutamicum strain bearing only two nucleotide substitutions mimics the IolR loss-of-function phenotype and allows for a high growth rate on d-xylose-containing media (µmax = 0.24 ± 0.01 h−1).

Introduction

Corynebacterium glutamicum is employed for the microbial production of amino acids such as l-glutamate and l-lysine on a large industrial scale of several million tons per year (Eggeling and Bott, 2015). In addition, engineered C. glutamicum strains for the production of various organic acids, alcohols and natural products such as carotenoids, stilbenes and flavonoids are available, rendering this microorganism a versatile microbial platform for a broad range of future biotechnological applications (Heider et al., 2012, Kallscheuer et al., 2016, Kallscheuer et al., 2017, Litsanov et al., 2012, Vogt et al., 2016).

Traditionally, d-glucose and d-fructose, mostly derived from starch hydrolysates or molasses, serve as feedstocks for large-scale productions employing C. glutamicum (Blombach and Seibold, 2010). With the aim to minimize competition for these hexoses between the industrial sectors of human nutrition and industrial biotechnology, C. glutamicum strains utilizing cellobiose, l-arabinose or d-xylose have been engineered, as these carbohydrates constitute a large portion of typical agro-waste streams (Kawaguchi et al., 2006, Kotrba et al., 2003, Radek et al., 2014, Schneider et al., 2011). In case of d-xylose, two different metabolic strategies have been implemented in C. glutamicum as this bacterium cannot naturally metabolize this pentose. In the isomerase pathway, d-xylose is first converted to d-xylulose by a heterologous xylose isomerase (encoded by xylA from either Escherichia coli or Xanthomonas campestris) and subsequently phosphorylated by an endogenous xylulokinase (encoded by xylB) yielding xylulose-5-phosphate (Fig. 1A) (Kawaguchi et al., 2006, Meiswinkel et al., 2013).

As this compound is an intermediate of the pentose phosphate pathway, it can be rapidly metabolized by C. glutamicum. Engineered strains using this pathway for d-xylose utilization allowed for the microbial synthesis of the amino acids l-lysine, l-glutamate and l-ornithine and the diamine putrescine from d-xylose (Meiswinkel et al., 2013). However, a significant fraction of the d-xylose-derived carbon is lost in the form of CO2 during synthesis of α-ketoglutarate-derived products such as l-glutamate, lowering the overall product yield when d-xylose is metabolized by the isomerase pathway.

In contrast, the Weimberg pathway as alternative strategy for d-xylose-metabolization offers the possibility to directly convert d-xylose to the citric acid cycle intermediate and direct l-glutamate precursor α-ketoglutarate without loss of any carbon (Fig. 1B) (Johnsen et al., 2009, Stephens et al., 2007, Weimberg, 1961). In this pathway d-xylose is first oxidized by a xylose dehydrogenase (encoded by xylB) yielding d-xylonolactone, which is subsequently hydrolyzed by a d-xylonolactonase (encoded by xylC) to d-xylonate. In the two following reaction steps two molecules of water are successively eliminated by a d-xylonate dehydratase (encoded by xylD) and a 2-keto-3-deoxyxylonate dehydratase (encoded by xylX) yielding α-ketoglutarate semialdehyde. Finally, this compound is oxidized to α-ketoglutarate by a α-ketoglutarate semialdehyde dehydrogenase (encoded by xylA). Establishing this five-step pathway, originally discovered in Pseudomonas fragi and later also in Haloferax volcanii and Caulobacter crescentus, could be achieved by heterologous expression of the xylXABCD-operon from C. crescentus in C. glutamicum (Radek et al., 2014). Presence and activity of the Weimberg pathway enabled growth of C. glutamicum pEKEx3-xylXABCDCc on d-xylose as sole carbon and energy source with µmax = 0.07 ± 0.01 h−1 (Radek et al., 2014).

