Direct carbon capture for production of high-performance biodegradable plastic by cyanobacterial cell factory

2021 ◽  
Author(s):  
Chunlin Tan ◽  
Fei Tao ◽  
Ping Xu
Keyword(s):  
Metabolic Engineering ◽  
Carbon Capture ◽  
De Novo ◽  
Pha Synthase ◽  
Cell Factory ◽  
Cyanobacterial Cell ◽  
Plastic Pollution ◽  
Cell Factories ◽  
Coa Transferase ◽  
Dry Cell Weight

Plastic pollution has become one of the most pressing environmental issues today, leading to an urgent need to develop biodegradable plastics1-3. Polylactic acid (PLA) is one of the most promising biodegradable materials because of its potential applications in disposable packaging, agriculture, medicine, and printing filaments for 3D printers4-6. However, current biosynthesis of PLA entirely uses edible biomass as feedstock, which leads to competition for resources between material production and food supply7,8. Meanwhile, excessive emission of CO2 that is the most abundant carbon source aggravates global warming, and climate instability. Herein, we first developed a cyanobacterial cell factory for the de novo biosynthesis of PLA directly from CO2, using a combinational strategy of metabolic engineering and high-density cultivation (HDC). Firstly, the heterologous pathway for PLA production, which involves engineered D-lactic dehydrogenase (LDH), propionate CoA-transferase (PCT), and polyhydroxyalkanoate (PHA) synthase, was introduced into Synechococcus elongatus PCC7942. Subsequently, different metabolic engineering strategies, including pathway debottlenecking, acetyl-CoA self-circulation, and carbon-flux redirection, were systematically applied, resulting in approximately 19-fold increase to 15 mg/g dry cell weight (DCW) PLA compared to the control. In addition, HDC increased cell density by 10-fold. Finally, the PLA titer of 108 mg/L (corresponding to 23 mg/g DCW) was obtained, approximately 270 times higher than that obtained from the initially constructed strain. Moreover, molecular weight (Mw, 62.5 kDa; Mn, 32.8 kDa) of PLA produced by this strategy was among the highest reported levels. This study sheds a bright light on the prospects of plastic production from CO2 using cyanobacterial cell factories.

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

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

2019 ◽  
Vol 7 (10) ◽  
pp. 472 ◽  
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.

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

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.

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

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.

scholarly journals In silico Design for Systems-Based Metabolic Engineering for the Bioconversion of Valuable Compounds From Industrial By-Products

2021 ◽  
Vol 12 ◽  
Author(s):  
Albert Enrique Tafur Rangel ◽  
Wendy Ríos ◽  
Daisy Mejía ◽  
Carmen Ojeda ◽  
Ross Carlson ◽  
...  
Keyword(s):  
Machine Learning ◽  
Metabolic Engineering ◽  
Succinic Acid ◽  
In Silico ◽  
Industrial Biotechnology ◽  
Cell Factory ◽  
Cell Factories ◽  
Transcriptomics Data ◽  
Valuable Compounds ◽  
By Products

Selecting appropriate metabolic engineering targets to build efficient cell factories maximizing the bioconversion of industrial by-products to valuable compounds taking into account time restrictions is a significant challenge in industrial biotechnology. Microbial metabolism engineering following a rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, it is important to use tools that allow predicting gene deletions. An in silico experiment was performed to model and understand the metabolic engineering effects on the cell factory considering a second complexity level by transcriptomics data integration. In this study, a systems-based metabolic engineering target prediction was used to increase glycerol bioconversion to succinic acid based on Escherichia coli. Transcriptomics analysis suggests insights on how to increase cell glycerol utilization to further design efficient cell factories. Three E. coli models were used: a core model, a second model based on the integration of transcriptomics data obtained from growth in an optimized culture media, and a third one obtained after integration of transcriptomics data from adaptive laboratory evolution (ALE) experiments. A total of 2,402 strains were obtained with fumarase and pyruvate dehydrogenase being frequently predicted for all the models, suggesting these reactions as essential to increase succinic acid production. Finally, based on using flux balance analysis (FBA) results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of the importance of each knockout’s (feature’s) contribution. Glycerol has become an interesting carbon source for industrial processes due to biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. The combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed the versatility of computational models to predict key metabolic engineering targets in a less cost-, time-, and laborious-intensive process. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work as a guide and platform for the selection/engineering of microorganisms for the production of interesting chemical compounds.

