Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories

In silico evolution of Aspergillus niger organic acid production suggests strategies for switching acid output

“…Malic acid production evolved primarily through a partial disruption of citrate synthase, which all solutions shared in common, and could be achieved via a specific constraint on citric production as malic production is the next most efficient means of acidification in its absence. A notable study reported recently successfully engineered A. niger ATCC1015 to produce significant quantities of malic acid via the targeting of four steps using a Cre-loxP based gene editing system [39]. The knockout of oxaloacetate hydrolase together with the overexpression of pyruvate carboxylase, malate dehydrogenase and a C4-dicarboxylate transporter achieved 120 g/L malic acid in shake flask culture and 200 g/L malic acid in fedbatch fermentation [39].…”

“…A notable study reported recently successfully engineered A. niger ATCC1015 to produce significant quantities of malic acid via the targeting of four steps using a Cre-loxP based gene editing system [39]. The knockout of oxaloacetate hydrolase together with the overexpression of pyruvate carboxylase, malate dehydrogenase and a C4-dicarboxylate transporter achieved 120 g/L malic acid in shake flask culture and 200 g/L malic acid in fedbatch fermentation [39]. Although the yield of the target product was very high in the engineered strain, citric acid was still produced at 28 g/L showing that a complete switch of acid output had not occurred.…”

Accumulation of Malic Acid and Other Industrially Important Organic Acids by Aspergillus Tubingensis An1257 / Produção Biotecnológica De Ácido Málico Por Aspergillus Tubingensis An1257

“…The question is why pH near 6 or 7 is better, once high activation of OAH could deplete oxaloacetate to form oxalate. Indeed, manipulation of A. niger to contain several copies of an OAH-encoding gene increased oxalic acid production, while deletion of this gene plus insertion of the genes encoding pyruvate carboxylase and malate dehydrogenase resulted in a strain overproducing malic acid (Xu et al, 2019 It could also be formed from oxaloglycolate, yet by catalysis of a tartrate dehydrogenase, or from meso-tartrate, which are both linked to ascorbate metabolism. The ascorbate route is important for production of tartrate in higher plants, including grapes, where tartaric acid contributes to the acidic component and confers microbiological stability.…”

“…Genetic engineering of A. niger allowed the production of malic acid in elevated quantities, attaining almost 70% the theoretical yield of bioconversion from glucose (Brown et al, 2013). Other authors also applied genetic engineering of A. niger to produce high amounts of malic acid in fed-batch processes (Xu et al, 2019). However, complex media were used for production, requiring not only glucose in high concentration and regular supplementation, but also an expensive supplement like peptone, and micro-nutrients.…”

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

“…Arguably, the first and most important technology for genetic manipulation of any given A. niger isolate is development of an efficient selection marker for transformation. The orotidine-5′-decarboxylase encoding gene pyrG has been widely applied as a recyclable transformation marker in many fungal species [8,11,[20][21][22][23]. The use of this system requires that a strain of interest firstly has pyrG disrupted/deleted, generating a uridine auxotrophy as the orotidine-5′-monophosphate (OMP) decarboxylase.…”