Metabolic Engineering of Brassica Seeds Oils: Improvement of Oil Quality and Quantity and Alteration of Carbon Flux

Abstract Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable.Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol.Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

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ATP drives efficient terpene biosynthesis in marine thraustochytrids

10.1101/2020.11.20.391870 ◽

2020 ◽

Author(s):

Aiqing Zhang ◽

Kaya Mernitz ◽

Chao Wu ◽

Wei Xiong ◽

Yaodong He ◽

...

Keyword(s):

Lipid Metabolism ◽

Metabolic Engineering ◽

Energy Metabolism ◽

Carbon Flux ◽

Driving Force ◽

Mevalonate Pathway ◽

Terpene Biosynthesis ◽

Acetyl Coa ◽

Essential Question ◽

Thermodynamic Driving Force

ABSTRACTUnderstanding carbon flux-controlling mechanisms in a tangled metabolic network is an essential question of cell metabolism. Secondary metabolism, such as terpene biosynthesis, has evolved with low carbon flux due to inherent pathway constraints. Thraustochytrids are a group of heterotrophic marine unicellular protists, and can accumulate terpenoids under the high salt condition in their natural environment. However, the mechanism behind the terpene accumulation is not well understood. Here we show that terpene biosynthesis in Thraustochytrium sp. ATCC 26185 is constrained by local thermodynamics in the mevalonate pathway. Thermodynamic analysis reveals the metabolite limitation in the nondecarboxylative Claisen condensation of acetyl-CoA to acetoacetyl-CoA step catalyzed by the acetyl-CoA acetyltransferase (ACAT). Through a sodium elicited mechanism, higher respiration leads to increased ATP investment into the mevalonate pathway, providing a strong thermodynamic driving force for enhanced terpene biosynthesis. The proteomic analysis further indicates that the increased ATP demands are fulfilled by shifting energy generation from carbohydrate to lipid metabolism. This study demonstrates a unique strategy in nature using ATP to drive a low-flux metabolic pathway, providing an alternative solution for efficient terpene metabolic engineering.IMPORTANCETerpenoids are a large class of lipid molecules with important biological functions, and diverse industrial and medicinal applications. Metabolic engineering for terpene production has been hindered by the low flux distribution to its biosynthesis pathway. In practice, a high substrate load is generally required to reach high product titers. Here we show that the mevalonate-derived terpene biosynthesis is constrained by local pathway thermodynamics, which can only be partially relieved by increasing substrate levels. Through comparative proteomic and biochemical analyses, we discovered a unique mechanism for high terpene accumulation in marine protists thraustochytrids. Through a sodium induced mechanism, thraustochytrids shift their energy metabolism from carbohydrate to lipid metabolism for enhanced ATP production, providing a strong thermodynamic driving force for efficient terpene biosynthesis. This study reveals an important mechanism in eukaryotes to overcome the thermodynamic constraint in low-flux pathways by increased ATP consumption. Engineering energy metabolism thus provides an important alternative to relieve flux constraints in low-flux and energy-consuming pathways.

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Metabolic Engineering Design Strategies for Increasing Acetyl-CoA Flux

Metabolites ◽

10.3390/metabo10040166 ◽

2020 ◽

Vol 10 (4) ◽

pp. 166 ◽

Cited By ~ 3

Author(s):

Jason T. Ku ◽

Arvin Y. Chen ◽

Ethan I. Lan

Keyword(s):

Amino Acids ◽

Metabolic Engineering ◽

Engineering Design ◽

Carbon Flux ◽

Design Strategies ◽

Acetyl Coa

Acetyl-CoA is a key metabolite precursor for the biosynthesis of lipids, polyketides, isoprenoids, amino acids, and numerous other bioproducts which are used in various industries. Metabolic engineering efforts aim to increase carbon flux towards acetyl-CoA in order to achieve higher productivities of its downstream products. In this review, we summarize the strategies that have been implemented for increasing acetyl-CoA flux and concentration, and discuss their effects. Furthermore, recent works have developed synthetic acetyl-CoA biosynthesis routes that achieve higher stoichiometric yield of acetyl-CoA from glycolytic substrates.

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Efficient production of myo-inositol in Escherichia coli through metabolic engineering

10.21203/rs.3.rs-17410/v1 ◽

2020 ◽

Author(s):

Ran You ◽

Lei Wang ◽

Congrong Shi ◽

Hao Chen ◽

Shasha Zhang ◽

...

