Saccharomyces cerevisiae spores have emerged as a novel surface display host and have been successfully applied in the biosynthesis of useful products. To construct a dual-enzyme co-display and assembly system on the surface of S. cerevisiae spores, the fluorescent proteins were initially used as the model proteins to prove the feasibility. The result of the in vitro assembly experiment of L-arabinose isomerase from Bacillus coagulans (BcAI) and mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GtMPI) confirmed that the assembled complex could significantly improve the biosynthetic efficiency of the rare sugar L-ribose. Subsequently, BcAI and GtMPI were co-displayed on the spore surface and further assembled via the SpyTag/SpyCatcher system to realize the biosynthesis of L-ribose. The maximum conversion rate of L-ribose reached approximately 16% with 10 g/L L-arabinose as the substrate. Nevertheless, the achieved L-ribose conversion rate of 16% is moderate compared with previously reported values. Even with this limitation, this study establishes a feasible spore-surface assembly strategy for multi-enzyme cascades, laying a technical foundation for the biosynthesis of high-value products.

Uridine diphosphate galactose (UDP-Gal) provides galactosyl units for active carbohydrate biosynthesis; however, limited availability and high costs hamper large-scale applications. In the two-enzyme cascade system of UDP-Gal synthesis, the pH conflict between UDP-galactose 4-epimerase (GALE) and sucrose synthase (Susy) blocks UDP-Gal production. Therefore, surface charge engineering was conducted to obtain a variant (GALEM2) with improved acid resistance. GALEM2 enzyme activity reached 214.26 ± 0.20% that of wild-type GALE at pH 6.5. Its half-life time increased by 2 h at pH 6.5, and the pH resistance range was widened effectively with local surface charge reshaping and a decreased isoelectric point. An improved flexibility of the substrate entrance enhanced the catalytic performance under acidic conditions. Cascading GALEM2 and SusyM6 yielded UDP-Gal (24.5 mM) with a space-time yield of 12 g/L/h within 1.25 h, demonstrating the robust route of short reaction time and high efficiency, for rapid UDP-Gal synthesis from readily available sucrose via cascade catalysis.

For bioethanol production from lignocellulosic materials, an ideal microorganism must possess both excellent xylose utilization and a high tolerance to inhibitory compounds. However, these two traits often exhibit antagonism in recombinant xylose-utilizing Saccharomyces cerevisiae strains. In this study, we developed a quantitative metric using an aggregated parameter to evaluate the degree of this antagonism and applied it to evaluate the antagonism of three strains (LF1, LF1-6M, and 6M-15), which had been iteratively evolved in xylose and hydrolyzate environments. Transcriptomic analysis revealed that the yeast strain elevates the alert level to stresses related to DNA replication, unfolded protein, starvation, and hyperosmosis, and reduces the uptake of unimportant nutrients to have a higher acetic acid tolerance during adaptive evolution in hydrolyzate. Additionally, the Snf1p-Mig1p signaling pathway was reprogrammed, enabling the strain to utilize xylose more efficiently during adaptive evolution in xylose. We also confirmed that disruption of the glyceraldehyde-3-phosphate dehydrogenase gene TDH1 significantly shortened the time required for glucose and/or xylose cofermentation under acetic acid stress by reducing reactive oxygen species accumulation and increasing ATP production. This study offers valuable insights for developing robust and efficient S. cerevisiae strains capable of glucose/xylose cofermentation.

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield. However, glucose and xylose share flux in the pentose phosphate pathway (PPP) and glycolysis pathway (EMP), with glucose having a competitive advantage in the shared metabolic pathways. In this work, we knocked down ZWF1 to preclude glucose from entering the PPP. This reduced the [NADPH] level and disturbed growth on both glucose or xylose, confirming that the oxidative PPP, which begins with Zwf1p and ultimately leads to CO2 production, is the primary source of NADPH in both glucose and xylose. Upon glucose depletion, gluconeogenesis is necessary to generate glucose-6-phosphate, the substrate of Zwf1p. We re-established the NADPH regeneration pathway by replacing the endogenous NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene TDH3 with heterogenous NADP + -GAPDH genes GDH, gapB, and GDP1. Among the resulting strains, the strain BZP1 (zwf1Δ, tdh3::GDP1) exhibited a similar xylose consumption rate before glucose depletion, but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001, and the ethanol yield for total consumed sugars of BZP1 was 13.5% higher than BSGX001. This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO2 release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1, therefore, to determine the competitive equilibrium between glucose-xylose co-metabolism. This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.

