Structural assembly of the nucleic-acid-binding Thp3-Csn12-Sem1 complex functioning in mRNA splicing.

PCI domain proteins play important roles in post-transcriptional gene regulation. In the TREX-2 complex, PCI domain-containing Sac3 and Thp1 proteins and accessory Sem1 protein form a ternary complex required for mRNA nuclear export. In contrast, structurally related Thp3-Csn12-Sem1 complex mediates pre-mRNA splicing. In this study, we determined the structure of yeast Thp3186-470-Csn12-Sem1 ternary complex at 2.9 Å resolution. Both Thp3 and Csn12 structures have a typical PCI structural fold, characterized by a stack of α-helices capped by a C-terminal winged-helix (WH) domain. The overall structure of Thp3186-470-Csn12-Sem1 complex has an inverted V-shape with Thp3 and Csn12 forming the two sides. A fishhook-shaped Sem1 makes extensive contacts on Csn12 to stabilize its conformation. The overall structure of Thp3186-470-Csn12-Sem1 complex resembles the previously reported Sac3-Thp1-Sem1 complex, but also has significant structural differences. The C-terminal WH domains of Thp3 and Csn12 form a continuous surface to bind different forms of nucleic acids with micromolar affinity. Mutation of the basic residues in the WH domains of Thp3 and Csn12 affects nucleic acid binding in vitro and mRNA splicing in vivo. The Thp3-Csn12-Sem1 structure provides a foundation for further exploring the structural elements required for its specific recruitment to spliceosome for pre-mRNA splicing.

The novel properties of Kluyveromyces marxianus glucose sensor/receptor repressor pathway and the construction of glucose repression-released strains.

BACKGROUND: Glucose repression in yeast leads to the sequential or diauxic utilization of mixed sugars and reduces the co-utilization of glucose and xylose from lignocellulosic biomasses. Study of the glucose sensing pathway helps to construct glucose repression-released yeast strains and enhance the utilization of lignocellulosic biomasses. RESULTS: Herein, the glucose sensor/receptor repressor (SRR) pathway of Kluyveromyces marxianus which mainly consisted of KmSnf3, KmGrr1, KmMth1, and KmRgt1 was studied. The disruption of KmSNF3 led to a release of glucose repression, enhanced xylose consumption and did not result in deficient glucose utilization. Over-expression of glucose transporter gene restored the mild decrease of glucose utilization ability of Kmsnf3 strain to a similar level of the wildtype strain but did not restore glucose repression. Therefore, the repression on glucose transporter is parallel to glucose repression to xylose and other alternative carbon utilization. KmGRR1 disruption also released glucose repression and kept glucose utilization ability, although its xylose utilization ability was very weak with xylose as sole carbon source. The stable mutant of KmMth1-ΔT enabled the release of glucose repression irrespective that the genetic background was Kmsnf3, Kmmth1, or wildtype. Disruption of KmSNF1 in the Kmsnf3 strain or KmMTH1-ΔT overexpression in Kmsnf1 strain kept constitutive glucose repression, indicating that KmSNF1 was necessary to release the glucose repression in both SRR and Mig1-Hxk2 pathway. Finally, overexpression of KmMTH1-ΔT released the glucose repression to xylose utilization in S. cerevisiae. CONCLUSION: The glucose repression-released K. marxianus strains constructed via a modified glucose SRR pathway did not lead to a deficiency in the utilization ability of sugar. The obtained thermotolerant, glucose repression-released, and xylose utilization-enhanced strains are good platforms for the construction of efficient lignocellulosic biomass utilization yeast strains.

Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.

BACKGROUND: Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance. RESULTS: In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors. CONCLUSION: This study demonstrated that KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.

Release of glucose repression on xylose utilization in Kluyveromyces marxianus to enhance glucose-xylose co-utilization and xylitol production from corncob hydrolysate.

