Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y-48190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9–39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70% v/v concentrated hydrolysate. However, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

In the present study, the economic evaluation of fermentation produced butyric acid (HBu) from corn was carried out (capacity 40 × 103 metric tons HBu∙yr-1) and two recovery processes (distillation and adsorption) were compared. Distillation is a longstanding process that has been effectively used to recover volatile chemicals. Recently, we developed an adsorption process that can be used to recover HBu cost competitively. The direct fixed capital (DFC) of the two processes (distillation & adsorption) were (in US dollars) $73.96 × 106 and $43.91 × 106, respectively. It is reported that the production of HBu from corn requires supplementing with amylase enzymes to the corn mash. For the two processes (distillation & adsorption) the annual operating costs were $37.67 × 106 and $31.83 × 106, respectively. The utilities annual costs for the two processes were $47.38 × 106 and $5.91 × 106, respectively. Corn price is one of the most important factors that influence HBu selling price, but other factors that could lower production costs include plant capacity. Factors such as tax on profit, interest rates on borrowed capital and plant life have marginal or low impact on HBu selling price. The significance of this paper is that HBu produced from corn and recovered by adsorption could be sold for $1.22 to 0.85∙kg-1. The selling price of fermentation produced HBu could further be reduced considerably if agricultural biomass such as wheat straw is used for production. One sentence summary Butyric acid production by fermentation and process economics.

Deconstruction of plant xylan is challenging due to the structural complexity arising from side groups on the polymer. One of the side groups, glucuronic acid, can be further modified with a methyl group at the 4-hydroxy position, as well as modification by the addition of sugar, acetate, and/or ferulate. This heterogeneity necessitates multiple enzymes capable of recognizing and hydrolyzing each side group type for complete hydrolysis of xylan to xylose. In this study, four fungal α-glucuronidase genes were identified and cloned into Coniochaeta ligniaria, a robust lignocellulose-degrading ascomycete isolated from soil. An E. coli-C. ligniaria shuttle vector was constructed with transcription promoter and termination sequences, a signal sequence directing enzymes for secretion, and a his6-tag sequence for antibody recognition and affinity purification. This cloning and expression strategy allowed secretion of heterologous fungal proteins in a microbe well-suited for growth in a biomass environment. α-glucuronidase enzymes were successfully expressed and secreted by C. ligniaria grown under four media conditions: defined mineral medium with glucose or acetate as sole carbon source; rich medium containing peptone and yeast extract; and corn stover hydrolysate. The four recombinant glycoside hydrolases were incubated with beech xylan, and α-glucuronidase activity for all four enzymes was detected as release of 4-methylglucuronic acid monomers from xylan. Hydrolysis of 4-methylglucuronic acid from xylan polymer is consistent with designation of the enzymes in glycoside hydrolase family 115.

Microcystins (MCs) are toxins produced by cyanobacteria, posing a significant emerging threat to human and public health. Therefore, control strategies combining frequent toxin monitoring with removal techniques are urgently needed. In this context, microcystin degradation using the bacterial enzyme microcystinase A, originally derived from Sphingosinicella microcystinivorans B9, has been identified as a sustainable and effective approach. To facilitate access to the enzyme, the gene encoding microcystinase A was successfully expressed in the Saccharomyces cerevisiae PE-2 strain. The recombinant microcystinase A was produced as an intracellular enzyme and applied in MC degradation assays. Optimal conditions for enzymatic activity were identified at 42.2°C and pH 6.3. The maximum degradation rate of microcystin was determined to be 3.09 mg/L/h, and a Km of 2.81 μM was obtained when assays were performed at 37°C and pH 7.4. The recombinant microcystinase A remained fully active for 2 h at 20°C. Exposure to 50°C for 1 h resulted in 60% residual activity, while 30 min at 65°C led to complete inactivation. The enzyme was also denatured when exposed to alkaline pH conditions. Therefore, this study provides key data on recombinant microcystinase A, supporting further investigations into its potential applications for MC degradation, particularly under mildly acidic conditions and temperatures up to 45°C.

