Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

Background As the second most abundant polysaccharide in nature, hemicellulose can be degraded to xylose as the feedstock for bioconversion to fuels and chemicals. To enhance xylose conversion, the engineered Saccharomyces cerevisiae with xylose metabolic pathway is usually adapted with xylose as the carbon source in the laboratory. However, the mechanism under the adaptation phenomena of the engineered strain is still unclear. Results In this study, xylose-utilizing S. cerevisiae was constructed and used for the adaptation study. It was found that xylose consumption rate increased 1.24-fold in the second incubation of the yYST12 strain in synthetic complete-xylose medium compared with the first incubation. The study figured out that it was observed at the single-cell level that the stagnation time for xylose utilization was reduced after adaptation with xylose medium in the microfluidic device. Such transient memory of xylose metabolism after adaptation with xylose medium, named “xylose consumption memory”, was observed in the strains with both xylose isomerase pathway and xylose reductase and xylitol dehydrogenase pathways. In further, the proteomic acetylation of the strains before and after adaptation was investigated, and it was revealed that H4K5 was one of the most differential acetylation sites related to xylose consumption memory of engineered S. cerevisiae. We tested 8 genes encoding acetylase or deacetylase, and it was found that the knockout of the GCN5 and HPA2 encoding acetylases enhanced the xylose consumption memory. Conclusions The behavior of xylose consumption memory in engineered S. cerevisiae can be successfully induced with xylose in the adaptation. H4K5Ac and two genes of GCN5 and HPA2 are related to xylose consumption memory of engineered S. cerevisiae during adaptation. This study provides valuable insights into the xylose adaptation of engineered S. cerevisiae.

Novel Propagation Strategy of Saccharomyces cerevisiae for Enhanced Xylose Metabolism during Fermentation on Softwood Hydrolysate

An economically viable production of second-generation bioethanol by recombinant xylose-fermenting Saccharomyces cerevisiae requires higher xylose fermentation rates and improved glucose–xylose co-consumption. Moreover, xylose-fermenting S. cerevisiae recognises xylose as a non-fermentable rather than a fermentable carbon source, which might partly explain why xylose is not fermented into ethanol as efficiently as glucose. This study proposes propagating S. cerevisiae on non-fermentable carbon sources to enhance xylose metabolism during fermentation. When compared to yeast grown on sucrose, cells propagated on a mix of ethanol and glycerol in shake flasks showed up to 50% higher xylose utilisation rate (in a defined xylose medium) and a double maximum fermentation rate, together with an improved C5/C6 co-consumption (on an industrial softwood hydrolysate). Based on these results, an automated propagation protocol was developed, using a fed-batch approach and the respiratory quotient to guide the ethanol and glycerol-containing feed. This successfully produced 71.29 ± 0.91 g/L yeast with an average productivity of 1.03 ± 0.05 g/L/h. These empirical findings provide the basis for the design of a simple, yet effective yeast production strategy to be used in the second-generation bioethanol industry for increased fermentation efficiency.

Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

Background Lignocellulosic biomass upgrading has become a promising alternative route to produce transportation fuels in response to energy security and environmental concerns. As the second most abundant polysaccharide in nature, hemicellulose mainly containing xylose is an important carbon source that can be used for the bioconversion to fuels and chemicals. However, the adaptation phenomena could appear and influence the bioconversion performance of xylose when Saccharomyces cerevisiae strain was transferred from the glucose to the xylose environment. Therefore, it is crucial to elucidate the mechanism of this adaptation phenomena, which can guide the strategy exploration to improve the efficiency of xylose utilization. Results In this study, xylose-utilizing strains had been constructed to effectively consume xylose. It is found that the second incubation of yYST218 strain in synthetic complete-xylose medium resulted in a 1.24-fold increase in xylose consumption ability as compared with the first incubation in synthetic complete-xylose medium. The results clearly showed that growing S. cerevisiae again in synthetic complete-xylose medium can significantly reduce the stagnation time and thus achieved a faster growth rate, by comparing the growth status of the strain in synthetic complete-xylose medium for the first and second time at the single-cell level through Microfluidic technology. Although these xylose-utilizing strains possessed different xylose metabolism pathways, they exhibited the “transient memory” phenomenon of xylose metabolism after changing the culture environment to synthetic complete-xylose medium, which named ‘xylose consumption memory (XCM)’ of S. cerevisiae in this study. According to the identification of protein acetylation, partial least squares analysis and the confirmatory test had verified that H4K5Ac affected the state of “XCM” in S. cerevisiae. Knockout of the acetylase-encoding genes GCN5 and HPA2 enhanced the “XCM” of the strain. Protein acetylation analysis suggested that xylose induced perturbation in S. cerevisiae stimulated the rapid adaptation of strains to xylose environment by regulating the level of acetylation. Conclusions All these results indicated protein acetylation modification is an important aspect that protein acetylation regulated the state of “XCM” in S. cerevisiae and thus determine the environmental adaptation of S. cerevisiae. Systematically exploiting the regulation approach of protein acetylation in S. cerevisiae could provide valuable insights into the adaptation phenomena of microorganisms in complex industrial environments.

