Metabolic engineering of Rhizopus oryzae for the production of platform chemicals

Abstract

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes . It is among others known for its ability to produce the sustainable platform chemicals l -(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l -(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.

Organic acid production in Aspergillus niger and other filamentous fungi

“…However, the ∆ r G' for the conversion of malate to fumarate at physiological conditions is 3.5 ± 0.6 kJ·mol -1 , suggesting higher concentrations of malate than fumarate at equilibrium (Meussen et al, 2012). Thus, accumulation of high amounts of fumarate by R. delemar could not unambiguously be explained by the properties of the characterised FumR.…”

“…by Meussen et al (2012). In summary, the reaction kinetics of FumR favour the conversion of fumarate to L-malate rather than the reverse direction, and FumR activity of acid-producing mycelium is completely blocked in the presence of 2 mM fumarate (note that the data on FumR inhibition have never been published, but that this finding is cited by Goldberg et al (2006); Meussen et al (2012).…”

“…Although technically not as mandatory a step as substrate import and conversion, citrate export is suggested to be the bottleneck of A. niger citrate production (Karaffa and Kubicek, 2003). Similarly, production of the C4-dicarboxylic acids fumarate, malate and succinate is suggested to be bottlenecked at the transport level across various cell-organelles and into the extracellular medium (Meussen et al, 2012).…”

“…Reductive and oxidative TCA cycles are connected by the pyruvate branch-point, which is an important cross-road for many metabolic engineering approaches as, depending on the culture conditions, pyruvate will be converted into a number of different compounds (Meussen et al, 2012).…”

“…To increase the amount of fumarate produced, R. delemar has been genetically modified (Meussen et al, 2012;Zhang et al, 2012;Zhang and Yang, 2012), but the occurrence of an ancestral whole-genome duplication as well as more recent gene-duplication events complicate the genetic engineering of R. delemar (Ma et al, 2009). A more promising approach for biological fumarate production would thus be rewiring the metabolism of a genetically more amenable cell-factory, based on R. delemar fumarate synthesis pathways.…”