Evolutionary engineering improved D-glucose/xylose co-fermentation of Yarrowia lipolytica

Abstract Background: Yarrowia lipolytica is considered as a promising biorefinery chassis for production of microbial lipids, the important precursors of advanced biofuels. Unfortunately, wild Yarrowia lipolytica is unable to consume xylose, the major pentose in lignocellulosic hydrolysates. A recombinant strain Yarrowia lipolytica yl-XYL+ can utilize xylose to produce microbial lipids efficiently, but its xylose uptake is severely delayed in the presentence of D-glucose. Therefore, it is critical to develop co-fermenting D-glucose and xylose strains and study the underlying mechanisms.Results: In this study, an adaptive laboratory evolution (ALE) is performed to engineering the strains in the medium containing xylose and D-glucose analog 2-deoxyglucose (dG). After four stages of evolution over a total of 64 days, we obtained for the first time a strain of Y. lipolytica (yl-XYL+*04*10) with derepressed xylose metabolism. Xylose uptake kinetics showed that it could efficiently utilize xylose in the presence of 10 g/L dG or D-glucose. Transcriptional profiling analysis revealed that relative expression level of YALI0_C04730g and YALI0_D00363g (both encoding xylose-specific transporter) was significantly up-regulated. Besides, we found that missense mutations N373T and G270A in YALI0_E23287g (encoding a D-glucose transporter) and YALI0_E15488g (encoding a hexokinase) respectively.Conclusions: These results indicate that these are important gene targets responsible for improved xylose utilization in the evolved Yarrowia lipolytica. Our work provides a new approach for breeding Yarrowia lipolytica and paved the way for future pentose metabolic engineering.

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Adaptive Laboratory Evolution for Multistress Tolerance, including Fermentability at High Glucose Concentrations in Thermotolerant Candida tropicalis

Energies ◽

10.3390/en15020561 ◽

2022 ◽

Vol 15 (2) ◽

pp. 561

Author(s):

Koudkeo Phommachan ◽

Chansom Keo-oudone ◽

Mochamad Nurcholis ◽

Nookhao Vongvilaisak ◽

Mingkhuan Chanhming ◽

...

Keyword(s):

High Temperatures ◽

Candida Tropicalis ◽

High Glucose ◽

Glucose Transporter ◽

Major Facilitator Superfamily ◽

Cellulosic Biomass ◽

High Concentration ◽

Adaptive Laboratory Evolution ◽

Membrane Spanning ◽

Major Facilitator

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

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Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway

Biotechnology for Biofuels ◽

10.1186/s13068-016-0562-6 ◽

2016 ◽

Vol 9 (1) ◽

Cited By ~ 59

Author(s):

Gabriel M. Rodriguez ◽

Murtaza Shabbir Hussain ◽

Lauren Gambill ◽

Difeng Gao ◽

Allison Yaguchi ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Xylose Utilization

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Reciprocal Regulation of l-Arabinose and d-Xylose Metabolism in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00709-15 ◽

2015 ◽

Vol 198 (3) ◽

pp. 386-393 ◽

Cited By ~ 21

Author(s):

Santosh Koirala ◽

Xiaoyi Wang ◽

Christopher V. Rao

Keyword(s):

