Vol.:(0123456789) 1 3 Journal of Industrial Microbiology & Biotechnology (2019) 46:159–169 https://doi.org/10.1007/s10295-018-2117-2 METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - ORIGINAL PAPER Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane‑bound pyridine nucleotide transhydrogenase Rhudith B. Cabulong 1  · Kris Niño G. Valdehuesa 1  · Angelo B. Bañares 1  · Kristine Rose M. Ramos 1  · Grace M. Nisola 1  · Won‑Keun Lee 2  · Wook‑Jin Chung 1 Received: 29 October 2018 / Accepted: 27 November 2018 / Published online: 15 December 2018 © Society for Industrial Microbiology and Biotechnology 2018 Abstract The non-conventional D-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce pyruvate and glycolaldehyde has been previously engineered in Escherichia coli. Strains that rely on this pathway exhibit lower growth rates which were initially attributed to the perturbed redox homeostasis as evidenced by the lower intracellular NADPH concentrations during exponential growth phase. NADPH-regenerating systems were then tested to restore the redox homeostasis. The membrane-bound pyridine nucleotide transhydrogenase, PntAB, was overexpressed and resulted to a signifcant increase in biomass and glycolic acid titer and yield. Furthermore, expression of PntAB in an optimized glycolic acid-producing strain improved the growth and product titer signifcantly. This work demonstrated that compensating for the NADPH demand can be achieved by overexpression of PntAB in E. coli strains assimilating D-xylose through the Dahms pathway. Consequently, increase in biomass accumulation and product concentration was also observed. Keywords NADPH · PntAB · Glycolic acid · Escherichia coli · Dahms pathway · Cofactor regeneration Introduction The conventional D-xylose metabolism through the isomer- ase (XIP) or oxo-reductive pathways (ORP) have been well studied in the metabolic engineering of microorgan- isms [28]. In recent years, interest in employing the xylose oxidative pathway (XOP) has emerged. Unlike the XIP and ORP, XOP involves D-xylose oxidation to D-xylonate (Fig. 1). Dehydratase and aldolase reactions further con- vert D-xylonate to pyruvate and glycolaldehyde [8]. Alter- natively, D-xylonate undergoes two dehydration steps and another oxidation reaction to form 2-ketoglutarate [32]. The former is regarded as the Dahms pathway, while the latter is called the Weimberg pathway. The Dahms pathway has been previously exploited in the biosynthesis of ethylene glycol (EG), glycolic acid (GA), 1,2,4-butanetriol, D-xylonate, and polyhydroxyalkanoates in recombinant Escherichia coli [4–6, 16, 27]. The key advan- tage of the Dahms pathway compared to the XIP and ORP is its shorter path for D-xylose conversion to pyruvate or 2-ketoglutarate, which are both essential intermediates for cell growth [28]. However, E. coli strains operating through the Dahms pathway often show low biomass concentrations [4, 5, 16]. The possible major contributing factors to such phenotype include slow and incomplete carbon assimilation due to D-xylonate accumulation, low levels of expression or activity of downstream enzymes, and intracellular redox cofactor imbalance. Lowering expression levels of xylose dehydrogenase by placing xdh under the control of a consti- tutive weak promoter has been proven successful in mini- mizing D-xylonate accumulation [5]. Meanwhile, selection Rhudith B. Cabulong and Kris Niño G. Valdehuesa have contributed equally to this work. * Won-Keun Lee wklee@mju.ac.kr * Wook-Jin Chung wjc0828@gmail.com 1 Department of Energy Science and Technology (DEST), Energy and Environment Fusion Technology Center (E2FTC), Myongji University, Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do 170-58, South Korea 2 Division of Bioscience and Bioinformatics, Myongji University, Myongji-ro 116, Cheoin-gu, Yongin, Gyeonggi-do 170-58, South Korea Downloaded from https://academic.oup.com/jimb/article/46/2/159/5997155 by guest on 29 October 2022