BACKGROUND: Lignocellulosic biomass is one of the most abundant materials for biochemicals production. However, efficient co-utilization of glucose and xylose from the lignocellulosic biomass is a challenge due to the glucose repression in microorganisms. Kluyveromyces marxianus is a thermotolerant and efficient xylose-utilizing yeast. To realize the glucose-xylose co-utilization, analyzing the glucose repression of xylose utilization in K. marxianus is necessary. In addition, a glucose-xylose co-utilization platform strain will facilitate the construction of lignocellulosic biomass-utilizing strains. RESULTS: Through gene disruption, hexokinase 1 (KmHXK1) and sucrose non-fermenting 1 (KmSNF1) were proved to be involved in the glucose repression of xylose utilization while disruption of the downstream genes of cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway or sucrose non-fermenting 3 (SNF3) glucose-sensing pathway did not alleviate the repression. Furthermore, disruption of the gene of multicopy inhibitor of GAL gene expression (KmMIG1) alleviated the glucose repression on some nonglucose sugars (galactose, sucrose, and raffinose) but still kept glucose repression of xylose utilization. Real-time PCR analysis of the xylose utilization related genes transcription confirmed these results, and besides, revealed that xylitol dehydrogenase gene (KmXYL2) was the critical gene for xylose utilization and stringently regulated by glucose repression. Many other genes of candidate targets interacting with SNF1 were also evaluated by disruption, but none proved to be the key regulator in the pathway of the glucose repression on xylose utilization. Therefore, there may exist other signaling pathway(s) for glucose repression on xylose consumption. Based on these results, a thermotolerant xylose-glucose co-consumption platform strain of K. marxianus was constructed. Then, exogenous xylose reductase and xylose-specific transporter genes were overexpressed in the platform strain to obtain YHY013. The YHY013 could efficiently co-utilized the glucose and xylose from corncob hydrolysate or xylose mother liquor for xylitol production (> 100 g/L) even with inexpensive organic nitrogen sources. CONCLUSIONS: The analysis of the glucose repression in K. marxianus laid the foundation for construction of the glucose-xylose co-utilizing platform strain. The efficient xylitol production strain further verified the potential of the platform strain in exploitation of lignocellulosic biomass.

Recurring sequence-structure motifs in (ßα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs.

An interesting way of generating novel artificial proteins is to combine sequence motifs from natural proteins, mimicking the evolutionary path suggested by natural proteins comprising recurring motifs. We analyzed the ßα and αß modules of TIM barrel proteins by structure alignment-based sequence clustering. A number of preferred motifs were identified. A chimeric TIM was designed by using recurring elements as mutually compatible interfaces. The foldability of the designed TIM protein was then significantly improved by six rounds of directed evolution. The melting temperature has been improved by more than 20°C. A variety of characteristics suggested that the resulting protein is well-folded. Our analysis provided a library of peptide motifs that is potentially useful for different protein engineering studies. The protein engineering strategy of using recurring motifs as interfaces to connect partial natural proteins may be applied to other protein folds.

Metabolic engineering of indole pyruvic acid biosynthesis in Escherichia coli with tdiD.