Recently, a C. glutamicum strain with a plasmid bearing the five genes of the Weimberg pathway as codon-optimized versions for expression in C. glutamicum and organized as a synthetic operon was subjected to an adaptive laboratory evolution experiment to improve growth on d-xylose as sole carbon and energy source (Radek et al., 2017). The best two strains allowed for growth at a maximum growth rate of µmax = 0.26 ± 0.02 h−1 and genome sequencing of these strains revealed several mutations potentially contributing to improved growth on d-xylose, but a detailed analysis has not been performed yet.

In this study, we present a detailed characterization of one of these two C. glutamicum strains bearing a large deletion in the iolR gene, which encodes for a repressor involved in myo-inositol catabolism, and investigate its impact on d-xylose uptake in different C. glutamicum strains. Furthermore, we describe the rational reengineering of a C. glutamicum strain mimicking the iolR loss-of-function mutation.

Section snippets

Bacterial strains, plasmids, media, and growth conditions

All used bacterial strains and plasmids are listed in Table 1. Escherichia coli DH5α was used for cloning purposes and was grown aerobically on a rotary shaker (170 rpm) at 37 °C in 5 mL Lysis Broth (LB) medium (Bertani, 1951) or on LB agar plates (LB medium with 1.8% [wt/vol] agar). All C. glutamicum strains are derived from C. glutamicum ATCC 13032 (Abe et al., 1967) and were routinely pre-cultivated aerobically in Brain Heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) on a

Deletion of iolR improves growth via the Weimberg pathway on d-xylose

Adaptive laboratory evolution of C. glutamicum ATCC 13032 pEKEx3-xylXABCDCc-opt (WMB2), engineered for the plasmid-based expression of the codon-optimized xylXABCD-operon from C. crescentus, yielded the strain C. glutamicum WMB2evo with the highest reported growth rate of 0.26 ± 0.02 h−1 on d-xylose as sole carbon and energy source (Radek et al., 2017). Genome sequencing identified a larger deletion of 98 base pairs from position 133 to 232 relative to the start codon in the open-reading frame

Conclusions

Loss of the transcriptional repressor IolR in C. glutamicum strains either employing the isomerase- or the Weimberg pathway improves growth on defined d-xylose containing media. Reason for this is an increased expression of iolT1, which encodes a permease contributing to d-xylose uptake in this bacterium. Introduction of two point mutations into the proximal IolR-binding site of the iolT1-promoter mimics the IolR loss-of function phenotype and allows for high growth rates on d-xylose containing

Acknowledgements

This work was funded by the German Federal Ministry of Education and Research (BMBF, Grant. No. 031L0015) as part of the project “XyloCut – Shortcut to the carbon efficient microbial production of chemical building blocks from lignocellulose-derived d-xylose”, which is embedded in the ERASysAPP framework.

References (38)

  • M. Vogt et al.

    Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

    Metab. Eng.

    (2016)
  • R. Weimberg

    Pentose oxidation by Pseudomonas fragi

    J. Biol. Chem.

    (1961)
  • S. Abe et al.

    Taxonomical studies on glutamic acid-producing bacteria

    J. Gen. Appl. Microbiol.

    (1967)
  • C. Bäumchen et al.

    Myo-inositol facilitators IolT1 and IolT2 enhance d-mannitol formation from d-fructose in Corynebacterium glutamicum

    FEMS Microbiol. Lett.

    (2009)
  • G. Bertani

    Studies on Lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli

    J. Bacteriol.

    (1951)
  • B. Blombach et al.

    Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of l-lysine production strains

    Appl. Microbiol. Biotechnol.

    (2010)
  • L. Eggeling et al.

    Handbook of Corynebacterium glutamicum

    (2005)
  • L. Eggeling et al.

    A giant market and a powerful metabolism: l-lysin provided by Corynebacterium glutamicum

    Appl. Microbiol. Biotechnol.

    (2015)
  • R. Gande et al.

    The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis

    J. Bacteriol.

    (2007)
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