Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

Abstract Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

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Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

Biotechnology for Biofuels ◽

10.1186/s13068-021-02090-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yong-Shui Tan ◽

Li Wang ◽

Ying-Ying Wang ◽

Qi-En He ◽

Zhi-Hua Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Xylose Reductase ◽

Xylose Isomerase ◽

Xylose Utilization ◽

Xylose Metabolism ◽

Xylose Consumption ◽

Synthetic Complete ◽

Before And After ◽

Genes Encoding ◽

Fuels And Chemicals

Abstract 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.

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Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01419-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5708-5716 ◽

Cited By ~ 96

Author(s):

Sun-Mi Lee ◽

Taylor Jellison ◽

Hal S. Alper

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Directed Evolution ◽

Ethanol Yield ◽

Xylose Isomerase ◽

Mutant Enzyme ◽

Xylose Utilization ◽

Xylose Consumption ◽

Content Type ◽

Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

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Efficient anaerobic consumption of D-xylose by E. coli BL21(DE3) via xylR adaptive mutation

BMC Microbiology ◽

10.1186/s12866-021-02395-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jung Min Heo ◽

Hyun Ju Kim ◽

Sang Jun Lee

Keyword(s):

Consumption Rate ◽

Xylose Isomerase ◽

Fold Increase ◽

Adaptive Mutation ◽

Growth Media ◽

Missense Mutations ◽

Xylose Consumption ◽

E Coli ◽

Fermentation Technology ◽

Xylose Transporter

Abstract Background Microorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly. Results When BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption. Conclusions Anaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.

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Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Biotechnology for Biofuels ◽

10.1186/s13068-021-02073-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jung-Hoon Bae ◽

Mi-Jin Kim ◽

Bong Hyun Sung ◽

Yong-Su Jin ◽

Jung-Hoon Sohn

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Lignocellulosic Biomass ◽

Xylose Fermentation ◽

Xylose Isomerase ◽

Carbon Substrate ◽

Yeast Fermentation ◽

Xylose Utilization ◽

Secretory Expression ◽

Xylose Consumption

Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

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Efficient Anaerobic Consumption of D-xylose by E. coli BL21(DE3) via xylR Adaptive Mutation

10.21203/rs.3.rs-375356/v1 ◽

2021 ◽

Author(s):

Jung Min Heo ◽

Hyun Ju Kim ◽

Sang Jun Lee

Keyword(s):

Consumption Rate ◽

Xylose Isomerase ◽

Fold Increase ◽

Adaptive Mutation ◽

Growth Media ◽

Missense Mutations ◽

Xylose Consumption ◽

E Coli ◽

Fermentation Technology ◽

Xylose Transporter

Abstract Background: Microorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both glucose and xylose under anaerobic conditions, glucose is consumed first, whereas xylose is consumed very slowly.Results: When BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption. Conclusions: We obtained anaerobically-adapted BL21(DE3) cells through short-term adaptive evolution and identified xylR mutations responsible for faster xylose consumption, which may facilitate the improvement of microbial fermentation technology.

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Cloning and Characterization of Transcription of the xylAB Operon in Thermoanaerobacter ethanolicus

Journal of Bacteriology ◽

10.1128/jb.180.5.1103-1109.1998 ◽

1998 ◽

Vol 180 (5) ◽

pp. 1103-1109 ◽

Cited By ~ 17

Author(s):

Milutin Erbeznik ◽

Karl A. Dawson ◽

Herbert J. Strobel

Keyword(s):

Amino Acid ◽

Transcription Initiation ◽

Xylose Isomerase ◽

Initiation Site ◽

Xylose Utilization ◽

Terminal Portion ◽

Thermoanaerobacter Ethanolicus ◽

Genes Encoding ◽

Xylulose Kinase

ABSTRACT The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobeThermoanaerobacter ethanolicus were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221–227, 1991) promoter region. The bicistronic xylAB mRNA was processed by cleavage within the 5′-terminal portion of the XylB-coding sequence. Transcription ofxylAB was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative xyl operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The T. ethanolicus xylB gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an xylB nucleotide sequence and an xylAB operon from a thermophilic anaerobic bacterium.

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The assembled and annotated genome of the pigeon louse Columbicola columbae, a model ectoparasite

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab009 ◽

2021 ◽

Vol 11 (2) ◽

Author(s):

James G Baldwin-Brown ◽

Scott M Villa ◽

Anna I Vickrey ◽

Kevin P Johnson ◽

Sarah E Bush ◽

...

