CAZyme Prediction in Ascomycetous Yeast Genomes Guides Discovery of Novel Xylanolytic Species with Diverse Capacities for Hemicellulose Hydrolysis

Abstract Background Ascomycetous yeasts from the kingdom fungi inhabit every biome in Nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the Ascomycetous yeast genomes. We identified a total of 224 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species contained several putative xylanolytic enzymes, including the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30 that appear attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possess novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

Download Full-text

CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis

Biotechnology for Biofuels ◽

10.1186/s13068-021-01995-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jonas L. Ravn ◽

Martin K. M. Engqvist ◽

Johan Larsbrink ◽

Cecilia Geijer

Keyword(s):

Biomass Conversion ◽

Enzyme Characterization ◽

Enzymatic Activities ◽

Cell Factory ◽

Ascomycetous Yeast ◽

Substrate Preferences ◽

Cell Factories ◽

Complex Polymers ◽

Ascomycetous Yeasts ◽

Yeast Genomes

Abstract Background Ascomycetous yeasts from the kingdom fungi inhabit every biome in nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the ascomycetous yeast genomes. We identified a total of 217 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, mixed-linkage β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species possess genes for several putative xylanolytic enzymes, including ones from the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30, which appear to be attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possesses novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

Download Full-text

Competence ofCorynebacterium glutamicumas a host for the production of type I polyketides

10.1101/622399 ◽

2019 ◽

Author(s):

Nicolai Kallscheuer ◽

Hirokazu Kage ◽

Lars Milke ◽

Markus Nett ◽

Jan Marienhagen

Keyword(s):

Enzymatic Activities ◽

Microbial Cell ◽

Cell Factory ◽

Type I ◽

Microbial Cell Factory ◽

Acetyl Coa ◽

Microbial Cell Factories ◽

Cell Factories ◽

C Terminus ◽

Malonyl Coa

AbstractType I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4’-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineeredCorynebacterium glutamicumstrain as a novel microbial cell factory for type I PKS-derived products. Suitability ofC. glutamicumfor polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 fromStreptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sato the C-terminus of the maltose-binding protein MalE fromEscherichia coli. Surprisingly, ChlB1Sawas also active in absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACgofC. glutamicumcan also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACgofC. glutamicumcan also activate nonribosomal peptide synthetases (NRPSs), renderingC. glutamicuma promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

Download Full-text

Nanocellulose-based mechanically stable immobilization matrix for enhanced ethylene production: A framework for photosynthetic solid-state cell factories

Green Chemistry ◽

10.1039/d1gc00502b ◽

2021 ◽

Author(s):

Ville Rissanen ◽

Sindhujaa Vajravel ◽

Sergey Kosourov ◽

Suvi Arola ◽

Eero Kontturi ◽

...

Keyword(s):

Solid State ◽

Ethylene Production ◽

Cell Immobilization ◽

Cell Factory ◽

Cell Factories ◽

Solid State Cell ◽

Immobilization Matrix

Cell immobilization is a promising approach to create efficient photosynthetic cell factories for sustainable chemicals production. Here, we demonstrate a novel photosynthetic solid-state cell factory design for sustainable biocatalytic ethylene...

Download Full-text

Computational Analysis of Dynamic Light Exposure of Unicellular Algal Cells in a Flat-Panel Photobioreactor to Support Light-Induced CO2 Bioprocess Development

Frontiers in Microbiology ◽

10.3389/fmicb.2021.639482 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nicolò S. Vasile ◽

Alessandro Cordara ◽

Giulia Usai ◽

Angela Re

Keyword(s):

Light Transmission ◽

Light Exposure ◽

Model Organism ◽

Cell Factory ◽

Bioprocess Development ◽

Cultivation Conditions ◽

Flat Panel ◽

Modeling Framework ◽

Cell Factories ◽

Cell Trajectories

Cyanobacterial cell factories trace a vibrant pathway to climate change neutrality and sustainable development owing to their ability to turn carbon dioxide-rich waste into a broad portfolio of renewable compounds, which are deemed valuable in green chemistry cross-sectorial applications. Cell factory design requires to define the optimal operational and cultivation conditions. The paramount parameter in biomass cultivation in photobioreactors is the light intensity since it impacts cellular physiology and productivity. Our modeling framework provides a basis for the predictive control of light-limited, light-saturated, and light-inhibited growth of the Synechocystis sp. PCC 6803 model organism in a flat-panel photobioreactor. The model here presented couples computational fluid dynamics, light transmission, kinetic modeling, and the reconstruction of single cell trajectories in differently irradiated areas of the photobioreactor to relate key physiological parameters to the multi-faceted processes occurring in the cultivation environment. Furthermore, our analysis highlights the need for properly constraining the model with decisive qualitative and quantitative data related to light calibration and light measurements both at the inlet and outlet of the photobioreactor in order to boost the accuracy and extrapolation capabilities of the model.

Download Full-text

Intelligent microbial cell factory with genetic pH shooting (GPS) for cell self-responsive base/acid regulation

Microbial Cell Factories ◽

10.1186/s12934-020-01457-3 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Chenyi Li ◽

Xiaopeng Gao ◽

Xiao Peng ◽

Jinlin Li ◽

Wenxin Bai ◽

...