scholarly journals Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity

2021 ◽  
Author(s):  
Nils Jonathan Helmuth Averesch ◽  
Vincent Evan Pane ◽  
Frauke Kracke ◽  
Marika Ziesack ◽  
Shannon Noel Nangle ◽  
...  
Keyword(s):  
High Performance ◽  
Durable Goods ◽  
De Novo ◽  
Cupriavidus Necator ◽  
Pha Synthase ◽  
Mode Analysis ◽  
Modern World ◽  
Knock Out ◽  
Cell Factories ◽  
Aromatic Polyesters

Synthetic materials are integral components of consumables and durable goods and indispensable in our modern world. Polyesters are the most versatile bulk- and specialty-polymers, but their production is not sustainable, and their fate at end-of-life of great concern. Bioplastics are highly regarded alternatives but have shortcomings in material properties and commercial competitiveness with conventional synthetic plastics. These constraints have limited the success in global markets. Enabling bio-production of advanced bioplastics with superior properties from waste-derived feedstocks could change this. We have created microbial cell factories that can produce a range of aliphatic and aromatic polyesters. A DphaC1 mutant of Cupriavidus necator H16 was complemented with hydroxyacyl-CoA transferases from either Clostridium propionicum (pct540) or Clostridium difficile (hadA), respectively. These were combined with a mutant PHA synthase (phaC1437) from Pseudomonas sp. MBEL 6 19, which rescued the PHA- phenotype of the knock-out mutant and allowed polymerization of various hydroxy carboxylates, including phloretic acid. This is the first-time, incorporation of an aromatic ring in the backbone of a biological polyester was achieved. Polymers contain para-hydroxyphenyl subunits are structurally analogous to synthetic aromatic polyesters like PET and high-strength polyarylates. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters from H2 and O2 generated in situ, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility of de novo production of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of high-performance thermoplastics and thermosets.

scholarly journals High-Efficiency Production of the Bisabolene from Waste Cooking Oil By Metabolically Engineered Yarrowia Lipolytica

2020 ◽  
Author(s):  
Yakun Zhao ◽  
Kun Zhu ◽  
Jian Li ◽  
Yu Zhao ◽  
Shenglong Li ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
High Efficiency ◽  
De Novo ◽  
Mevalonate Pathway ◽  
Zingiber Officinale ◽  
Waste Cooking Oil ◽  
Cooking Oil ◽  
Cell Factories ◽  
Wide Range

Abstract Background: The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders their isolation from plant sources economically inviable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene.Results: In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, β-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the β-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus, respectively. Subsequently, metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize de novo bisabolene production by the engineered Y. lipolytica strains from glucose. Our engineering strategies have led to engineered Y. lipolytica strains that produce 282.6 mg/L α-bisabolene, 48.3 mg/L β-bisabolene and 5.3 mg/L γ-bisabolene. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from waste cooking oil. The results showed that α-bisabolene, β-bisabolene and γ-bisabolene could be produced at the respective titers of 973.0 mg/L, 68.2 mg/L, 20.2 mg/L in shake flasks. These titers correspond to 2433-fold, 340-fold and 404-fold enhancement in bisabolene production, respectively, over the parent strain.Conclusions: To our knowledge, this is the first report of bisabolene production in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

scholarly journals Metabolic engineering strategy for synthetizing trans-4-hydroxy-l-proline in microorganisms

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenyu Zhang ◽  
Pengfu Liu ◽  
Weike Su ◽  
Huawei Zhang ◽  
Wenqian Xu ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Industrial Applications ◽  
Microbial Cell Factories ◽  
Important Amino Acid ◽  
Cell Factories ◽  
Complex Process ◽  
Engineering Strategy ◽  
Hydrolysis Of ◽  
New Strategies

AbstractTrans-4-hydroxy-l-proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans-4-hydroxy-l-proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low productivity, a complex process and heavy environmental pollution. Presently, trans-4-hydroxy-l-proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to effectively construct microbial cell factories that have improved the trans-4-hydroxy-l-proline biosynthetic pathway. To probe the potential of microorganisms for trans-4-hydroxy-l-proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans-4-hydroxy-l-proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production.

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