scholarly journals Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts

2020 ◽  
Vol 12 (12) ◽  
pp. 5125
Author(s):  
Neha Arora ◽  
Hong-Wei Yen ◽  
George P. Philippidis
Keyword(s):  
Genetic Engineering ◽  
Lipid Production ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Production Costs ◽  
Screening Methods ◽  
Scale Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Oleaginous Microalgae

Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. Strain improvement can play an essential role in the development of such industrial microorganisms by increasing lipid production and hence reducing production costs. The main means of strain improvement are random mutagenesis, adaptive laboratory evolution (ALE), and rational genetic engineering. Among these, random mutagenesis and ALE are straight forward, low-cost, and do not require thorough knowledge of the microorganism’s genetic composition. This paper reviews available mutagenesis and ALE techniques and screening methods to effectively select for oleaginous microalgae and yeasts with enhanced lipid yield and understand the alterations caused to metabolic pathways, which could subsequently serve as the basis for further targeted genetic engineering.

scholarly journals Application of Microalgal Stress Responses in Industrial Microalgal Production Systems

Marine Drugs ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 30
Author(s):  
Jia Wang ◽  
Yuxin Wang ◽  
Yijian Wu ◽  
Yuwei Fan ◽  
Changliang Zhu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Production Systems ◽  
Strain Improvement ◽  
Value Added ◽  
Product Yield ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Theoretical Support ◽  
Further Development ◽  
Value Added Products

Adaptive laboratory evolution (ALE) has been widely utilized as a tool for developing new biological and phenotypic functions to explore strain improvement for microalgal production. Specifically, ALE has been utilized to evolve strains to better adapt to defined conditions. It has become a new solution to improve the performance of strains in microalgae biotechnology. This review mainly summarizes the key results from recent microalgal ALE studies in industrial production. ALE designed for improving cell growth rate, product yield, environmental tolerance and wastewater treatment is discussed to exploit microalgae in various applications. Further development of ALE is proposed, to provide theoretical support for producing the high value-added products from microalgal production.

scholarly journals Genetic engineering of microalgae lipid biosynthesis for sustainable biodiesel production

2021 ◽  
Vol 11 (3) ◽  
pp. 072-077
Author(s):  
Siti Zulaiha
Keyword(s):  
Genetic Engineering ◽  
Metabolic Pathways ◽  
Alternative Energy ◽  
Fossil Fuels ◽  
Lipid Production ◽  
High Growth ◽  
Lipid Biosynthesis ◽  
Production Costs ◽  
Biodiesel Production ◽  
Promising Alternative

Biofuel is one of the most promising alternative energy sources for reducing human reliance on fossil fuels. Microalgae has recently emerged as the most promising biofuel source. However, biofuels from microalgae are still not feasible to replace fossil fuels because of their high production costs, therefore, it is necessary to pick microalgae species with high growth rates and lipid content. Overexpression of lipid biosynthesis enzymes and inhibition of competitive metabolic pathways are two genetic engineering strategies that can be developed to assess microalgae lipid production. Malate and multienzyme enzymes (GPAT, LPAAT and DGAT) can be overexpressed in microalgae to boost lipid production. The strategy of blocking competitive metabolic pathways can be carried out through suppression of starch metabolism and lipid catabolism. The strategy of blocking competitive metabolic pathways has been carried out in several microalgae and is effective for enhancing lipid biosynthesis. Several mutations that block both the starch metabolic and lipid catabolic pathways can result in increased levels of microalgal lipid accumulation.

scholarly journals Thermotolerant Yeast Strains Adapted by Laboratory Evolution Show Trade-Off at Ancestral Temperatures and Preadaptation to Other Stresses

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Luis Caspeta ◽  
Jens Nielsen
Keyword(s):  
Ethanol Production ◽  
Production Costs ◽  
Thermotolerant Yeast ◽  
Adaptive Laboratory Evolution ◽  
Genetic Changes ◽  
Laboratory Evolution ◽  
Trade Off ◽  
Thermal Niche ◽  
Production Processes ◽  
Yeast Strains

