Engineering cytoplasmic acetyl-CoA synthesis decouples lipid production from nitrogen starvation in the oleaginous yeast Rhodosporidium azoricum

Abstract

BackgroundOleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful.ResultsIn this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to simulate an industrial process for lipid production from lignocellulosic materials, we obtained an increase of 89% in final lipid concentration by the engineered strain in comparison to the wild type. This indicates that the transformed strain can produce higher cellular biomass with a high lipid content than the wild type. The transformed strain furthermore evidenced the advantage over the wild type in performing this process, being the lipid yields 0.13 and 0.05, respectively.ConclusionOur results show that the overexpression of homologous Xfpk and heterologous Pta activities in R. azoricum creates a new cytosolic AcCoA supply that decouples lipid production from nitrogen starvation. This metabolic modification allows improving lipid production in cultural conditions that can be suitable for the development of industrial bioprocesses using lignocellulosic hydrolysates.

Recycling Industrial Food Wastes for Lipids Production by Oleaginous Yeasts Rhodosporidiobolus Azoricus and Cutaneotrichosporon Oleaginosum.

“…The main differences are found in terms of nal amount of produced biomass and in biomass yield, indicating that fructose is metabolized in a more e cient way than glucose. These results were unexpected, because the growth of R. azoricus had been previously documented as occurring very e ciently on glucose-based media, especially for lipids production (Galafassi et al, 2012;Capusoni et al, 2017;Donzella et al, 2019). However, the media used for lipids production, containing yeast extract or corn steep, are not exclusively mineral as the one used in the present study.…”

“…Typically, when nitrogen level is low and the sugar content is still high (high C/N ratio), the carbon ux is diverted from acetyl-CoA (via citric acid) towards fatty acid biosynthesis (Ageitos et al, 2011;Vorapreeda et al, 2012). Remarkably, given their increasing role played in the eld of microbial oils production, some oleaginous species have been engineered in order to by-pass strict metabolic regulation mechanisms, to enlarge their substrate range, as well as to modify their FAs composition (Tai and Stephanopoulos, 2012;Qiao et al, 2017;Donzella et al, 2019;Chattopadhyay et al 2021). Oleaginous yeasts show ability to grow using a variety of carbon sources, including those present in waste streams from the agro-food sector (Huang et al, 2013;Qin et al, 2017).…”

Prospect of metabolic engineering in enhanced microbial lipid production: review

“…Studies in which the newly developed genetic tools have been utilised primarily includes the oleaginous yeast R. azoricum, in which the homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta) genes were overexpressed in order to introduce a new cytoplasmic route for the formation of acetyl-CoA. The engineered strain when grown in a bioreactor in the presence of a glucose/ xylose mixture produced a higher lipid concentration with an increase of 89% in comparison to the wild type [110]. In the case of Candida phangngensis-a phylogenetic relative of Y. lipolytica, genetic tools like promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system established in the latter proved to be transferable in the yeast requiring little to no modifications.…”

Bioprocesses with Reduced Ecological Footprint by Marine Debaryomyces hansenii Strain for Potential Applications in Circular Economy

“…The DNA was washed with 70% ethanol and dissolved in 50 µL of TE RNAsi (10 mMTris-HCl, 1 mM EDTA, pH 7.5 RNAsi 100 µg/mL). Samples were incubated at 37 • C for 30 min [27].…”

“…By using a rich medium, the protein content of D. hansenii Mo40 was comparable to well established meat alternatives such as Quorn by Fusarium venenatum [38] and oncom by Neurospora intermedia [39]. In order to evaluate the ability to produce SCO, D. hansenii Mo40 cells were cultured on lipidogenic medium characterized by low nitrogen content, known to induce lipids accumulation [27]. Under these conditions (Figure 3), after 24 h of cultivation, due to nitrogen exhaustion, lipids started to accumulate and after 72 h of cultivation the biomass reached a lipid content of 20.7%.…”