BACKGROUND: Indole pyruvic acid (IPA) is a versatile platform intermediate and building block for a number of high-value products in the pharmaceutical and food industries. It also has a wide range of applications, such as drugs for the nervous system, cosmetics, and luminophores. Chemical synthesis of IPA is a complicated and costly process. Moreover, through the biosynthesis route employing L-amino acid oxidase, the byproduct hydrogen peroxide leads the degradation of IPA. TdiD, identified as a specific tryptophan aminotransferase, could be an alternative solution for efficient IPA biosynthesis. RESULTS: Escherichia coli strain W3110, which demonstrates basic production when supplied with tryptophan, was engineered for IPA biosynthesis. Several strategies were implemented to improve IPA production. First, through incorporating the codon-optimized tdiD into W3110, IPA levels increased from 41.54 ± 1.26 to 52.54 ± 2.08 mg/L. Second, after verifying the benefit of an increased phenylpyruvate pool, a YL03 strain was constructed based on a previously reported mutant strain of W3110 with a plasmid carrying aroF fbr and pheA fbr to further improve IPA production. The recombinant YL03 strain accumulated IPA at 158.85 ± 5.36 mg/L, which was 3.82-fold higher than that of the wild-type W3110 strain. Third, optimization of tdiD co expression was carried out by replacing the Trc promoter with a series of constitutively active promoters along with increasing the plasmid copy numbers. The highest IPA production was observed in YL08, which achieved 236.42 ± 17.66 mg/L and represented a greater than 5-fold increase as compared to W3110. Finally, the effects of deletion and overexpression of tnaA on IPA biosynthesis were evaluated. The removal of tnaA led to slightly reduced IPA levels, whereas the overexpression of tnaA resulted in a considerable decline in production. CONCLUSIONS: This study illustrates the feasibility of IPA biosynthesis in E. coli through tdiD. An efficient IPA producing strain, YL08, was developed, which provides a new possibility for biosynthesis of IPA. Although the final production was limited, this study demonstrates a convenient method of IPA synthesis.

Error removal in microchip-synthesized DNA using immobilized MutS.

The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP(+)-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.

Characterizing yeast promoters used in Kluyveromyces marxianus.

Fermentation at higher temperatures can potentially reduce the cooling cost in large-scale fermentation and reduce the contamination risk. Thus, the thermotolerant yeast, Kluyveromyces marxianus, which can grow and ferment at elevated temperatures, is a promising biotechnological tool for future applications. However, the promoters used in K. marxianus are not well characterized, especially at elevated temperatures, which is important in efficient metabolic pathway construction. In this study, six constitutive promoters (P(TDH3), P(PGK), and P(ADH1) from both Saccharomyces cerevisiae and K. marxianus) were evaluated in K. marxianus through the heterologous expression of the KlLAC4, GUSA, and SH BLE genes at various temperatures, with various carbon sources and oxygen conditions. The expression was evaluated at the transcription and protein level using real-time PCR and protein activity determination to eliminate the effect of heterologous protein stability. While the transcription of all the promoters decreased at higher temperatures, the order of their promoting strength at various temperatures with glucose as the carbon source was P(KmPGK) > P(KmTDH3) > P(ScPGK) > P(ScTDH3) > P(KmADH1) > P(ScADH1). When glycerol or xylose was supplied as the carbon source at 42 °C, the order of promoter strength was P(KmPGK) > P(ScPGK) > P(KmADH1) > P(ScADH1) > P(ScTDH3) > P(KmTDH3). The promoter activity of P TDH3 decreased significantly, while the promoter activity of both of the P(ADH1) promoters increased. Oxygen conditions had non-significant effect. The results of this study provide important information for fine-tuned pathway construction for the metabolic engineering of K. marxianus.

Structural Insights into the Catalytic Activity of Cyclobacterium marinum N-Acetylglucosamine Deacetylase.

N-Acetylglucosamine deacetylase from Cyclobacterium marinum (CmCBDA) is a highly effective and selective biocatalyst for the production of d-glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). However, the underlying catalytic mechanism remains elusive. Here, we show that CmCBDA is a metalloenzyme with a preference for Ni2+ over Mn2+. Crystal structures of CmCBDA in complex with Ni2+ and Mn2+ revealed slight remodeling of the CmCBDA active site by the metal ions. We also demonstrate that CmCBDA exists as a mixture of homodimers and monomers in solution, and dimerization is indispensable for catalytic activity. A mutagenesis analysis also indicated that the active site residues Asp22, His72, and His143 as well as the residues involved in dimerization, Pro52, Trp53, and Tyr55, are essential for catalytic activity. Furthermore, a mutation on the protein surface, Lys219Glu, resulted in a 2.3-fold improvement in the deacetylation activity toward GlcNAc. Mechanistic insights obtained here may facilitate the development of CmCBDA variants with higher activities.