Keyword(s):

Metabolic Engineering ◽

Carbon Flux ◽

Scale Up ◽

Value Added ◽

Efficient Production ◽

Synthetic Pathway ◽

E Coli ◽

Mixed Carbon Source ◽

Glycolysis Pathway

Abstract Background The biosynthesis of high value-added compounds through metabolically engineered strains has received widely attention in recent years. As an effective compound in pharmaceutical, cosmetic and food industry, myo-inositol (inositol) is mainly produced via a harsh set of chemical reactions from phytate. The proper distribution of carbon flux between cell growth and inositol production was a major challenge for constructing an efficient inositol-synthetic pathway. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce inositol, yet with a low yield. Therefore, construction of E. coli metabolically engineered strains with high stoichiometric yield will be attractive. Results In the present study, the recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained to efficiently synthesize inositol. Inositol was successfully biosynthesized after introducing two crucial enzymes, inositol-3-phosphate synthase (IPS) from Trypanosoma brucei , and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild type) and SG104 ( ΔptsG::glk , ΔgalR::zglf , ΔpoxB::acs ), a series of engineered strains for inositol production were constructed by deleting the key genes pgi, pfkA or pykF . Furthermore, the plasmid expression systems of IPS and IMP were optimized, and the gene zwf was regulated to enhance stoichiometric yield. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved with the combined strain R15 of SG104, Δpgi , Δpgm , and RBSL5-zwf. Simultaneously, the engineered strain R04 reached high-density fermentation level in a 1-L fermenter by using glucose and glycerol as mixed carbon source. In the scale-up bioconversion in situ with R04, 0.82 mol inositol/mol glucose was produced by fed-batch within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM). Conclusions The biosynthetic pathway of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provided an essential reference to obtain a suitable distribution of carbon flux between glycolysis pathway and product synthetic pathway.

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Metabolic engineering of Bacillus subtilis for redistributing the carbon flux to 2,3-butanediol by manipulating NADH levels

Biotechnology for Biofuels ◽

10.1186/s13068-015-0320-1 ◽

2015 ◽

Vol 8 (1) ◽

Cited By ~ 15

Author(s):

Taowei Yang ◽

Zhiming Rao ◽

Guiyuan Hu ◽

Xian Zhang ◽

Mei Liu ◽

...

Keyword(s):

Bacillus Subtilis ◽

Metabolic Engineering ◽

Carbon Flux

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Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

10.21203/rs.2.18380/v1 ◽

2019 ◽

Author(s):

Huiling Liu ◽

Shuangying Liu ◽

Fengyu Xie ◽

Xian Zhang ◽

Meijuan Xu ◽

...

Keyword(s):

Bacillus Subtilis ◽

Metabolic Engineering ◽

Carbon Flux ◽

Glucose Dehydrogenase ◽

Transcriptional Regulator ◽

Pentose Phosphate ◽

Regeneration System ◽

Nadph Regeneration ◽

By Products ◽

Regeneration Systems

Abstract Background: Generally, glucose is transformed into pyruvate from glycolysis before the target products acetoin and 2,3-butanediol (2,3-BDO) are formed. Pentose Phosphate Pathway (PPP) is an inefficient synthetic pathway for pyruvate production from glucose in Bacillus subtilis. Previously, it was found that engineered PPP in B. subtilis unbalanced NADH and NADPH regeneration systems and affected acetoin and 2,3 -BDO production.Results: In this study, metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BDO via reconstructing intracellular cofactors regeneration systems. Firstly, extra copies of glucose dehydrogenase (GDH)and an exogenous NADPH-dependent 2,3-BDO dehydrogenase (TDH) were introduced into the GRAS strain B. subtilis 168 to introduce an exogenous NADPH/NADP + regeneration system and broaden 2,3-BDO production pathway. It was found that overexpressing the NADPH/NADP + regeneration system effectively improved 2,3-BDO production and inhibited NADH-dependent by-products accumulation. Subsequently, the disruption of lactate dehydrogenase (encoded by ldh ) by insertion of the transcriptional regulator ALsR, essential for the expression of alsSD (encoding two key enzymes for the conversion of pyruvate to acetoin) in B. subtilis, resulted in the recombinant strain in which alsSD was overexpressed and the pathway to lactate was blocked simultaneously. On fermentation by the result engineered strain, the highest 2,3-BDO concentration increased by18.43%, while the titers of main byproducts acetoin and lactate decreased by 22.03% and 64%, respectively.Conclusion: In this study, it shows that engineering PPP and reconstructing intracellular cofactors regeneration system could be an alternative strategy in the metabolic engineering of 2,3-BDO production in B. subtilis .

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Efficient production of myo-inositol in Escherichia coli through metabolic engineering

10.21203/rs.3.rs-17410/v2 ◽

2020 ◽

Author(s):

Ran You ◽

Lei Wang ◽

Congrong Shi ◽

Hao Chen ◽

Shasha Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Carbon Flux ◽

Value Added ◽

Efficient Production ◽

Wild Type ◽

Food Industries ◽

E Coli ◽

Mixed Carbon Source

Abstract Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable.Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol.Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

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Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

Author(s):

Qiong Wang ◽

Michael J. Betenbaugh

Keyword(s):

Metabolic Engineering ◽

Cho Cells ◽

Genetic Manipulation ◽

Chinese Hamster ◽

Post Translational Modification ◽

Effector Functions ◽

Recombinant Glycoproteins ◽

Production Platform ◽

Chemical Inhibitors ◽

Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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