scholarly journals Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

2021 ◽  
Vol 8 ◽  
Author(s):  
Peng-Wei Huang ◽  
Ying-Shuang Xu ◽  
Xiao-Man Sun ◽  
Tian-Qiong Shi ◽  
Yang Gu ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Homologous Recombination ◽  
Gene Editing ◽  
Saturated Fatty Acids ◽  
Cell Factory ◽  
Terpenoid Biosynthesis ◽  
Acetyl Coa ◽  
Knock Out ◽  
Cell Factories ◽  
Efficient Gene

Schizochytrium sp. HX-308 is a marine microalga with fast growth and high lipid content, which has potential as microbial cell factories for lipid compound biosynthesis. It is significant to develop efficient genetic editing tool and discover molecular target in Schizochytrium sp. HX-308 for lipid compound biosynthesis. In this study, we developed an efficient gene editing tool in HX-308 which was mediated by Agrobacterium tumefaciens AGL-1. Results showed that the random integration efficiency reached 100%, and the homologous recombination efficiency reached about 30%. Furthermore, the metabolic pathway of lipid and terpenoid biosynthesis were engineered. Firstly, the acetyl-CoA c-acetyltransferase was overexpressed in HX-308 with a strong constitutive promoter. With the overexpression of acetyl-CoA c-acetyltransferase, more acetyl-CoA was used to synthesize terpenoids, and the production of squalene, β-carotene and astaxanthin was increased 5.4, 1.8, and 2.4 times, respectively. Interestingly, the production of saturated fatty acids and polyunsaturated fatty acids also changed. Moreover, three Acyl-CoA oxidase genes which catalyze the first step of β-oxidation were knocked out using homologous recombination. Results showed that the production of lipids increased in the three knock-out strains. Our results demonstrated that the A. tumefaciens-mediated transformation method will be of great use for the study of function genes, as well as developing Schizochytrium sp. as a strong cell factory for producing high value products.

scholarly journals Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Yiming Zhang ◽  
Mo Su ◽  
Ning Qin ◽  
Jens Nielsen ◽  
Zihe Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Unsaturated Fatty Acids ◽  
Pyruvate Dehydrogenase Complex ◽  
Cell Factory ◽  
Acetyl Coa ◽  
Fatty Acid Production

Abstract Background Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.

scholarly journals Expressing a Cytosolic Pyruvate Dehydrogenase Complex to Increase Free Fatty Acid Production in Saccharomyces Cerevisiae

2020 ◽  
Author(s):  
Yiming Zhang ◽  
Mo Su ◽  
Ning Qin ◽  
Jens Nielsen ◽  
Zihe Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Unsaturated Fatty Acids ◽  
Pyruvate Dehydrogenase Complex ◽  
Cell Factory ◽  
Acetyl Coa ◽  
Fatty Acid Production

Abstract Background Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The final strain with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acid in shake flask, which was 83.2% higher than the control strain. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.

Engineering outer membrane could facilitate better bacterial performance and effectively enhance poly-3-hydroxybutyrate accumulation

2021 ◽  
Author(s):  
Jianli Wang ◽  
Wenjian Ma ◽  
Yu Fang ◽  
Hailing Zhang ◽  
Hao Liang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Inclusion Bodies ◽  
Cellular Metabolism ◽  
Gene Clusters ◽  
Microbial Cell ◽  
Cell Factory ◽  
Acetyl Coa ◽  
E Coli ◽  
Cell Factories ◽  
Phb Production

Poly-3-hydroxybutyrate (PHB) is an environmentally friendly polymer and can be produced in Escherichia coli cells after overexpressing the heterologous gene cluster phaCAB . The biosynthesis of outer membrane (OM) consumes lots of nutrients and influences cell morphology. Here we engineered OM by disrupting all gene clusters relevant to polysaccharide portion of LPS, colanic acid (CA), flagella or/and fimbria in E. coli W3110. All these disruptions benefited PHB production. Especially, disrupting all these OM components improved PHB content to 83.0 wt%, while the wild-type control produced only 1.5 wt% PHB. The improvement was mainly due to the LPS truncation to Kdo 2 -lipid A, which facilitated 82.0 wt% PHB with 25-fold larger cell volume; and disrupting CA facilitated 57.8 wt% PHB. In addition, disrupting LPS facilitated advantageous fermentation features including 69.1% less acetate, 550% higher percentage of autoaggregated cells among the total culture cells, 69.1% less biofilm and higher cellular broken ratio. Further detailed mechanism investigations showed that disrupting LPS caused global regulations on envelope and cellular metabolism: (i) sharply decrease of flagella, fimbria and secretions; (ii) more elastic cell; (iii) much more carbon flux towards acetyl-CoA and cofactors supply including NADP, NAD and ATP; (iv) decrease of byproduct acids but increase of γ-aminobutyric acid by activating σ E factor. Disrupting CA, flagella and fimbria also improved the levels of acetyl-CoA and cofactors. The results indicated that engineering OM is an effective strategy to enhance PHB production, and highlighted the applicability of OM engineering to increase microbial cell factory performance. Importance Understanding the detailed influence of OM on cell envelope and cellular metabolism is important for optimizing E. coli cell factory and many other microorganisms. This study revealed the applicability of remodeling OM to enhance PHB accumulation as representative inclusion bodies. The knowledge generated in this study provided insights concerning the influence and application of OM engineering, and gave essential references for producing other inclusion bodies or chemicals derived from acetyl-CoA or with the need of cofactor NADPH, NADH or ATP supply, and reducing byproduct acids. This study is promising to provide new ideas for the improvement of microbial cell factories.

