Studies on the mechanism of synthesis of ethyl acetate in Kluyveromyces marxianus DSM 5422

Abstract

Kluyveromyces marxianus converts whey-borne sugar into ethyl acetate, an environmentally friendly solvent with many applications. K. marxianus DSM 5422 presumably synthesizes ethyl acetate from acetyl-SCoA. Iron limitation as a trigger for this synthesis is explained by a diminished aconitase and succinate dehydrogenase activity (both enzymes depend on iron) causing diversion of acetyl-SCoA from the tricarboxic acid cycle to ester synthesis. Copper limitation as another trigger for ester synthesis in this yeast refers to involvement of the electron transport chain (all ETC complexes depend on iron and complex IV requires copper). This hypothesis was checked by using several ETC inhibitors. Malonate was ineffective but carboxin partially inhibited complex II and initiated ester synthesis. Antimycin A and cyanide as complexes III and IV inhibitors initiated ester synthesis only at moderate levels while higher concentrations disrupted all respiration and caused ethanol formation. A restricted supply of oxygen (the terminal electron acceptor) also initiated some ester synthesis but primarily forced ethanol production. A switch from aerobic to anaerobic conditions nearly stopped ester synthesis and induced ethanol formation. Iron-limited ester formation was compared with anaerobic ethanol production; the ester yield was lower than the ethanol yield but a higher market price, a reduced number of process stages, a faster process, and decreased expenses for product recovery by stripping favor biotechnological ester production.

CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

“…Atf activity was observed when lysates were supplemented with ethanol and acetyl-CoA, while Adh activity was observed when supplementing with hemiacetal and NAD + cofactor. These results are in contrast to prior reports that describe Atf and reverse esterase activities as the only reactions responsible for ethyl acetate biosynthesis in K. marxianus DSM 5422 and other strains [17, 18]. In the CBS 6556 strain, we found no reverse esterase activity, but did identify Atf and Adh activities as possible routes for ethyl acetate production.…”

“…Adh1 from S. cerevisiae ( Sc Adh1) and an Adh from Neurospora crassa are also known to exhibit hemiacetal oxidation activity [15, 16], a reaction that is thought to be involved in aldehyde detoxification under conditions of low NADH availability [7]. In K. marxianus , both Atf and reverse esterase activities have been identified, and Atf activity is thought to be primarily responsible for ethyl acetate biosynthesis [17, 18]. However, the metabolism of K. marxianus is less well understood than that of S. cerevisiae , and promiscuous Adh activity toward hemiacetal oxidation and the roles of different Adh enzymes in ester and alcohol metabolism are not yet completely understood.…”

Efficient growth of Kluyveromyces marxianus biomass used as a biocatalyst in the sustainable production of ethyl acetate

“…This acidification was caused by consumption of ammonium without an equivalent uptake of sulfate. The use of ammonium sulfate as an N source calls for buffered medium (in shake-flask experiments [12,14]) or for pH control (in bioreactor experiments [10,11,13,16,22]). …”

“…Ammonium is usually supplemented in the form of ammonium sulfate where ammonium is intensively consumed while most of the sulfate remains in the medium and causes an ionic imbalance and acidification [15,[38][39][40][41]. Such acidification can be inhibitory to K. marxianus since its growth rate is distinctly reduced at pH ≤3.5 [22,28,42,43]. Supplementation of whey with (NH 4 ) 2 SO 4 or NH 4 Cl requires cost-intensive pH correction with NaOH or KOH which in turn increases the salt load and thus inhibits yeast growth [31,42] and creates waste-water problems.…”

“…Ethanol is another product of microbial sugar conversion, but several factors favor microbial ester over ethanol production [22]: the higher market price of the ester, a reduced number of process stages, a faster process, and a cost-saving product recovery.…”