Wheat Bran Hydrolysate Culture Medium Design for Talaromyces Purpureogenus CFRM02 Pigment Production and Colour Characteristics

Wheat bran hydrolysate (WBH) in combination with carbon and nitrogen was utilised as substrate for pigment production by Talaromyces purpureogenus CFRM02. Pigment yield was significantly increased (≈ 3 fold: OD units and ≈ 2 fold: a* value) by xylose supplementation with WBH compared to other carbon sources. Whereas 1% xylose supplementation increased pigment production (1.57 ± 0.05 OD Units and 49 ± 1.62 a* value). Pigment yield was low when WBH supplemented with 0.3% nitrogen sources. However significant increase (≈ 2-2.5 fold, OD units and a* value) was observed, when yeast extract (1.2%), nitrate of sodium (1.2%) and potassium (1.6%) were supplemented. Accordingly, 16 WBH media were designed by supplementing carbon and nitrogen. Interestingly the pigment production was significantly increased (1.59 OD units and 32 a* value) in the medium supplemented with 4% carbon and 0.9–1.2% nitrogen. T. purpureogenus CFRM02 was able to co-utilize xylose, fructose and glucose in WBH medium. The CIE Lab values indicated that, pigment characteristics differed significantly among the media. Apparently, T. purpureogenus CFRM02 possess alternative gene(s) or pathway(s) for xylose metabolism and channelled towards pigment biosynthesis. Comparative results revealed that, 1% xylose supplementation to WBH makes the fermentation process economically competitive for pigment production.

Xylose Metabolism in Bacteria—Opportunities and Challenges towards Efficient Lignocellulosic Biomass-Based Biorefineries

In a sustainable society based on circular economy, the use of waste lignocellulosic biomass (LB) as feedstock for biorefineries is a promising solution, since LB is the world’s most abundant renewable and non-edible raw material. LB is available as a by-product from agricultural and forestry processes, and its main components are cellulose, hemicellulose, and lignin. Following suitable physical, enzymatic, and chemical steps, the different fractions can be processed and/or converted to value-added products such as fuels and biochemicals used in several branches of industry through the implementation of the biorefinery concept. Upon hydrolysis, the carbohydrate-rich fraction may comprise several simple sugars (e.g., glucose, xylose, arabinose, and mannose) that can then be fed to fermentation units. Unlike pentoses, glucose and other hexoses are readily processed by microorganisms. Some wild-type and genetically modified bacteria can metabolize xylose through three different main pathways of metabolism: xylose isomerase pathway, oxidoreductase pathway, and non-phosphorylative pathway (including Weimberg and Dahms pathways). Two of the commercially interesting intermediates of these pathways are xylitol and xylonic acid, which can accumulate in the medium either through manipulation of the culture conditions or through genetic modification of the bacteria. This paper provides a state-of-the art perspective regarding the current knowledge on xylose transport and metabolism in bacteria as well as envisaged strategies to further increase xylose conversion into valuable products.

