Abstract
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 120gL−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 134gL−1 malate and biomass (73gL−1), succinate (20gL−1), CO2 (17gL−1), and α-ketoglutarate (8gL−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.
Highlights
The biotechnological production of chemicals has gained great interest in the last decades
Wildtype strain RK089 adapted to glycerol by adaptive laboratory evolution RK089 with genomic integration of pSMUT; hygromycin resistant RK089 episomally expressing pNEBUC; carboxin resistant RK089 episomally expressing pNEBUN; nourseothricin resistant RK089 episomally expressing pNEBUP; phleomycin resistant TZ1 with genomic integration of pUTr01; carboxin resistant TZ1 with genomic integration of pUTr01-malate dehydrogenases UMAG_11161 (Mdh1); carboxin resistant TZ1 with genomic integration of pUTr01-Mdh2 (m); carboxin resistant TZ1 with genomic integration of pUTr01-Mdh2 (c); carboxin resistant TZ1 with genomic integration of pUTr01-pyruvate carboxylase UMAG_01054 (Pyc); carboxin resistant TZ1 with genomic integration of pUTr01-Ssu1; carboxin resistant TZ1 with genomic integration of pUTr01-Ssu2; carboxin resistant
In S. cerevisiae combined overexpression of the native pyruvate carboxylase gene pyc2, an allele of the peroxisomal malate dehydrogenase gene mdh3, which had been retargeted to the cytosol by deletion of the C-terminal targeting sequence, and expression of the Schizosaccharomyces pombe malate transporter gene mae1 resulted in a malic acid titer of 59 g L−1 produced with a yield of 0.42 molmal molglu−1 (Zelle et al, 2008)
Summary
The biotechnological production of chemicals has gained great interest in the last decades. The final strain overexpressing all conversion and transport steps from pyruvate to extracellular malic acid reached a 2.6-fold increased titer of 154 g L−1 produced at a rate of 0.94 g L−1 h−1 reaching a yield of 1.38 mol mol−1 on glucose (Brown et al, 2013). This clearly demonstrates the importance of the reductive tricarboxylic acid (rTCA) pathway among the five identified possible microbial production pathways for malic acid (Zelle et al, 2008). In other organisms, such as different Aspergillus species and R. oryzae, this pathway has been shown to be essential in efficient malic acid production (Bercovitz et al, 1990; Goldberg et al, 2006; Osmani and Scrutton, 1983; Peleg et al, 1988)
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