Enhancing simultaneous saccharification and co-fermentation of corncob by Kluyveromyces marxianus through overexpression of putative transcription regulator.

Overexpression of a gene with unknown function in Kluyveromyces marxianus markedly improved tolerance to lignocellulosic biomass-derived inhibitors. This overexpression also enhanced tolerance to elevated temperatures, ethanol, and high concentrations of NaCl and glucose. Inhibitor degradation and transcriptome analyses related this K. marxianusMultiple Stress Resistance (KmMSR) gene to the robustness of yeast cells. Nuclear localization and DNA-binding domain analyses indicate that KmMsr is a putative transcriptional regulator. Overexpression of a mutant protein with deletion in the flexible region between amino acids 100 and 150 further enhanced tolerance to multiple inhibitors during fermentation, with ethanol production and productivity increasing by 36.31 % and 80.22 %, respectively. In simultaneous saccharification co-fermentation of corncob without detoxification, expression of KmMSR with the deleted flexible region improved ethanol production by 5-fold at 42 °C and 2-fold at 37 °C. Overexpression of the KmMSR mutant provides a strategy for constructing robust lignocellulosic biomass using strains.

Effect of Caproicibacterium lactatifermentans inoculation on the microbial succession and flavor formation of pit mud used in Chinese Baijiu fermentation.

Caproicibacterium lactatifermentans is a major caproate-producing bacterium in high-quality pit mud and has an impact on the synthesis of fatty acids during Baijiu fermentation. To develop an effective method for cultivating high-quality pit mud, we explored the role of Caproicibacterium lactatifermentans inoculation. The inoculation resulted in a high level of Caproicibacterium lactatifermentans (29.16%) and fortified pit mud produced abundant fatty acids and ethyl esters in short-term usage. Rare microbes, such as Hazenella coriacea, promoted the production of fatty acids. After long-term usage, changes in physicochemical properties led to a decrease in caproate-producing bacterium, namely Clostridium and Caproicibacterium, and an increase in microbes with limited fatty acid biosynthesis capability, including Proteiniphilum, Fastidiosipila, and Caldicoprobacter. These alterations ultimately led to a decrease in fatty acids and ethyl esters. In summary, Caproicibacterium lactatifermentans inoculation exhibited positive outcomes in obtaining high-quality pit mud. However, the maintenance of functional microbes necessitates further investigation.

Unveiling malic acid biorefinery: Comprehensive insights into feedstocks, microbial strains, and metabolic pathways.

The over-reliance on fossil fuels and resultant environmental issues necessitate sustainable alternatives. Microbial fermentation of biomass for malic acid production offers a viable, eco-friendly solution, enhancing resource efficiency and minimizing ecological damage. This review covers three core aspects of malic acid biorefining: feedstocks, microbial strains, and metabolic pathways. It emphasizes the significance of utilizing biomass sugars, including the co-fermentation of different sugar types to improve feedstock efficiency. The review discusses microbial strains for malic acid fermentation, addressing challenges related to by-products from biomass breakdown and strategies for overcoming them. It delves into the crucial pathways and enzymes for malic acid production, outlining methods to optimize its metabolism, focusing on enzyme regulation, energy balance, and yield enhancement. These insights contribute to advancing the field of consolidated bioprocessing in malic acid biorefining.

Deciphering layer formation in Red Heart Qu: A comprehensive study of metabolite profile and microbial community influenced by raw materials and environmental factors.

Environmental-origin microbiota significantly influences Red Heart Qu (RH_Qu) stratification, but their microbial migration and metabolic mechanisms remain unclear. Using high-throughput sequencing and metabolomics, we divided the stratification of RH_Qu into three temperature-based stages. Phase I features rising temperatures, causing microbial proliferation and a two-layer division. Phase II, characterized by peak temperatures, sees the establishment of thermotolerant species like Bacillus, Thermoactinomyces, Rhodococcus, and Thermoascus, forming four distinct layers and markedly altering metabolite profiles. The Huo Quan (HQ), developing from the Pi Zhang (PZ), is driven by the tyrosine-melanin pathway and increased MRPs (Maillard reaction products). The Hong Xin evolves from the Rang, associated with the phenylalanine-coumarin pathway and QCs (Quinone Compounds) production. Phase III involves the stabilization of the microbial and metabolic profile as temperatures decline. These findings enhance our understanding of RH_Qu stratification and offer guidance for quality control in its fermentation process.