scholarly journals Continuous photoproduction of hydrocarbon drop-in fuel by microbial cell factories

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Solène Moulin ◽  
Bertrand Légeret ◽  
Stéphanie Blangy ◽  
Damien Sorigué ◽  
Adrien Burlacot ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Gas Phase ◽  
Low Cost ◽  
Continuous Production ◽  
Production Costs ◽  
Cell Factory ◽  
Medium Chain ◽  
Continuous Release ◽  
Cell Factories

Abstract Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

A POSSIBLE MECHANISM FOR FATTY ACID INHIBITION OF CHOLESTEROL SYNTHESIS

1957 ◽  
Vol 35 (1) ◽  
pp. 645-653
Author(s):  
J. D. Wood ◽  
B. B. Migicovsky
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cholesterol Synthesis ◽  
Unsaturated Fatty Acids ◽  
Cholesterol Biosynthesis ◽  
Saturated Fatty Acids ◽  
Liver Homogenate ◽  
Acid Inhibition ◽  
Acetyl Coa ◽  
Fatty Acid Inhibition

Further investigations have been carried out on the fatty acid inhibition of cholesterol biosynthesis in rat liver homogenates. A correlation appears to exist between cholesterol inhibition and the elongation of the carbon chain of saturated fatty acids containing an even number of carbon atoms. Neither saturated nor unsaturated fatty acids interfere with the formation of acetyl CoA by liver homogenate. The stage where acetoacetate is formed from acetyl CoA is suggested as a possible site for inhibition of cholesterol synthesis by fatty acids.

scholarly journals Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Yiming Zhang ◽  
Mo Su ◽  
Ning Qin ◽  
Jens Nielsen ◽  
Zihe Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Unsaturated Fatty Acids ◽  
Pyruvate Dehydrogenase Complex ◽  
Cell Factory ◽  
Acetyl Coa ◽  
Fatty Acid Production

Abstract Background Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD + -dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP + -dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.

A POSSIBLE MECHANISM FOR FATTY ACID INHIBITION OF CHOLESTEROL SYNTHESIS

1957 ◽  
Vol 35 (8) ◽  
pp. 645-653
Author(s):  
J. D. Wood ◽  
B. B. Migicovsky
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cholesterol Synthesis ◽  
Unsaturated Fatty Acids ◽  
Cholesterol Biosynthesis ◽  
Saturated Fatty Acids ◽  
Liver Homogenate ◽  
Acid Inhibition ◽  
Acetyl Coa ◽  
Fatty Acid Inhibition

Further investigations have been carried out on the fatty acid inhibition of cholesterol biosynthesis in rat liver homogenates. A correlation appears to exist between cholesterol inhibition and the elongation of the carbon chain of saturated fatty acids containing an even number of carbon atoms. Neither saturated nor unsaturated fatty acids interfere with the formation of acetyl CoA by liver homogenate. The stage where acetoacetate is formed from acetyl CoA is suggested as a possible site for inhibition of cholesterol synthesis by fatty acids.

scholarly journals gcFront: a tool for determining a Pareto front of growth-coupled cell factory designs

2021 ◽  
Author(s):  
Laurence Legon ◽  
Christophe Corre ◽  
Declan G. Bates ◽  
Ahmad A. Mannan
Keyword(s):  
Cell Growth ◽  
Coupling Strength ◽  
Rational Design ◽  
Pareto Front ◽  
Software Tool ◽  
Cell Factory ◽  
Knock Out ◽  
Cell Factories ◽  
Growth Product ◽  
Genome Scale

Motivation: A widely applicable strategy for developing evolutionarily robust cell factories is to knock out (KO) genes or reactions to couple chemical synthesis with cell growth. Genome-scale metabolic models enable their rational design, but KOs that provide growth-coupling (gc) are rare in the immense design space, making searching difficult and slow, and though several measures determine the utility of those strains, few drive the search. Results: To address these issues we developed a software tool named gcFront - using a genetic algorithm it explores KOs that maximise key performance objectives: cell growth, product synthesis, and coupling strength. Our measure of coupling strength facilitates the search, so gcFront not only finds a gc-design in minutes but also outputs many alternative Pareto optimal gc-designs from a single run - granting users freedom to select designs to take to the lab.

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

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.

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