Keyword(s):

Ph Regulation ◽

Self Regulation ◽

Scale Up ◽

Microbial Cell ◽

Production Costs ◽

Cell Factory ◽

Ph Sensing ◽

Genetic Circuits ◽

Microbial Cell Factory ◽

Cell Factories

Abstract Background In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called “Genetic pH Shooting (GPS)” to realize microbial self-regulation of pH. Results In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. Conclusions The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

Download Full-text

Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway

Microorganisms ◽

10.3390/microorganisms7100472 ◽

2019 ◽

Vol 7 (10) ◽

pp. 472 ◽

Cited By ~ 9

Author(s):

Larissa Ribeiro Ramos Tramontin ◽

Kanchana Rueksomtawin Kildegaard ◽

Suresh Sudarsan ◽

Irina Borodina

Keyword(s):

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Small Scale ◽

Cell Factory ◽

Standard Equipment ◽

Cell Factories ◽

Dry Cell Weight ◽

Astaxanthin Production ◽

Yeast Yarrowia Lipolytica ◽

Β Carotene

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

Download Full-text

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

10.1101/2021.10.04.462994 ◽

2021 ◽

Author(s):

Eline Postma ◽

Else-Jasmijn Hassing ◽

Venda Mangkusaputra ◽

Jordi Geelhoed ◽

Pilar de la Torre ◽

...

Keyword(s):

Copy Number ◽

Large Scale ◽

Metabolic Networks ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Microbial Cell Factory ◽

Microbial Cell Factories ◽

Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Download Full-text

Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation

Frontiers in Microbiology ◽

10.3389/fmicb.2021.647164 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shanshan Zhang ◽

Sini Zheng ◽

Jiahui Sun ◽

Xuexia Zeng ◽

Yangkai Duan ◽

...

Keyword(s):

Carbon Dioxide ◽

High Temperature ◽

Solar Energy ◽

High Light ◽

Cell Factory ◽

Cyanobacterial Cell ◽

Cell Factories ◽

Green Production ◽

Free Mode ◽

Pcc 7942

Photosynthetic biomanufacturing is a promising route for green production of biofuels and biochemicals utilizing carbon dioxide and solar energy. Cyanobacteria are important microbial platforms for constructing photosynthetic cell factories. Toward scaled outdoor cultivations in the future, high light and high temperature tolerances of cyanobacterial chassis strains and cell factories would be determinant properties to be optimized. We proposed a convenient strategy for rapidly improving high light and high temperature tolerances of an important cyanobacterial chassis Synechococcus elongatus PCC 7942 and the derived cell factories. Through introduction and isolation of an AtpA-C252F mutation, PCC 7942 mutants with improved high light and high temperature tolerances could be obtained in only 4 days with an antibiotics-free mode. Adopting this strategy, cellular robustness and sucrose synthesizing capacities of a PCC 7942 cell factory were successfully improved.

Download Full-text

Data-driven rational biosynthesis design: from molecules to cell factories

Briefings in Bioinformatics ◽

10.1093/bib/bbz065 ◽

2019 ◽

Vol 21 (4) ◽

pp. 1238-1248

Author(s):

Fu Chen ◽

Le Yuan ◽

Shaozhen Ding ◽

Yu Tian ◽

Qian-Nan Hu

Keyword(s):

Large Scale ◽

Protein Domain ◽

Data Driven ◽

Cell Factory ◽

Dna Assembly ◽

Metabolic Reaction ◽

Cell Factories ◽

Metabolic Systems ◽

One Stop ◽

Target Molecules

Abstract A proliferation of chemical, reaction and enzyme databases, new computational methods and software tools for data-driven rational biosynthesis design have emerged in recent years. With the coming of the era of big data, particularly in the bio-medical field, data-driven rational biosynthesis design could potentially be useful to construct target-oriented chassis organisms. Engineering the complicated metabolic systems of chassis organisms to biosynthesize target molecules from inexpensive biomass is the main goal of cell factory design. The process of data-driven cell factory design could be divided into several parts: (1) target molecule selection; (2) metabolic reaction and pathway design; (3) prediction of novel enzymes based on protein domain and structure transformation of biosynthetic reactions; (4) construction of large-scale DNA for metabolic pathways; and (5) DNA assembly methods and visualization tools. The construction of a one-stop cell factory system could achieve automated design from the molecule level to the chassis level. In this article, we outline data-driven rational biosynthesis design steps and provide an overview of related tools in individual steps.

Download Full-text

Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency

Cell Discovery ◽

10.1038/s41421-018-0075-5 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 24

Author(s):

Pingping Wang ◽

Wei Wei ◽

Wei Ye ◽

Xiaodong Li ◽

Wenfang Zhao ◽

...

Keyword(s):

Yeast Cell ◽

Batch Fermentation ◽

Cell Factory ◽

Ginsenoside Rh2 ◽

Fed Batch ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Shake Flasks ◽

Produce Plant

AbstractSynthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

Download Full-text