Uridine/guanosine diphosphate-mannose (UDP/GDP-Man) is the major mannosyl donor in producing mannose-containing oligo/polysaccharides. Its acquisition is greatly limited by its complex and costly synthetic process, which requires multiple substrates and enzymes. The natural UDP/GDP-glucose 2-epimerase functioning C2 epimerization between UDP/GDP-Glc and UDP/GDP-Man remains unreported which is the main hurdle to realize concise production of UDP/GDP-Man. Here, the UDP-glucose 2-epimerase (Glc2E), which behaves like a naturally evolved enzyme, is created and exhibits high-efficient catalysis in producing UDP-Man. Multidimensional engineering, including redesigning the nucleobase recognition region, displacement of the substrate tunnel entrance, and expansion of space for sugar ring rotation, is employed to develop Glc2E from CDP-tyvelose 2-epimerase. Glc2E converts 55.63% of UDP-Glc to UDP-Man, a trace value for the initial enzyme, stTyvE, and its aptitude for GDP-Glc epimerization evolves from unobserved activity to 23.94% conversion. Coupling sucrose synthase with Glc2E achieves the theoretical synthase-epimerase route for UDP/GDP-Man production from inexpensive sucrose. The space-time-yield of UDP-Man is maximized to 8.05 g/L/h within 2.5 h, with a final titer of 22.54 g/L, demonstrating competitive application potential. Moreover, the GDP-Man is synthesized successfully at a titer of 3.49 g/L. Our work inspires the enzyme engineering for epimerases and glycosyltransferases that catalyze nucleotide sugars. The application of Glc2E in the synthase-epimerase route unlocks a concise and feasible synthetic approach for producing cost-competitive mannosyl donors.

The one-step deconstruction of sucrose into uridine 5'-diphosphate-glucose (UDP-Glc), an important sugar donor for transglycosylation, employing sucrose synthase (Susy) is emerging as a valuable sucrose utilization process. The insufficient activity and stability of Susy limit the productivity of UDP-Glc from sucrose. Here, an engineered Susy (SusyM6) that counteracted the activity-stability trade-off was developed with the half-life time and activity being 43-fold and 1.4-fold of wild-type, respectively. Tighter hydrophobic patches and stabilization of the SSN2 domain contributed to greater activity and stability. The use of SusyM6 in UDP-Glc production resulted in a satisfactory space-time yield of 73 g/L/h within 1 h. The cascade of different biocatalysts with SusyM6, focusing on utilizing two products of sucrose decomposition, fructose and UDP-Glc, expanded sucrose utilization, efficiently promoting the UDP-Glc productivity and giving a cost-effective method for UDP-galactose (UDP-Gal) synthesis. This study demonstrated promising green pathways for producing multiple value-added products from sucrose using Susy.

The use of agricultural biomass-based fertilizers, and the release of feces into the environment leads to lastlasting pollution of antibiotic resistance genes that cannot be removed from waters via traditional methods, resulting in significant health threats. To solve this issue, an antibiotic resistance gene removal method was proposed and tested that used sequence-specific DNA-binding designer zinc finger proteins, which target an 18bp DNA sequence for specific antibiotic resistance gene binding and removal. Targeting the sulfonamide-resistant sul1 gene, sul1-binding zinc-finger protein was designed, overexpressed, and purified. This protein showed specific binding with sul1 over tetA that do not have the targeted sequence. This protein was further immobilized on agarose-based resins to prepare a sul1-removal column. When loaded with 10 mg protein, this column can remove over 99 % sul1 in water, suggesting high efficiency. This work presents a new method attempting to eliminate environmental and health threats posed by antibiotic resistance genes.

Nucleotide sugars are essential precursors for carbohydrate synthesis but are in scarce supply. Uridine diphosphate (UDP)-glucose is a core building block in nucleotide sugar preparation, making its efficient synthesis critical. Here, a process for producing valuable UDP-glucose and functional mannose from sucrose was established and improved via a semirational sucrose synthase (SuSy) design and the accurate D-mannose isomerase (MIase) cascade. Engineered SuSy exhibited enzyme activity 2.2-fold greater than that of the WT. The structural analysis identified a latch-hinge combination as the hotspot for enhancing enzyme activity. Coupling MIase, process optimization, and reaction kinetic analysis revealed that MIase addition during the high-speed UDP-glucose synthesis phase distinctly accelerated the entire process. The simultaneous triggering of enzyme modules halved the reaction time and significantly increased the UDP-glucose yield. A maximum UDP-glucose yield of 83%, space-time yield of 70 g/L/h, and mannose yield of 32% were achieved. This novel and efficient strategy for sucrose value-added exploitation has industrial promise.