ABSTRACT A major challenge for the production of ethanol from biomass-derived feedstocks is to develop yeasts that can sustain growth under the variety of inhibitory conditions present in the production process, e.g., high osmolality, high ethanol titers, and/or elevated temperatures (≥40°C). Using adaptive laboratory evolution, we previously isolated seven Saccharomyces cerevisiae strains with improved growth at 40°C. Here, we show that genetic adaptations to high temperature caused a growth trade-off at ancestral temperatures, reduced cellular functions, and improved tolerance of other stresses. Thermotolerant yeast strains showed horizontal displacement of their thermal reaction norms to higher temperatures. Hence, their optimal and maximum growth temperatures increased by about 3°C, whereas they showed a growth trade-off at temperatures below 34°C. Computational analysis of the physical properties of proteins showed that the lethal temperature for yeast is around 49°C, as a large fraction of the yeast proteins denature above this temperature. Our analysis also indicated that the number of functions involved in controlling the growth rate decreased in the thermotolerant strains compared with the number in the ancestral strain. The latter is an advantageous attribute for acquiring thermotolerance and correlates with the reduction of yeast functions associated with loss of respiration capacity. This trait caused glycerol overproduction that was associated with the growth trade-off at ancestral temperatures. In combination with altered sterol composition of cellular membranes, glycerol overproduction was also associated with yeast osmotolerance and improved tolerance of high concentrations of glucose and ethanol. Our study shows that thermal adaptation of yeast is suitable for improving yeast resistance to inhibitory conditions found in industrial ethanol production processes. IMPORTANCE Yeast thermotolerance can significantly reduce the production costs of biomass conversion to ethanol. However, little information is available about the underlying genetic changes and physiological functions required for yeast thermotolerance. We recently revealed the genetic changes of thermotolerance in thermotolerant yeast strains (TTSs) generated through adaptive laboratory evolution. Here, we examined these TTSs’ physiology and computed their proteome stability over the entire thermal niche, as well as their preadaptation to other stresses. Using this approach, we showed that TTSs exhibited evolutionary trade-offs in the ancestral thermal niche, as well as reduced numbers of growth functions and preadaptation to other stresses found in ethanol production processes. This information will be useful for rational engineering of yeast thermotolerance for the production of biofuels and chemicals.

Strain improvement of Chlorella sp. for phenol biodegradation by adaptive laboratory evolution

2016 ◽  
Vol 205 ◽  
pp. 264-268 ◽  
Author(s):  
Libo Wang ◽  
Chuizhao Xue ◽  
Liang Wang ◽  
Quanyu Zhao ◽  
Wei Wei ◽  
...  
Keyword(s):  
Strain Improvement ◽  
Adaptive Laboratory Evolution ◽  
Phenol Biodegradation ◽  
Laboratory Evolution ◽  
Chlorella Sp

13C-NMR, MS and metabolic flux balancing in biotechnology research

1998 ◽  
Vol 31 (1) ◽  
pp. 41-106 ◽  
Author(s):  
THOMAS SZYPERSKI
Keyword(s):  
Large Scale ◽  
Metabolic Flux ◽  
Raw Materials ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Living System ◽  
European Federation ◽  
Scale Production ◽  
Natural Scientist ◽  
Engineering Sciences

The European Federation of Biotechnology defines biotechnology as ‘the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services’. Biotechnology thus focuses on the industrial exploitation of biological systems and is based on their unique expertise in specific molecular recognition and catalysis. The enormous potential for drug synthesis, design of biomedical diagnostics, large-scale production of biochemicals including fuels, food production, degradation of resistant wastes and extraction of raw materials will very likely make biotechnology, along with electronics and material sciences, one of the key technologies of the 21st century. From the chemical engineer's point of view, the living system participating in a biotechnological process is the central unit that catalyses chemical reactions. It exhibits a complex dependence on the bioprocess parameters, and the engineer focuses on these parameters to achieve optimal control (Hamer, 1985; Bailey & Ollis, 1986). For the natural scientist, the living system itself is in the centre of interest, so that attempts to optimize a bioprocess aim at its appropriate redesign by genetic manipulations. The increase in penicillin production by strain improvement based on random mutagenesis, which was pursued from 1940 to the mid 1970s, represents an early contribution of life scientists to improve a bioprocess that is of utmost medical importance (Hardy & Oliver, 1985).

