Dataset Information

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

ABSTRACT: High costs and low production efficiency are a serious constraint to bio-based xylitol production. For industrial-scale production of xylitol, a plasmid-free Escherichia coli for arabitol-free xylitol production from corncob hemicellulosic hydrolysate has been constructed. Instead of being plasmid and inducer dependent, this strain relied on multiple-copy integration of xylose reductase (XR) genes into the chromosome, where their expression was controlled by the constitutive promoter P43. In addition, to minimize the flux from L-arabinose to arabitol, two strategies including low XR total activity and high selectivity of XR has been adopted. Arabitol was significantly decreased using plasmid-free strain which had lower XR total activity and an eight point-mutations of XR with a 27-fold lower enzyme activity toward L-arabinose was achieved. The plasmid-free strain in conjunction with this mutant XR can completely eliminate arabitol formation in xylitol production. In fed-batch fermentation, this plasmid-free strain produced 143.8?g L(-1) xylitol at 1.84?g L(-1) h(-1) from corncob hemicellulosic hydrolysate. From these results, we conclude that this route by plasmid-free E. coli has potential to become a commercially viable process for xylitol production.

SUBMITTER: Su B

PROVIDER: S-EPMC4880924 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Publications

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

Su Buli B Zhang Zhe Z Wu Mianbin M Lin Jianping J Yang Lirong L

Scientific reports 20160526

High costs and low production efficiency are a serious constraint to bio-based xylitol production. For industrial-scale production of xylitol, a plasmid-free Escherichia coli for arabitol-free xylitol production from corncob hemicellulosic hydrolysate has been constructed. Instead of being plasmid and inducer dependent, this strain relied on multiple-copy integration of xylose reductase (XR) genes into the chromosome, where their expression was controlled by the constitutive promoter P43. In add ...[more]

PMID: 27225023

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Project description: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.

Project description:Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h-1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h-1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL-1 and 1.72 ± 0.04 mgL-1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Dataset Information

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

Publications

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

Similar Datasets

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