Escherichia Coli ◽

Plant Biomass ◽

Single Cells ◽

Xylose Utilization ◽

Xylose Metabolism ◽

Reciprocal Regulation ◽

Content Type ◽

E Coli ◽

Metabolic Genes ◽

Mixed Populations

ABSTRACTGlucose is known to inhibit the transport and metabolism of many sugars inEscherichia coli. This mechanism leads to its preferential consumption. Far less is known about the preferential utilization of nonglucose sugars inE. coli. Two exceptions arel-arabinose andd-xylose. Previous studies have shown thatl-arabinose inhibitsd-xylose metabolism inEscherichia coli. This repression results froml-arabinose-bound AraC binding to the promoter of thed-xylose metabolic genes and inhibiting their expression. This mechanism, however, has not been explored in single cells. Both thel-arabinose andd-xylose utilization systems are known to exhibit a bimodal induction response to their cognate sugar, where mixed populations of cells either expressing the metabolic genes or not are observed at intermediate sugar concentrations. This suggests thatl-arabinose can only inhibitd-xylose metabolism inl-arabinose-induced cells. To understand how cross talk between these systems affects their response, we investigatedE. coliduring growth on mixtures ofl-arabinose andd-xylose at single-cell resolution. Our results showed that mixed, multimodal populations ofl-arabinose- andd-xylose-induced cells occurred at intermediate sugar concentrations. We also found thatd-xylose inhibited the expression of thel-arabinose metabolic genes and that this repression was due to XylR. These results demonstrate that a strict hierarchy does not exist betweenl-arabinose andd-xylose as previously thought. The results may also aid in the design ofE. colistrains capable of simultaneous sugar consumption.IMPORTANCEGlucose,d-xylose, andl-arabinose are the most abundant sugars in plant biomass. Developing efficient fermentation processes that convert these sugars into chemicals and fuels will require strains capable of coutilizing these sugars. Glucose has long been known to repress the expression of thel-arabinose andd-xylose metabolic genes inEscherichia coli. Recent studies found thatl-arabinose also represses the expression of thed-xylose metabolic genes. In the present study, we found thatd-xylose also represses the expression of thel-arabinose metabolic genes, leading to mixed populations of cells capable of utilizingl-arabinose andd-xylose. These results further our understanding of mixed-sugar utilization and may aid in strain design.

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Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains

Annals of Microbiology ◽

10.1007/s13213-016-1228-0 ◽

2016 ◽

Vol 66 (4) ◽

pp. 1409-1418 ◽

Cited By ~ 15

Author(s):

L. Canonico ◽

S. Ashoor ◽

M. Taccari ◽

F. Comitini ◽

M. Antonucci ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Microbial Lipids ◽

Raw Glycerol

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Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-013-1350-y ◽

2013 ◽

Vol 41 (1) ◽

pp. 27-39 ◽

Cited By ~ 37

Author(s):

Jian Zha ◽

Minghua Shen ◽

Menglong Hu ◽

Hao Song ◽

Yingjin Yuan

Keyword(s):

Saccharomyces Cerevisiae ◽

Pentose Phosphate Pathway ◽

Pentose Phosphate ◽

Oxidative Pentose Phosphate Pathway ◽

Evolutionary Engineering ◽

Xylose Metabolism ◽

Expression Of Genes ◽

Enhanced Expression

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Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain

FEMS Yeast Research ◽

10.1093/femsyr/foz083 ◽

2019 ◽

Vol 19 (8) ◽

Author(s):

Jeroen G Nijland ◽

Xiang Li ◽

Hyun Yong Shin ◽

Paul P de Waal ◽

Arnold J M Driessen

Keyword(s):

Saccharomyces Cerevisiae ◽

Morphological Analysis ◽

Membrane Transporters ◽

Evolutionary Engineering ◽

Xylose Consumption ◽

Xylose Transport ◽

Xylose Uptake ◽

Cost Efficient ◽

Engineered Saccharomyces Cerevisiae ◽

Selection Of

ABSTRACT Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113–7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

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Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

Biotechnology for Biofuels ◽

10.1186/s13068-019-1643-0 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 5

Author(s):

Misun Lee ◽

Henriëtte J. Rozeboom ◽

Eline Keuning ◽

Paul de Waal ◽

Dick B. Janssen

Keyword(s):

Xylose Isomerase ◽

Single Type ◽

Metal Availability ◽

Kinetic Properties ◽

Xylose Utilization ◽

Xylose Metabolism ◽

Vitro Assay ◽

In Vivo Selection

Abstract Background Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.

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Establishment of Oxidative d-Xylose Metabolism in Pseudomonas putida S12

Applied and Environmental Microbiology ◽

10.1128/aem.02713-08 ◽

2009 ◽

Vol 75 (9) ◽

pp. 2784-2791 ◽

Cited By ~ 43

Author(s):

Jean-Paul Meijnen ◽

Johannes H. de Winde ◽

Harald J. Ruijssenaars

Keyword(s):

Pseudomonas Putida ◽

Caulobacter Crescentus ◽

Glucose Dehydrogenase ◽

Dry Weight ◽

Maximum Growth ◽

Maximum Growth Rate ◽

Xylose Utilization ◽

Catabolic Pathway ◽

Xylose Metabolism ◽

Semialdehyde Dehydrogenase

ABSTRACT The oxidative d-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on d-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g d-xylose−1) and a maximum growth rate of 0.21 h−1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on d-xylose. Only the xylD gene, encoding d-xylonate dehydratase, proved to be essential for establishing an oxidative d-xylose catabolic pathway in P. putida S12. The growth performance on d-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-d-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative d-xylose utilization. Gcd activity not only contributes to d-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on d-xylose.

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