Improving ethanol and xylitol fermentation at elevated temperature through substitution of xylose reductase in Kluyveromyces marxianus.

Thermo-tolerant yeast Kluyveromyces marxianus is able to utilize a wide range of substrates, including xylose; however, the xylose fermentation ability is weak because of the redox imbalance under oxygen-limited conditions. Alleviating the intracellular redox imbalance through engineering the coenzyme specificity of NADPH-preferring xylose reductase (XR) and improving the expression of XR should promote xylose consumption and fermentation. In this study, the native xylose reductase gene (Kmxyl1) of the K. marxianus strain was substituted with XR or its mutant genes from Pichia stipitis (Scheffersomyces stipitis). The ability of the resultant recombinant strains to assimilate xylose to produce xylitol and ethanol at elevated temperature was greatly improved. The strain YZB014 expressing mutant PsXR N272D, which has a higher activity with both NADPH and NADH as the coenzyme, achieved the best results, and produced 3.55 g l(-1) ethanol and 11.32 g l(-1) xylitol-an increase of 12.24- and 2.70-fold in product at 42 °C, respectively. A 3.94-fold increase of xylose consumption was observed compared with the K. marxianus YHJ010 harboring KmXyl1. However, the strain YZB015 expressing a mutant PsXR K21A/N272D, with which co-enzyme preference was completely reversed from NADPH to NADH, failed to ferment due to the low expression. So in order to improve xylose consumption and fermentation in K. marxianus, both higher activity and co-enzyme specificity change are necessary.

Improved xylose fermentation of Kluyveromyces marxianus at elevated temperature through construction of a xylose isomerase pathway.

To improve the xylose fermentation ability of Kluyveromyces marxianus, a xylose assimilation pathway through xylose isomerase was constructed. The genes encoding xylose reductase (KmXyl1) and xylitol dehydrogenase (KmXyl2) were disrupted in K. marxianus YHJ010 and the resultant strain was named YRL002. A codon-optimized xylose isomerase gene from Orpinomyces was transformed into K. marxianus YRL002 and expressed under GAPDH promoter. The transformant was adapted in the SD medium containing 1 % casamino acid with 2 % xylose as sole carbon source. After 32 times of trans-inoculation, a strain named YRL005, which can grow at a specific growth rate of 0.137/h with xylose as carbon source, was obtained. K. marxianus YRL005 could ferment 30.15 g/l of xylose and produce 11.52 g/l ethanol with a yield of 0.38 g/g, production rate of 0.069 g/l/h at 42 °C, and also could ferment 16.60 g/l xylose to produce 5.21 g/l ethanol with a yield of 0.31 g/g, and production rate of 0.054 g/l h at 45 °C. Co-fermentation with 2 % glucose could not improve the amount and yield of ethanol fermented from xylose obviously, but it could improve the production rate. Furthermore, K. marxianus YRL005 can ferment with the corn cob hydrolysate, which contained 20.04 g/l xylose to produce 8.25 g/l ethanol. It is a good platform to construct thermo-tolerant xylose fermentation yeast.

MRSD: a web server for metabolic route search and design.

SUMMARY: We present a tool called MRSD (Metabolic Route Search and Design) to search and design routes based on the weighted compound transform diagraph. The search submodule returns routes between a source and product compound within seconds in the network of one or multiple organisms based on data from KEGG. The design submodule designs a route from an appointed compound in an interactive mode. The two complementary functions, Metabolic Route Search and Design, can be broadly used in biosynthesis, bio-pharmaceuticals and the other related fields. AVAILABILITY: bioinfo.ustc.edu.cn/softwares/MRSD/.