Acetyl-coenzyme A (Acetyl-CoA) and malonyl-coenzyme A (malonyl-CoA) are important precursors for producing various chemicals, and their availability affects the production of their downstream chemicals. Storage carbohydrates are considered important carbon and energy reservoirs. Herein, we find that regulating the storage carbohydrate synthesis improves metabolic fluxes toward malonyl-CoA. Interestingly, not only directly decreasing storage carbohydrate accumulation improved malonyl-CoA availability but also increasing the storage carbohydrate by UGP1 overexpression enables an even higher production of acetyl-CoA- and malonyl-CoA-derived chemicals. We find that Ugp1p overexpression dynamically regulates the carbon flux to storage carbohydrate synthesis. In early exponential phases, Ugp1 overexpression causes more storage carbohydrate accumulation, while the carbon flux is then redirected toward acetyl-CoA and malonyl-CoA in later phases, thereby contributing to the synthesis of their derived products. Our study demonstrates the importance of storage carbohydrates rearrangement for the availability of acetyl-CoA and malonyl-CoA and therefore will facilitate the synthesis of their derived chemicals.

The use of non-food lignocellulosic biomass to produce ethanol fits into the strategy of a global circular economy with low dependence on fossil energy resources. Xylose is the second most abundant sugar in lignocellulosic hydrolysate, and its utilization in fermentation is a key issue in making the full use of raw plant materials for ethanol production and reduce production costs. Saccharomyces cerevisiae is the best ethanol producer but the organism is not a native xylose user. In recent years, great efforts have been made in the construction of xylose utilizing S. cerevisiae strains by metabolic and evolutionary engineering approaches. In addition, managing global transcriptional regulation works provides an effective means to increase the xylose utilization capacity of recombinant strains. Here we review the common strategies and research advances in the research field in order to facilitate the researches in xylose metabolism and xylose-based fermentation.

The extracellular sucrase (SacC) gene of Zymomonas mobilis was overexpressed in Escherichia coli BL21 using the T7 polymerase expression system. A low cell density induction method was designed to have maximum expression, and the conditions (IPTG concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing SacC protein.

This erratum is being published to correct the fund number. On page 2410, funding information “National Key R&D Program of China, Grant/Award Number: 2018YFA090010” should read “National Key R&D Program of China, Grant/Award Number: 2018YFA0901700”. The authors regret these errors. The published article can be found here: https://onlinelibrary.wiley.com/doi/full/10.1002/bit.27370

Engineered Saccharomyces cerevisiae strains are good cell factories, and developing additional genetic manipulation tools will accelerate construction of metabolically engineered strains. Highly repetitive rDNA sequence is one of two main sites typically used for multicopy integration of genes. Here, we developed a simple and high-efficiency strategy for rDNA-mediated multicopy gene integration based on the dynamic balance of rDNA in S. cerevisiae. rDNA copy number was decreased by pre-treatment with hydroxyurea (HU). Then, heterologous genes were integrated into the rDNA sequence. The copy number of the integrated heterologous genes increased along with restoration of the copy number of rDNA. Our results demonstrated that HU pre-treatment doubled the number of integrated gene copies; moreover, compared with removing HU stress during transformation, removing HU stress after selection of transformants had a higher probability of resulting in transformants with high-copy integrated genes. Finally, we integrated 18.0 copies of the xylose isomerase gene into the S. cerevisiae genome in a single step. This novel rDNA-mediated multicopy genome integration strategy provides a convenient and efficient tool for further metabolic engineering of S. cerevisiae.

Lignin-carbohydrate complex (LCC) is the native existing form of major components in lignocellulose. In this study, the structural cross-link between lignin and polysaccharides in lignocellulose was quantitatively estimated with carboxymethylation-separation (CM-Sep) method, and its influence on lignocellulose pyrolysis was systematically investigated. The cross-linked lignin was found to positively correlate with the production of small molecules and furan derivatives while negatively affecting the generation of anhydrous sugars. Content of small molecules was increased by 97% while that of anhydrous sugars was decreased by 47% in pyrolytic products with levoglucosan yield lowered by 54 wt% in the existence of cross-linked lignin. Furthermore, the impact of cross-linked lignin was revealed to be significantly distinguished from free lignin. Impeded glycosidic end formation and boosted glycosyl ring scission as well as lignin fragmentation were responsible for the distinction. Excellent correlations between structural cross-link and lignocellulose pyrolytome could facilitate product prediction and process design.

