The myo-inositol/proton symporter IolT1 contributes to d-xylose uptake in Corynebacterium glutamicum
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.
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2021, Metabolic EngineeringCitation Excerpt :This difference is presumably caused by the strongly increased expression of iolT1 (>70-fold increased mRNA level) caused by the deletion of iolR (Fig. 1) encoding the transcriptional regulator IolR (Cg0196), which represses iolT1 in the absence of myo-inositol (Klaffl et al., 2013). IolT1 is a secondary transporter with a relaxed substrate specificity, which does not only transport myo-inositol, but also glucose (Ikeda et al., 2011; Lindner et al., 2011) and xylose (Brüsseler et al., 2018). IolT1 provides an additional pathway for glucose uptake besides the glucose-specific phosphotransferase system PtsG.
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2020, Metabolic EngineeringCitation Excerpt :Xylan breakdown by C. glutamicum strain was achieved with endoxylanase (XlnA) from Streptomyces coelicolor A3 and xylosidase (XynB) from Bacillus pumilus (Imao et al., 2017; Kuge et al., 2017; Watanabe et al., 2015; Yim et al., 2016). The generated xylose can be imported into the C. glutamicum cell via IolT1 and/or IolT2, permeases that facilitate uptake of myo-inositol (Krings et al., 2006), fructose (Bäumchen et al., 2009), glucose (Lindner et al., 2011) and xylose (Brüsseler et al., 2018) and derepression of their genes via deletion of repressor gene iolR accelerates xylose utilization (Klaffl et al., 2013). Xylose uptake was also supported by heterologous uptake systems: XylE from E. coli (Yim et al., 2017) or AraE from C. glutamicum ATCC31831 or B. subtilis (Mao et al., 2018; Sasaki et al., 2009).