Carotenoid production from nondetoxified xylose mother liquid or corncob hydrolysate with engineered Kluyveromyces marxianus.

Carotenoids are widely utilized in the food, pharmaceutical and nutraceutical industries. Here, Kluyveromyces marxianus was engineered to overproduce carotenoids from corncob hydrolysate or xylose mother liquid (XML, a byproduct of commercial xylose purification). First, the toxicity of fat-soluble carotenoids to cells was reduced by employing xylose inducible promoters using with a two-temperature strategy to separate cell growth and product accumulation. Then, through further engineering and optimization of the carotenoid biosynthesis pathway, 1506.7 mg/L lycopene, 988.5 mg/L ß-carotene or 142.9 mg/L astaxanthin were produced with glucose and xylose by K. marxianus. Finally, 397.7 mg/L and 279.7 mg/L lycopene, 297.3 mg/L and 108.8 mg/L ß-carotene, and 86.4 mg/L and 56.8 mg/L astaxanthin were produced with nonsterilized andnondetoxified XML or corncob hydrolysate after nitrogen source optimization. To our knowledge, the produced amounts of lycopene, ß-carotene and astaxanthin from lignocellulose biomass by yeast in this study were higher than those in previous reports.

Lignocellulosic xylitol production from corncob using engineered Kluyveromyces marxianus.

Xylitol production from lignocellulose hydrolysate is a sustainable and environment-friendly process. In this study, a systematic process of converting corncob waste into xylitol is described. First, the corncobs are hydrolyzed with acid to a hydrolysate. Second, Kluyveromyces marxianus YZJQ016 derived from K. marxianus YZJ074, constructed by overexpressing ScGAL2-N376F from Saccharomyces cerevisiae, CtXYL1 from Candida tropicalis, and KmZWF1 from K. marxianus, produces xylitol from the hydrolysate. A total of ten xylose reductase genes were evaluated, and CtXYL1 proved best by showing the highest catalytic activity under the control of the KmGAPDH promoter. A 5 L fermenter at 42°C produced 105.22 g/L xylitol using K. marxianus YZJQ016-the highest production reported to date from corncob hydrolysate. Finally, for crystallization of the xylitol, the best conditions were 50% (v/v) methanol as an antisolvent, at 25°C, with purity and yield of 99%-100% and 74%, respectively-the highest yield reported to date.

Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast.

Easy and low-cost protein purification methods for the mass production of commonly used enzymes that play important roles in biotechnology are in high demand. In this study, we developed a fast, low-cost recombinant protein purification system in the methylotrophic yeast Pichia pastoris using the family 3 cellulose-binding module (CBM3)-based affinity tag. The codon of the cbm3 gene from Clostridium thermocellum was optimized based on the codon usage of P. pastoris. The CBM3 tag was then fused with enhanced green fluorescent protein (CBM3-EGFP) or with inulinase and expressed in P. pastoris to demonstrate its ability to function as an affinity tag in a yeast expression system. We also examined the effects of glycosylation on the secreted CBM3-tag. The secreted wild-type CBM3-EGFP was glycosylated; however, this had little influence on the adsorption of the fusion protein to the regenerated amorphous cellulose (RAC; maximum adsorption capacity of 319 mg/g). Two CBM3-EGFP mutants lacking glycosylation sites were also constructed. The three CBM3-EGFPs expressed in P. pastoris and the CBM3-EGFP expressed in Escherichia coli all had similar RAC adsorption capacity. To construct a tag-free recombinant protein purification system based on CBM3, a CBM3-intein-EGFP fusion protein was expressed in P. pastoris. This fusion protein was stably expressed and the self-cleavage of intein was efficiently induced by DTT or L: -cysteine. In this study, we were able to purify the recombinant fusion protein with high efficiency using both intein and direct fusion-based strategies.