title: Fine-tuning of NADH oxidase decreases byproduct accumulation in respiration deficient xylose metabolic Saccharomyces cerevisiae.
abstract: BACKGROUND
  Efficiently utilizing all available carbon from lignocellulosic feedstock presents a major barrier to the production of economically feasible biofuel. Previously, to enable xylose utilization, we introduced a cofactor-dependent xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway, or a cofactor-independent xylose isomerase (XI) pathway, into Saccharomyces cerevisiae. The resulting strains metabolized xylose with high efficiency. However, in both pathway recombinant strains, the cofactor imbalance caused accumulation of the byproducts glycerol and/or xylitol and reduced the ethanol production efficiency.
  
  
  RESULTS
  In this study, we introduced NADH oxidase from Lactococcus lactis into both XI and XR-XDH pathway recombinant strains. To reduce byproduct accumulation while maintaining xylose metabolism, we optimized the expression level of NADH oxidase by comparing its expression under the control of different promoters and plasmids. In recombinant XI strains, NADH oxidase was expressed at different levels, regulated by the GPD2 promoter or TEF1 promoter in the 2 μ plasmid. The expression under the control of GPD2 promoter decreased glycerol production by 84% and increased the ethanol yield and specific growth rate by 8% and 12%, respectively. In contrast, in the recombinant XR-XDH strains, such expression level was not efficient enough to decrease the byproduct accumulation. Therefore, higher NADH oxidase expression levels were tested. In the strain expressing NADH oxidase under the control of the TEF1 promoter in the centromeric plasmids, xylitol and glycerol production were reduced by 60% and 83%, respectively, without significantly affecting xylose consumption.
  
  
  CONCLUSIONS
  By fine-tuning NADH oxidase expression, we decreased the glycerol or/and xylitol production in both recombinant XI and XR-XDH xylose-metabolizing yeast strains. The optimal NADH oxidase expression levels depend on metabolic pathways. Similar cofactor engineering strategies could maximize the production of other redox dependent metabolites.
authors: Jin
authors: J
authors: Hou
authors: Fan
authors: F
authors: Suo
authors: Chengqiang
authors: C
authors: Wang
authors: Xiaowei
authors: X
authors: Li
authors: Yu
authors: Y
authors: Shen
authors: State Key Laboratory of Microbial Technology, Shandong University, Shanda Nan Road 27, Jinan 250100, China. bxm@sdu.edu.cn.
authors: Xiaoming
authors: X
authors: Bao
mesh_terms: D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods; D005990:Glycerol / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D013294:Lactococcus lactis / Q000201:enzymology; D053858:Metabolic Networks and Pathways; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D000431:Ethanol; D014994:Xylose; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D005990:Glycerol