This study investigated the secretion of endolysin LysKB317 integrated into the HO locus of Saccharomyces cerevisiae strain NRRL Y-2034 to enable the yeast to simultaneously perform ethanol fermentation and control bacterial contaminants frequently present in ethanol refineries. The cell wall hydrolase gene was expressed using TEF1 and NAT5 promoter and terminator sequences with α-MF secretion signal and an N-terminus poly-histidine tag. LysKB317 was detectable by western blot analysis, which showed a molecular weight slightly larger than the 33 kDa native protein, presumably due to residual amino acids from the α-MF secretion signal peptide or S. cerevisiae glycosylation. Secreted LysKB317 was confirmed to be active using turbidity reduction and cell viability assay. Contaminated corn mash fermentations with yeast secreting LysKB317 demonstrated a significant reduction in bacterial contamination by at least 2-log compared to the contamination controls without LysKB317 expression. Moreover, LysKB317 expression led to a 73% decrease in acetic acid concentration and a 67% decrease in lactic acid levels. Contaminated fermentations with yeast expressing LysKB317 also exhibited a 16% improvement in ethanol production over the contamination controls without LysKB317, with no significant difference observed when compared to yeast-only controls during a 72-h corn mash fermentation. These findings suggest that a yeast endolysin secretion platform holds promise for mitigating bacterial contamination in biorefineries and potentially reducing reliance on antibiotics usage.

Butyric acid, a four-carbon fatty acid, is an important industrial chemical and feedstock. To produce this chemical, a control fermentation was run with a 126.5 g.L−1 glucose concentration in the feed medium. In this medium, the strain produced 44.8 g.L−1 total acid with a productivity of 0.23 g.L−1h−1 and a yield of 0.41 g.g−1. The strain (Clostridium tyrobutyricum ATCC 25755) was also able to utilize glucose and xylose simultaneously with similar fermentation performance. The culture was also used to produce butyric acid from wheat straw hydrolysate (WSH) employing a hot water pretreatment. In a batch system, the strain resulted in a productivity and yield of 0.27 g.L−1h−1 and 0.44 g.g−1, respectively, which was an improvement over the use of glucose or xylose alone or mixtures of both. To improve reactor productivity, a membrane cell recycle bioreactor was used which resulted in a productivity of 1.89 g.L−1h−1. This productivity was 822% of that achieved in the glucose or xylose batch fermentation. Furthermore, a butyric acid recovery method was developed using XAD-4 adsorbent resin. In this system, up to 206.1 g.L−1 of butyric acid was used in the feed and, as a result of the quick adsorption, the residual butyric acid concentration was 29.5 g.L−1. In this experiment, the rate of acid removal of 1059.4 g.L−1h−1 was achieved.

Background Significant genetic diversity exists across Saccharomyces strains. Natural isolates and domesticated brewery and industrial strains are typically more robust than laboratory strains when challenged with inhibitory lignocellulosic hydrolysates. These strains also contain genes that are not present in lab strains and likely contribute to their superior inhibitor tolerance. However, many of these strains have poor sporulation efficiencies and low spore viability making subsequent gene analysis, further metabolic engineering, and genomic analyses of the strains challenging. This work aimed to develop an inhibitor tolerant haploid with stable mating type from S. cerevisiae YB-2625, which was originally isolated from bagasse. Results Haploid spores isolated from four tetrads from strain YB-2625 were tested for tolerance to furfural and HMF. Due to natural mutations present in the HO-endonuclease, all haploid strains maintained a stable mating type. One of the haploids, YRH1946, did not flocculate and showed enhanced tolerance to furfural and HMF. The tolerant haploid strain was further engineered for xylose fermentation by integration of the genes for xylose metabolism at two separate genomic locations ( ho ∆ and pho13 ∆). In fermentations supplemented with inhibitors from acid hydrolyzed corn stover, the engineered haploid strain derived from YB-2625 was able to ferment all of the glucose and 19% of the xylose, whereas the engineered lab strains performed poorly in fermentations. Conclusions Understanding the molecular mechanisms of inhibitor tolerance will aid in developing strains with improved growth and fermentation performance using biomass-derived sugars. The inhibitor tolerant, xylose fermenting, haploid strain described in this work has potential to serve as a platform strain for identifying pathways required for inhibitor tolerance, and for metabolic engineering to produce fuels and chemicals from undiluted lignocellulosic hydrolysates.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33-9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032-0.131 (g/g) and lipid contents were 13.5-53.7% (g/g). Four strains had significantly higher lipid yields and contents. One of these strains, which had among the highest lipid yield in this study (0.131 +/- 0.007 g/g), has not been previously described in the literature.Summary The yeast Rhodotorula toruloides was used to produce oil using sugars extracted from a bioenergy grass. Graphical Abstract