scholarly journals Enhanced inhibitor tolerance and increased lipid productivity through adaptive laboratory evolution in the oleaginous yeast Metshnikowia pulcherrima

2020 ◽  
Author(s):  
Robert H. Hicks ◽  
Yuxin Sze ◽  
Christopher J. Chuck ◽  
Daniel A. Henk
Keyword(s):  
Formic Acid ◽  
Oleaginous Yeast ◽  
Lipid Production ◽  
Dry Weight ◽  
Lipid Productivity ◽  
Adaptive Laboratory Evolution ◽  
Stirred Tank Bioreactor ◽  
Inhibitor Tolerance ◽  
Laboratory Evolution ◽  
Wide Range

AbstractMicrobial lipid production from second generation feedstocks presents a sustainable route to future fuels, foods and bulk chemicals. The oleaginous yeast Metshnikowia pulcherrima has previously been investigated as a potential platform organism for lipid production due to its ability to be grown in non-sterile conditions and metabolising a wide range of oligo- and monosaccharide carbon sources within lignocellulosic hydrolysates. However, the generation of inhibitors from depolymerisation causes downstream bioprocessing complications, and despite M. pulcherrima’s comparative tolerance, their presence is deleterious to both biomass and lipid formation. Using either a single inhibitor (formic acid) or an inhibitor cocktail (formic acid, acetic acid, fufural and HMF), two strategies of adaptive laboratory evolution were performed to improve M. pulcherrima’s fermentation inhibitor tolerance. Using a sequential batch culturing approach, the resulting strains from both strategies had increased growth rates and reduced lag times under inhibiting conditions versus the progenitor. Interestingly, the lipid production of the inhibitor cocktail evolved strains markedly increased, with one strain producing 41% lipid by dry weight compared to 22% of the progenitor. The evolved species was cultured in a non-sterile 2L stirred tank bioreactor and accumulated lipid rapidly, yielding 6.1 g/L of lipid (35% cell dry weight) within 48 hours; a lipid productivity of 0.128 g L-1 h-1. Furthermore, the lipid profile was analogous to palm oil, consisting of 39% C16:0 and 56% C18:1 after 48 hours.

scholarly journals Adaptive laboratory evolution of β-caryophyllene producing Saccharomyces cerevisiae

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Avinash Godara ◽  
Katy C. Kao
Keyword(s):  
Oxidative Stress ◽  
Yeast Strain ◽  
Strain Improvement ◽  
Genomic Analysis ◽  
Pathway Engineering ◽  
Selection Strategy ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Fourfold Increase ◽  
Extracellular Product

Abstract Background β-Caryophyllene is a plant terpenoid with therapeutic and biofuel properties. Production of terpenoids through microbial cells is a potentially sustainable alternative for production. Adaptive laboratory evolution is a complementary technique to metabolic engineering for strain improvement, if the product-of-interest is coupled with growth. Here we use a combination of pathway engineering and adaptive laboratory evolution to improve the production of β-caryophyllene, an extracellular product, by leveraging the antioxidant potential of the compound. Results Using oxidative stress as selective pressure, we developed an adaptive laboratory evolution that worked to evolve an engineered β-caryophyllene producing yeast strain for improved production within a few generations. This strategy resulted in fourfold increase in production in isolated mutants. Further increasing the flux to β-caryophyllene in the best evolved mutant achieved a titer of 104.7 ± 6.2 mg/L product. Genomic analysis revealed a gain-of-function mutation in the a-factor exporter STE6 was identified to be involved in significantly increased production, likely as a result of increased product export. Conclusion An optimized selection strategy based on oxidative stress was developed to improve the production of the extracellular product β-caryophyllene in an engineered yeast strain. Application of the selection strategy in adaptive laboratory evolution resulted in mutants with significantly increased production and identification of novel responsible mutations.

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