Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production
Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process p...
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doaj-5cc052bd87b8474b9ba785711f3695e02020-11-24T23:16:50ZengElsevierMetabolic Engineering Communications2214-03012017-06-014C122110.1016/j.meteno.2017.01.002Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid productionThiemo Zambanini0Hamed Hosseinpour Tehrani1Elena Geiser2Christiane K. Sonntag3Joerg M. Buescher4Guido Meurer5Nick Wierckx6Lars M. Blank7iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyiAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyiAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyiAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyBRAIN AG, Darmstädter Straße 34-36, D-64673 Zwingenberg, GermanyBRAIN AG, Darmstädter Straße 34-36, D-64673 Zwingenberg, GermanyiAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyiAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, GermanyUstilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L−1 malate and biomass (73 g L−1), succinate (20 g L−1), CO2 (17 g L−1), and α-ketoglutarate (8 g L−1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.http://www.sciencedirect.com/science/article/pii/S2214030116300414Carbon-balanceGlycerolMalateMetabolic engineeringOverexpressionUstilago trichophora |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Thiemo Zambanini Hamed Hosseinpour Tehrani Elena Geiser Christiane K. Sonntag Joerg M. Buescher Guido Meurer Nick Wierckx Lars M. Blank |
spellingShingle |
Thiemo Zambanini Hamed Hosseinpour Tehrani Elena Geiser Christiane K. Sonntag Joerg M. Buescher Guido Meurer Nick Wierckx Lars M. Blank Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production Metabolic Engineering Communications Carbon-balance Glycerol Malate Metabolic engineering Overexpression Ustilago trichophora |
author_facet |
Thiemo Zambanini Hamed Hosseinpour Tehrani Elena Geiser Christiane K. Sonntag Joerg M. Buescher Guido Meurer Nick Wierckx Lars M. Blank |
author_sort |
Thiemo Zambanini |
title |
Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production |
title_short |
Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production |
title_full |
Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production |
title_fullStr |
Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production |
title_full_unstemmed |
Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production |
title_sort |
metabolic engineering of ustilago trichophora tz1 for improved malic acid production |
publisher |
Elsevier |
series |
Metabolic Engineering Communications |
issn |
2214-0301 |
publishDate |
2017-06-01 |
description |
Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species.
Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L−1 malate and biomass (73 g L−1), succinate (20 g L−1), CO2 (17 g L−1), and α-ketoglutarate (8 g L−1) as main by-products.
These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation. |
topic |
Carbon-balance Glycerol Malate Metabolic engineering Overexpression Ustilago trichophora |
url |
http://www.sciencedirect.com/science/article/pii/S2214030116300414 |
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