Conversion of anaerobic fermentation derived volatile fatty acids to biodiesel - Comparative Transcriptomic and Proteomic Analysis of the Lipid Producing Yeast Cryptococcus albidus

Abstract Oleaginous yeast Cryptococcus albidus has demonstrated the potential to efficiently convert ‘non-ideal’ carbon sources such as volatile fatty acids (VFA) generated from anaerobic fermentation of organic waste streams into lipids [1]. However, the underlying metabolic pathways and factors affecting the lipid productivity (oleaginity) of C. albidus and other oleaginous microorganism are poorly understood. Relative carbon to nitrogen ratio (COD:N) has long been implicated to play a role in oleaginity and based primarily on empirical evidence, it is assumed that nitrogen acts as a metabolic ‘switch’ to divert carbon away from catabolic tricarboxylic acid (TCA) cycle towards anabolic fatty acid biosynthesis under nitrogen limited growth conditions. This phenomena has been reported in several studies[2, 3] utilizing a diverse array of feed stocks and microorganisms, including our previous study, where the intracellular lipid content of C. albidus cultures subjected to nitrogen limitation increased by 52% w/w (from 19% to 29%) [1].Several studies[3-6] have attempted to elucidate the underlying biochemical response of oleaginous microorganisms to nitrogen limitation induced stress. However, nearly all attempts at such characterization have utilized glucose as the carbon and energy source and therefore, the current understanding of lipogenesis is heavily skewed towards the changes in carbohydrate metabolism. However, from a practical perspective, glucose is far too expensive to support any large-scale application of such platforms and therefore, it is necessary to understand the metabolism of alternate cheaper carbon sources. To our knowledge, no literature pertaining to - omics of oleaginity in microbes utilizing VFA or other less ideal carbon sources currently exists. Here, we report for the first time, a comprehensive account of the transcriptome and proteome level changes in continuous cultures of Cryptococcus albidus in response to nitrogen limitation.The overall picture obtained was that nitrogen limitation resulted in a complete redistribution of carbon flux throughout the cellular processes, including TCA, gluconeogenesis, pentose phosphate pathway, nitrogenous compound recycling, autophagy and, nucleic acid and ribosome biosynthesis. C. albidus attempts to minimize its nitrogen usage and conserve its ammonium inventory by salvaging it from any non-essential metabolites. In purview of the global –omic response, the most highly up-regulated genes and proteins were involved in nitrogen uptake, protein turnover and autophagy to utilize assimilated nitrogen (Figure 1). We also highlight the interconnectedness of the global carbon and nitrogen cycles and the overexpression of genes influencing the distribution of carbon flux between oxidative and assimilative pathways under nitrogen limitation. Differential gene expression data revealed complete suppression of catabolic pathways including the TCA cycle processes under nitrogen limited conditions with nearly all the related genes undergoing a negative log fold change (log2FC) compared to sufficient nitrogen conditions, indicating their down-regulation. We also report the metabolic basis of differences in uptake and assimilation of various VFA, resulting in their differential uptake rate with cells demonstrating a preference for acetate over other VFA. Lipid accumulation by C. albidus however, does not seem to involve transcriptional regulation but is a passive consequence of carbon flux redistribution and excess availability of cytosolic acetyl-CoA during nitrogen limitation.In conclusion, the catabolic and anabolic fluxes of carbon in C. albidus seem to be intricately linked through nitrogen availability and it was quite evident that the nitrogen limitation had a severe impact on the over all carbon flux into various cellular processes, causing the carbon to accumulate in the cytosol and be channeled towards lipid storage. Although, this study reaffirms the classical opinion of nitrogen-mediated lipid biogenesis, it also highlights the significant differences in carbon metabolism under growth on alternate carbon sources and, by integrating the global transcriptomic and proteomic responses, it presents a much broader and clearer view of oleaginity of C. albidus.This comparative transcriptome and proteome data is expected to help elucidate factors driving lipid accumulation in C. albidus and contribute toward bioprocess development and for optimization engineered lipid production from ‘waste’ streams.