Identification and engineering a C 4 -dicarboxylate transporter for improvement of malic acid production in Aspergillus
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APPLIED MICROBIAL AND CELL PHYSIOLOGY
Identification and engineering a C4-dicarboxylate transporter for improvement of malic acid production in Aspergillus niger Wei Cao 1,2 & Luwen Yan 1 & Mengxin Li 1 & Xinyuan Liu 1 & Yongxue Xu 1 & Zhoujie Xie 1,2 & Hao Liu 1,2 Received: 20 February 2020 / Revised: 12 August 2020 / Accepted: 23 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Modification of C4-dicarboxylate transport processes is an important strategy for the development of efficient malic acid producing cell factory in Aspergillus niger. However, there is a lack of identification and functional research of malic acid transport proteins, which seriously hinders the construction of high-yield malic acid metabolic engineering strains. A C4dicarboxylate transport protein (DCT) DCT1 is identified as major malic acid transport protein and exhibits significant elevation in malic acid production when overexpressed. DCT1 is found by homology searches and domain analyses with SpMAE1 from Schizosaccharomyces pombe as the template. Phylogenetic and domain analyses show that DCTs belong to voltage-dependent slow-anion channel transporter (SLAC1) family and are members of Tellurite-resistance/Dicarboxylate Transporter (TDT) Family. DCT1 disruption dramatically decreases malic acid titer by about 85.6% and 96.2% at 3 days and 5 days compared with the parent strain, respectively. Meanwhile, the citric acid titers increase by 36.4% and 13.7% at 3 days and 5 days upon DCT1 deficiency. These results suggest that DCT1 is the major malic acid transporter in A. niger. Overexpression of dct1 with its native promoter significantly improves malic acid production yielding up to 13.86 g/L and 30.79 g/L at 3 days and 5 days, respectively, which is 36.8% and 22.8% higher than those in the parent strain. However, the citric acid has no significant change during the 5-day fermentation. These results demonstrate the importance of C4-dicarboxylate transporters for the efficient production of malic acid. Furthermore, enhancement of malic acid transport process is a feasible approach of efficient malic acid production in this citric acid producing A. niger strain. Key points • A dicarboxylate transporter DCT1 is identified as a major malic acid transporter. • DCT1 deficiency results in significant decrease of malic acid. • DCT1 overexpression leads to increased titers of malic acid. • Enhancement of malic acid transport is vital for malic acid production in A. niger. Keywords Dicarboxylate . Transport . Export . Malic acid . Aspergillus niger
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10932-1) contains supplementary material, which is available to authorized users. * Hao Liu [email protected] 1
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic
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