Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents.

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from ß-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress.

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner-Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer's productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

Microbial electrochemical ammonia recovery from anaerobic digester centrate and subsequent application to fertilize Arabidopsis thaliana.

Although ammonia recovery from wastewater can be environmentally friendly and energy efficient compared to the conventional Haber-Bosch process, there is a lack of research on the reuse of the recovered ammonia to exhibit a complete picture of resource recovery. In this study, a microbial electrochemical system (MES) was used to recover ammonia from a mixture of anaerobic digester (AD) centrate and food wastewater at a volume ratio of 3:1. More than 60% of ammonia nitrogen was recovered with energy consumption of 2.7 kWh kg-1 N. The catholyte of the MES, which contained the recovered ammonia, was used to prepare fertilizers to support the growth of a model plant Arabidopsis thaliana. It was observed that A. thaliana grown on the MES generated fertilizer amended with extra potassium, phosphorus, and trace elements showed comparable sizes and an even lower death rate (0%) than the control group (24%) that was added with a commercial fertilizer. RNA-Seq analyses were used to examine A. thaliana genetic responses to the MES generated fertilizers or the commercial counterpart. The comparative study offered metabolic insights into A. thaliana physiologies subject to the recovered nitrogen fertilizers. The results of this study have demonstrated the potential application of using the recovered ammonia from AD centrate as a nitrogen source in fertilizer and identified the necessity of supplementing other nutrient elements.

Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock.

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a ß-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best ß-carotene titer (121 ±â€¯13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

Biosynthesis of terpene compounds using the non-model yeast Yarrowia lipolytica: grand challenges and a few perspectives.

Yarrowia lipolytica has emerged as an important non-model host for terpene production. However, three main challenges remain in industrial production using this yeast. First, considerable knowledge gaps exist in metabolic flux across multiple compartments, cofactor generation, and catabolism of non-sugar carbon sources. Second, many enzymatic steps in the complex-terpene synthesis pathway can pose rate-limitations, causing accumulation of toxic intermediates and increased metabolic burdens. Third, metabolic shifts, morphological changes, and genetic mutations are poorly characterized under industrial fermentation conditions. To overcome these challenges, systems metabolic analysis, protein engineering, novel pathway engineering, model-guided strain design, and fermentation optimization have been attempted with some successes. Further developments that address these challenges are needed to advance the Yarrowia lipolytica platform for industrial-scale production of high-value terpenes, including those with highly complex structures such as anticancer molecules withanolides and insecticidal limonoids.