Genome Mining Discovery of Hydrogen Production Pathway of Klebsiella sp. WL1316 Fermenting Cotton Stalk Hydrolysate

Genome sequencing was used to identify key genes for the generation of hydrogen gas through cotton stalk hydrolysate fermentation by Klebsiella sp. WL1316. Genome annotation indicated that the genome size was 5.2 Mb with GC content 57.6%. Xylose was metabolised in the pentose phosphate pathway via the conversion of xylose to xylulose in Klebsiella sp. WL1316. This strain contained diverse formate-hydrogen lyases and hydrogenases with gene numbers higher than closely related species. A metabolic network involving glucose, xylose utilisation, and fermentative hydrogen production was reconstructed. Metabolic analysis of key node metabolites showed that glucose and xylose metabolism influenced biomass synthesis and biohydrogen production. Formic acid accumulated during fermentation at 24–48 h but decreased sharply after 48 h, illustrating the splitting of formic acid to hydrogen gas during early-to-mid fermentation. The Kreb’s cycle was the main competitive metabolic branch of biohydrogen synthesis at 24 h of fermentation. Lactic and acetic acid fermentation and late ethanol accumulation competed the carbon skeleton of biohydrogen synthesis after 72 h of fermentation, indicating that these competitive pathways are regulated in middle-to-late fermentation (48–96 h). This study is the first to elucidate the metabolic mechanisms of mixed sugar utilisation and biohydrogen synthesis based on genomic information.

Microbial Community Dysbiosis and Functional Gene Content Changes in Apple Flowers due to Fire Blight

Despite the plant microbiota plays an important role in plant health, little is known about the potential interactions of the flower microbiota with pathogens. In this study, we investigated the microbial community of apple blossoms when infected with Erwinia amylovora. The long-read sequencing technology, which significantly increased the genome sequence resolution, thus enabling the characterization of fire blight-induced changes in the flower microbial community. Each sample showed a unique microbial community at the species level. Pantoea agglomerans and P. allii were the most predominant bacteria in healthy flowers, whereas E. amylovora comprised more than 90% of the microbial population in diseased flowers. Furthermore, gene function analysis revealed that glucose and xylose metabolism were enriched in diseased flowers. Overall, our results showed that the microbiome of apple blossoms is rich in specific bacteria, and the nutritional composition of flowers is important for the incidence and spread of bacterial disease.

Thermodynamic and Kinetic Modeling of Co-utilization of Glucose and Xylose for 2,3-BDO Production by Zymomonas mobilis

Prior engineering of the ethanologen Zymomonas mobilis has enabled it to metabolize xylose and to produce 2,3-butanediol (2,3-BDO) as a dominant fermentation product. When co-fermenting with xylose, glucose is preferentially utilized, even though xylose metabolism generates ATP more efficiently during 2,3-BDO production on a BDO-mol basis. To gain a deeper understanding of Z. mobilis metabolism, we first estimated the kinetic parameters of the glucose facilitator protein of Z. mobilis by fitting a kinetic uptake model, which shows that the maximum transport capacity of glucose is seven times higher than that of xylose, and glucose is six times more affinitive to the transporter than xylose. With these estimated kinetic parameters, we further compared the thermodynamic driving force and enzyme protein cost of glucose and xylose metabolism. It is found that, although 20% more ATP can be yielded stoichiometrically during xylose utilization, glucose metabolism is thermodynamically more favorable with 6% greater cumulative Gibbs free energy change, more economical with 37% less enzyme cost required at the initial stage and sustains the advantage of the thermodynamic driving force and protein cost through the fermentation process until glucose is exhausted. Glucose-6-phosphate dehydrogenase (g6pdh), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and phosphoglycerate mutase (pgm) are identified as thermodynamic bottlenecks in glucose utilization pathway, as well as two more enzymes of xylose isomerase and ribulose-5-phosphate epimerase in xylose metabolism. Acetolactate synthase is found as potential engineering target for optimized protein cost supporting unit metabolic flux. Pathway analysis was then extended to the core stoichiometric matrix of Z. mobilis metabolism. Growth was simulated by dynamic flux balance analysis and the model was validated showing good agreement with experimental data. Dynamic FBA simulations suggest that a high agitation is preferable to increase 2,3-BDO productivity while a moderate agitation will benefit the 2,3-BDO titer. Taken together, this work provides thermodynamic and kinetic insights of Z. mobilis metabolism on dual substrates, and guidance of bioengineering efforts to increase hydrocarbon fuel production.