AbstractManufacturing fuels and chemicals from cellulose materials is a promising strategy to achieve carbon neutralization goals. In addition to the commonly used enzymatic hydrolysis by cellulase, rapid pyrolysis is another way to degrade cellulose. The sugar obtained by fast pyrolysis is not glucose, but rather its isomer, levoglucosan (LG). Here, we revealed that both levoglucosan kinase activity and the transportation of levoglucosan are bottlenecks for LG utilization in Saccharomyces cerevisiae, a widely used cell factory. We revealed that among six heterologous proteins that had levoglucosan kinase activity, the 1,6-anhydro-N-acetylmuramic acid kinase from Rhodotorula toruloides was the best choice to construct levoglucosan-utilizing S. cerevisiae strain. Furthermore, we revealed that the amino acid residue Q341 and W455, which were located in the middle of the transport channel closer to the exit, are the sterically hindered barrier to levoglucosan transportation in Gal2p, a hexose transporter. The engineered yeast strain expressing the genes encoding the 1,6-anhydro-N-acetylmuramic acid kinase from R. toruloides and transporter mutant Gal2pQ341A or Gal2pW455A consumed ~ 4.2 g L−1 LG in 48 h, which is the fastest LG-utilizing S. cerevisiae strain to date.

Studying the mechanisms of resistance to vanillin in microorganisms, which is derived from lignin and blocks a major pathway of DNA double-strand break repair in yeast, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. A high vanillin-tolerant Saccharomyces cerevisiae strain EMV-8 carrying site mutations compared to its parent strain NAN-27 was selected for the analyses. The dynamics of the chromatin structure of eukaryotic cells play a critical role in transcription and the regulation of gene expression and thus the phenotype. Consequently, Hi-C and transcriptome analyses were conducted in EMV-8 and NAN-27 in the log phase with or without vanillin stress to determine the effects of mutations and vanillin disturbance on the dynamics of three-dimensional chromosome organization and the influence of the organization on the transcriptome. The outcomes indicated that the chromosome interaction pattern disturbed by vanillin stress or genetic mutations in the log phase was similar to that in mouse cells. The short chromosomes contact the short chromosomes, and the long chromosomes contact the long chromosomes. In response to vanillin stress, the boundaries of the topologically associating domain (TAD) in the vanillin-tolerant strain EMV-8 were more stable than those in its parent strain NAN-27. The motifs of SFL1, STB3, and NHP6A/B were enriched at TAD boundaries in both EMV-8 and NAN-27 with or without vanillin, indicating that these four genes were probably related to TAD formation. The Indel mutation of YRR1, whose absence was confirmed to benefit vanillin tolerance in EMV-8, caused two new interaction sites that contained three genes, WTM2, PUP1, and ALE1, whose overexpression did not affect vanillin resistance in yeast. Overall, our results revealed that in the log phase, genetic mutations and vanillin disturbance have a negligible effect on three-dimensional chromosome organization, and the reformation or disappearance of TAD boundaries did not show an association with gene expression, which provides an example for studying yeast chromatin structure during stress tolerance using Hi-C technology.

A saltiness perception enhancement method of grass carp meat conducted by microwave heating was investigated. Ion chromatographic results demonstrated that all samples had the same sodium level retained in matrices after being treated by water bath (WBV) and microwave with different power of 2.5, 7.5, 10, and 12.5 W/g (MWV). However, the meat treated by microwave exhibited a higher salty intensity than that of WBV, particularly MWV-10 W/g and MWV-12.5 W/g. The enhanced saltiness perception of meat treated by microwave was attributed to the facilitated water and sodium mobility demonstrated by low field-NMR and pulse-field-gradient stimulated echo (PFG-STE) Na-23 NMR experiments. Furthermore, the enhancement was also related to the formation of microstructure favorable for sodium diffusion, originating from the insufficient denaturation and less exposure of hydrophobic groups of proteins induced by microwave heating. Therefore, microwave heating has the potential to enhance the saltiness perception of meat in the food industry.

BACKGROUND:Vanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin.RESULTS:In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae.CONCLUSIONS:Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Dynamic regulation has been widely applied to optimize metabolic flux distribution. However, compared with prokaryotes, quorum sensing-mediated pathway control is still very limited in Saccharomyces cerevisiae. In this study, we designed quorum sensing-regulated protein degradation circuits for dynamic metabolic pathway control in S. cerevisiae. The synthetic quorum sensing circuits were developed by integration of a plant hormone cytokinin system with the endogenous yeast Ypd1-Skn7 signal transduction pathway and the positive feedback circuits were optimized by promoter engineering. We then constructed an auxin-inducible protein degradation system and used quorum sensing circuits to regulate auxin synthesis to achieve dynamic control of protein degradation. As a demonstration, the circuits were applied to control Erg9 degradation to produce α-farnesene and the titer of α-farnesene increased by 80%. The population-regulated protein degradation system developed here extends dynamic regulation to the protein level in S. cerevisiae and is a promising approach for metabolic pathway control.

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 degrees C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 degrees C, and by 6 h incubation, the free amino acids and gamma-aminobutyric acid almost doubled.