Economic conversion of biomass to biofuels and chemicals requires efficient and complete utilization of xylose. Saccharomyces cerevisiae strains engineered for xylose utilization are still considerably limited in their overall ability to metabolize xylose. In this study, we identified causative mutations resulting in improved xylose fermentation of an adapted S. cerevisiae strain expressing codon-optimized xylose isomerase and xylulokinase genes from the rumen bacterium Prevotella ruminicola. Genome sequencing identified single-nucleotide polymorphisms in seven open reading frames. Tetrad analysis showed that mutations in both PBS2 and PHO13 genes were required for increased xylose utilization. Single deletion of either PBS2 or PHO13 did not improve xylose utilization in strains expressing the xylose isomerase pathway. Saccharomyces can also be engineered for xylose metabolism using the xylose reductase/xylitol dehydrogenase genes from Scheffersomyces stipitis. In strains expressing the xylose reductase pathway, single deletion of PHO13 did show a significant increase xylose utilization, and further improvement in growth and fermentation was seen when PBS2 was also deleted. These findings will extend the understanding of metabolic limitations for xylose utilization in S. cerevisiae as well as understanding of how they differ among strains engineered with two different xylose utilization pathways.

Degradation products from sugars and lignin are commonly generated as byproducts during pretreatment of biomass being processed for production of renewable fuels and chemicals. Many of the degradation products act as microbial inhibitors, including furanic and phenolic compounds and acetate, which is solubilized from hemicellulose. We previously identified a group of fungi, Coniochaeta species, that are intrinsically tolerant to and capable of mineralizing furans present in biomass hydrolysates. Here, we challenged 20 C. ligniaria and phylogenetically related isolates with acetate to test if the robustness phenotype extended to this important inhibitor as well, and all strains grew at concentrations up to 2.5% (w/v) sodium acetate. At the highest concentrations tested (5.0–7.5% w/v), some variation in growth on solid medium containing glucose plus acetate was apparent among the strains. The hardiness of four promising strains was further evaluated by challenging them (0.5% w/v sodium acetate) in mineral medium containing 10 or 15 mM furfural. The strains grew and consumed all of the acetate and furfural. At a higher (2.5% w/v) concentration, consumption of acetate varied among the strains: only one consumed any acetate in the presence of furfural, but all four strains consumed acetate provided that a small amount (0.2% w/v) of glucose was added. Finally, the four strains were evaluated for biological abatement of rice hull hydrolysates having elevated acetate content. The hardiest strains were also able to consume furfural and 5-hydroxymethylfurfural (HMF) within 24 h, followed by acetate within 40 h when grown in dilute acid pretreated rice hulls containing 0.55% acetate, 15 mM furfural, and 1.7 mM HMF. As such, these strains are expected to be helpful for abating non-desirable compounds from unrefined hydrolysates so as to enable their conversion to bioproducts.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg−1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g−1, and productivities of 0.39 and 0.29 g (L h)−1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g−1 and productivities from 0.14 to 0.23 g (L h)−1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Background Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern. Results A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion. Conclusion This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways. Graphic abstract

Background Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae. However, there has been no consensus on which of these possible bottlenecks are the most important to address. A previous strain characterization study from our lab suggested that insufficient NAD+ limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol. The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimizing bottleneck impact on process economics. Results A dynamic flux balance model was developed to identify bottlenecks in several strains of S. cerevisiae, both with wild-type pentose phosphate pathway expression and with the pathway over expressed. ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions. This pathway is used to regenerate NADPH. Under oxygen limiting conditions, respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor. Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH. The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance, increasing ethanol production by 50% while producing succinate as a coproduct at upwards of 20 g/l. Conclusion In the production of high value chemicals from biomass, the use of the respiratory metabolism is a waste of feedstock carbon. Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways. Focusing future efforts on the non-respiratory balancing of NAD+/NADH, perhaps through the glyoxylate pathway, would improve the economics of ethanol production both directly and through coproduct formation. Graphic abstract

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.

Water consumption is an important consideration for production of advanced biofuels and chemicals. Recycling process water to reduce water consumption concentrates inhibitors formed during biomass pretreatment processes. To lessen their impact, Coniochaeta ligniaria NRRL 30616 was used to biologically abate inhibitors in dilute-acid pretreated corn stover hydrolysate. Biological abatement of inhibitors enabled recombinant ethanologenic E. coli FBR5 to complete fermentation before cultures using untreated hydrolysate emerged from the lag phase. Biological abatement allowed 50% reuse of the liquor in subsequent fermentations, and hydrolysate could be recycled five times. Although growth of E. coli FBR5 was variable in fermentations using recycled hydrolysate, ethanol productivity was essentially the same as in single-use hydrolysate. In contrast to biological abatement, overliming did not adequately eliminate inhibitors and led to unsatisfactory fermentation results. Bioabatement also enabled recovery of failed fermentations and allowed recycling of spent liquor for use as process water in a corn stover pretreatment process.

Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

Additional file 2: Table S2. List of all proteins found in the 2T2.1 genome and their duplication status.