Keyword(s):

Low Cost ◽

Single Copy ◽

Odorant Receptors ◽

Rna Seq ◽

Pediculus Humanus ◽

Final Assembly ◽

Oxford Nanopore ◽

Genes Encoding ◽

Host Parasite ◽

G Protein Coupled

Abstract The pigeon louse Columbicola columbae is a longstanding and important model for studies of ectoparasitism and host-parasite coevolution. However, a deeper understanding of its evolution and capacity for rapid adaptation is limited by a lack of genomic resources. Here, we present a high-quality draft assembly of the C. columbae genome, produced using a combination of Oxford Nanopore, Illumina, and Hi-C technologies. The final assembly is 208 Mb in length, with 12 chromosome-size scaffolds representing 98.1% of the assembly. For gene model prediction, we used a novel clustering method (wavy_choose) for Oxford Nanopore RNA-seq reads to feed into the MAKER annotation pipeline. High recovery of conserved single-copy orthologs (BUSCOs) suggests that our assembly and annotation are both highly complete and highly accurate. Consistent with the results of the only other assembled louse genome, Pediculus humanus, we find that C. columbae has a relatively low density of repetitive elements, the majority of which are DNA transposons. Also similar to P. humanus, we find a reduced number of genes encoding opsins, G protein-coupled receptors, odorant receptors, insulin signaling pathway components, and detoxification proteins in the C. columbae genome, relative to other insects. We propose that such losses might characterize the genomes of obligate, permanent ectoparasites with predictable habitats, limited foraging complexity, and simple dietary regimes. The sequencing and analysis for this genome were relatively low cost, and took advantage of a new clustering technique for Oxford Nanopore RNAseq reads that will be useful to future genome projects.

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Analysis of saponins detoxification genes in Ilyonectria mors-panacis G3B inducing root rot of Panax notoginseng by RNA-Seq

Archives of Microbiology ◽

10.1007/s00203-021-02502-4 ◽

2021 ◽

Author(s):

Guohong Zeng ◽

Jin Li ◽

Yuxiu Ma ◽

Qian Pu ◽

Tian Xiao ◽

...

Keyword(s):

Root Rot ◽

Molecular Mechanisms ◽

Management Strategies ◽

Panax Notoginseng ◽

Glycoside Hydrolases ◽

Rna Seq ◽

Host Interaction ◽

Excellent Starting Point ◽

Starting Point ◽

Genes Encoding

AbstractSaponins are kinds of antifungal compounds produced by Panax notoginseng to resist invasion by pathogens. Ilyonectria mors-panacis G3B was the dominant pathogen inducing root rot of P. notoginseng, and the abilities to detoxify saponins were the key to infect P. notoginseng successfully. To research the molecular mechanisms of detoxifying saponins in I. mors-panacis G3B, we used high-throughput RNA-Seq to identify 557 and 1519 differential expression genes (DEGs) in I. mors-panacis G3B with saponins treatments for 4H (Hours) and 12H (Hours) compared with no saponins treatments, respectively. Among these DEGs, we found 93 genes which were simultaneously highly expressed in I. mors-panacis G3B with saponins treatments for 4H and 12H, they mainly belong to genes encoding transporters, glycoside hydrolases, oxidation–reduction enzymes, transcription factors and so on. In addition, there were 21 putative PHI (Pathogen–Host Interaction) genes out of those 93 up-regulated genes. In this report, we analyzed virulence-associated genes in I. mors-panacis G3B which may be related to detoxifying saponins to infect P. notoginseng successfully. They provided an excellent starting point for in-depth study on pathogenicity of I. mors-panacis G3B and developed appropriate root rot disease management strategies in the future.

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A Transcriptomic Approach to the Metabolism of Tetrapyrrolic Photosensitizers in a Marine Annelid

Molecules ◽

10.3390/molecules26133924 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3924

Author(s):

Maria Leonor Santos ◽

Mariaelena D’Ambrosio ◽

Ana P. Rodrigo ◽

A. Jorge Parola ◽

Pedro M. Costa

Keyword(s):

Metabolic Pathways ◽

Molecular Networks ◽

Bile Pigments ◽

Rna Seq ◽

The Past ◽

Wide Range ◽

Genes Encoding ◽

Organ Specific ◽

Bright Green ◽

Green Coloration

The past decade has seen growing interest in marine natural pigments for biotechnological applications. One of the most abundant classes of biological pigments is the tetrapyrroles, which are prized targets due their photodynamic properties; porphyrins are the best known examples of this group. Many animal porphyrinoids and other tetrapyrroles are produced through heme metabolic pathways, the best known of which are the bile pigments biliverdin and bilirubin. Eulalia is a marine Polychaeta characterized by its bright green coloration resulting from a remarkably wide range of greenish and yellowish tetrapyrroles, some of which have promising photodynamic properties. The present study combined metabolomics based on HPLC-DAD with RNA-seq transcriptomics to investigate the molecular pathways of porphyrinoid metabolism by comparing the worm’s proboscis and epidermis, which display distinct pigmentation patterns. The results showed that pigments are endogenous and seemingly heme-derived. The worm possesses homologs in both organs for genes encoding enzymes involved in heme metabolism such as ALAD, FECH, UROS, and PPOX. However, the findings also indicate that variants of the canonical enzymes of the heme biosynthesis pathway can be species- and organ-specific. These differences between molecular networks contribute to explain not only the differential pigmentation patterns between organs, but also the worm’s variety of novel endogenous tetrapyrrolic compounds.

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Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Fermentation ◽

10.3390/fermentation4030059 ◽

2018 ◽

Vol 4 (3) ◽

pp. 59 ◽

Cited By ~ 8

Author(s):

Tingting Liu ◽

Shuangcheng Huang ◽

Anli Geng

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcoholic Fermentation ◽

Ethanol Yield ◽

Xylose Isomerase ◽

Cost Effective ◽

Xylose Utilization ◽

Yeast Strains ◽

Multiple Copies ◽

Biomass Sugars ◽

Sugar Conversion

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.

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