Effects of Inhibitory Compounds Present in Lignocellulosic Biomass Hydrolysates on the Growth of Bacillus subtilis

This study evaluated the individual and combined effects of inhibitory compounds formed during pretreatment of lignocellulosic biomass on the growth of Bacillus subtilis. Ten inhibitory compounds commonly present in lignocellulosic hydrolysates were evaluated, which included sugar degradation products (furfural and 5-hydroxymethylfurfural), acetic acid, and seven phenolic compounds derived from lignin (benzoic acid, vanillin, vanillic acid, ferulic acid, p-coumaric acid, 4-hydroxybenzoic acid, and syringaldehyde). For the individual inhibitors, syringaldehyde showed the most toxic effect, completely inhibiting the strain growth at 0.1 g/L. In the sequence, assays using mixtures of the inhibitory compounds at a concentration of 12.5% of their IC50 value were performed to evaluate the combined effect of the inhibitors on the strain growth. These experiments were planned according to a Plackett–Burman experimental design. Statistical analysis of the results revealed that in a mixture, benzoic acid and furfural were the most potent inhibitors affecting the growth of B. subtilis. These results contribute to a better understanding of the individual and combined effects of inhibitory compounds present in biomass hydrolysates on the microbial performance of B. subtilis. Such knowledge is important to advance the development of sustainable biomanufacturing processes using this strain cultivated in complex media produced from lignocellulosic biomass, supporting the development of efficient bio-based processes using B. subtilis.

Engineering the Oleaginous Yeast Rhodosporidium toruloides for Improved Resistance Against Inhibitors in Biomass Hydrolysates

Conversion of lignocellulosic biomass into lipids and related chemicals has attracted much attention in the past two decades, and the oleaginous yeast Rhodosporidiumtoruloides has been widely used in this area. While R. toruloides species naturally have physiological advantages in terms of substrate utilization, lipid accumulation, and inhibitor resistance, reduced lipid production and cell growth are noticed when biomass hydrolysates are used as feedstocks. To improve the robustness of R. toruloides, here, we devised engineered strains by overexpressing genes responsible for phenolic compound degradation. Specifically, gene expression cassettes of the manganese peroxidase gene (MNP) and versatile peroxidase gene (VP) were constructed and integrated into the genome of R. toruloides NP11. A series of engineered strains were evaluated for lipid production in the presence of typical phenolic inhibitors. The results showed that R. toruloides strains with proper expression of MNP or VP indeed grew faster in the presence of vanillin and 5-hydroxymethylfurfural than the parental strain. When cultivated in concentrated mode biomass hydrolysates, the strain VP18 had improved performance as the cell mass and lipid content increased by 30% and 25%, respectively. This study provides more robust oleaginous yeast strains for microbial lipid production from lignocellulosic biomass, and similar efforts may be used to devise more advanced lipid producers.

A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveals Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

Yarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates.

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, β-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates.

Abatement of Inhibitors in Recycled Process Water from Biomass Fermentations Relieves Inhibition of a Saccharomyces cerevisiae Pentose Phosphate Pathway Mutant

Understanding the nature of fermentation inhibition in biomass hydrolysates and recycled fermentation process water is important for conversion of biomass to fuels and chemicals. This study used three mutants disrupted in genes important for tolerance to either oxidative stress, salinity, or osmolarity to ferment biomass hydrolysates in a xylose-fermenting Saccharomyces cerevisiae background. The S. cerevisiaeZWF1 mutant with heightened sensitivity to fermentation inhibitors was unable to ferment corn stover dilute-acid hydrolysate without conditioning of hydrolysate using a fungal strain, Coniochaeta ligniaria, to consume inhibitors. Growth of two other strains, a salt-sensitive HAL4 mutant and a GPD1 mutant sensitive to osmotic stress, was not negatively affected in hydrolysate compared to the parent xylose-metabolizing strain. In recycled fermentation process water, inhibition of the ZWF1 mutant could again be remediated by biological abatement, and no effect on growth was observed for any of the mutants compared to the parent strain.