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

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.

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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.

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

Microbial Cell Factories ◽

10.1186/s12934-021-01615-1 ◽

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.

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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.

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Tn-Core: context-specific reconstruction of core metabolic models using Tn-seq data

10.1101/221325 ◽

2017 ◽

Cited By ~ 2

Author(s):

George C diCenzo ◽

Alessio Mengoni ◽

Marco Fondi

Keyword(s):

Sinorhizobium Meliloti ◽

Metabolic Networks ◽

De Novo ◽

Supplementary Information ◽

Data Sets ◽

Microbial Cell Factories ◽

Cell Factories ◽

Functional Models ◽

Metabolic Models ◽

Context Specific

ABSTRACTMotivationTn-seq (transposon mutagenesis and sequencing) and constraint-based metabolic modelling represent highly complementary approaches. They can be used to probe the core genetic and metabolic networks underlying a biological process, revealing invaluable information for synthetic biology engineering of microbial cell factories. However, while algorithms exist for integration of –omics data sets with metabolic models, no method has been explicitly developed for integration of Tn-seq data with metabolic reconstructions.ResultsWe report the development of Tn-Core, a Matlab toolbox designed to generate gene-centric, context-specific core reconstructions consistent with experimental Tn-seq data. Extensions of this algorithm allow: i) the generation of context-specific functional models through integration of both Tn-seq and RNA-seq data; ii) to visualize redundancy in core metabolic processes; and iii) to assist in curation ofde novodraft metabolic models. The utility of Tn-Core is demonstrated primarily using aSinorhizobium melilotimodel as a case study.Availability and implementationThe software can be downloaded fromhttps://github.com/diCenzo-GC/Tn-Core. All results presented in this work have been obtained with Tn-Core v. [email protected],[email protected] informationSupplementary data are available at Bioinformatics online.

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Comparative genomics study reveals Red Sea Bacillus with characteristics associated with potential microbial cell factories (MCFs)

Scientific Reports ◽

10.1038/s41598-019-55726-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

G. Othoum ◽

S. Prigent ◽

A. Derouiche ◽

L. Shi ◽

A. Bokhari ◽

...

Keyword(s):

Red Sea ◽

Metabolic Networks ◽

Genomic Region ◽

Microbial Cell ◽

Microbial Cell Factories ◽

Cell Factories ◽

Ecological Features ◽

Bacillus Paralicheniformis ◽

Protein Secretion System ◽

Potential Use

AbstractRecent advancements in the use of microbial cells for scalable production of industrial enzymes encourage exploring new environments for efficient microbial cell factories (MCFs). Here, through a comparison study, ten newly sequenced Bacillus species, isolated from the Rabigh Harbor Lagoon on the Red Sea shoreline, were evaluated for their potential use as MCFs. Phylogenetic analysis of 40 representative genomes with phylogenetic relevance, including the ten Red Sea species, showed that the Red Sea species come from several colonization events and are not the result of a single colonization followed by speciation. Moreover, clustering reactions in reconstruct metabolic networks of these Bacillus species revealed that three metabolic clades do not fit the phylogenetic tree, a sign of convergent evolution of the metabolism of these species in response to special environmental adaptation. We further showed Red Sea strains Bacillus paralicheniformis (Bac48) and B. halosaccharovorans (Bac94) had twice as much secreted proteins than the model strain B. subtilis 168. Also, Bac94 was enriched with genes associated with the Tat and Sec protein secretion system and Bac48 has a hybrid PKS/NRPS cluster that is part of a horizontally transferred genomic region. These properties collectively hint towards the potential use of Red Sea Bacillus as efficient protein secreting microbial hosts, and that this characteristic of these strains may be a consequence of the unique ecological features of the isolation environment.

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Advances and Prospects of Phenolic Acids Production, Biorefinery and Analysis

Biomolecules ◽

10.3390/biom10060874 ◽

2020 ◽

Vol 10 (6) ◽

pp. 874 ◽

Cited By ~ 2

Author(s):

Egle Valanciene ◽

Ilona Jonuskiene ◽

Michail Syrpas ◽

Ernesta Augustiniene ◽

Paulius Matulis ◽

...

Keyword(s):

Phenolic Acids ◽

Large Scale ◽

Microbial Cell ◽

Free Radical Scavengers ◽

Scale Production ◽

Biotechnological Production ◽

Natural Sources ◽

Sustainable Technologies ◽

Microbial Cell Factories ◽

Cell Factories

Biotechnological production of phenolic acids is attracting increased interest due to their superior antioxidant activity, as well as other antimicrobial, dietary, and health benefits. As secondary metabolites, primarily found in plants and fungi, they are effective free radical scavengers due to the phenolic group available in their structure. Therefore, phenolic acids are widely utilised by pharmaceutical, food, cosmetic, and chemical industries. A demand for phenolic acids is mostly satisfied by utilising chemically synthesised compounds, with only a low quantity obtained from natural sources. As an alternative to chemical synthesis, environmentally friendly bio-based technologies are necessary for development in large-scale production. One of the most promising sustainable technologies is the utilisation of microbial cell factories for biosynthesis of phenolic acids. In this paper, we perform a systematic comparison of the best known natural sources of phenolic acids. The advances and prospects in the development of microbial cell factories for biosynthesis of these bioactive compounds are discussed in more detail. A special consideration is given to the modern production methods and analytics of phenolic acids.

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A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

10.1101/2020.02.18.954131 ◽

2020 ◽

Cited By ~ 1

Author(s):

Eline D. Postma ◽

Sofia Dashko ◽

Lars van Breemen ◽

Shannara K. Taylor Parkins ◽

Marcel van den Broek ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Engineering ◽

De Novo ◽

Transcriptional Unit ◽

Dna Assembly ◽

Cellular Processes ◽

Microbial Cell Factories ◽

Cell Factories ◽

Expression Platform

ABSTRACTThe construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

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A Glimpse into the Biosynthesis of Terpenoids

KnE Life Sciences ◽

10.18502/kls.v3i5.981 ◽

2017 ◽

Vol 3 (5) ◽

pp. 81 ◽

Cited By ~ 10

Author(s):

Ingy I. Abdallah ◽

Wim J. Quax

Keyword(s):

Catalytic Mechanism ◽

Production Method ◽

Microbial Cell ◽

Terpene Synthase ◽

Cell Factory ◽

Terpene Synthases ◽

Microbial Cell Factories ◽

Cell Factories ◽

Therapeutic Properties ◽

Insight Into

<p>Terpenoids represent the largest class of natural products with a diverse array of structures and functions. Many terpenoids have reported therapeutic properties such as antimicrobial, anti-inflammatory, immunomodulatory and chemotherapeutic properties making them of great interest in the medical field. Also, they are widely used in the flavors and fragrances industries, in addition to being a source of biofuels. Terpenoids suffer from low natural yields and complicated chemical synthesis, hence the need for a more sustainable production method. Metabolic engineering provide an excellent opportunity to construct microbial cell factories producing the desired terpenoids. The biosynthetic mevalonate and non-mevalonate pathways involved in the production of terpenoid precursors are fully characterized so exploring methods to improve their flux would be the first step in creating a successful cell factory. The complexity and diversity of terpenoid structures depends mainly on the action of the terpene synthases responsible for their synthesis. These enzymes are classified into different classes and gaining insight into their catalytic mechanism will be useful in designing approaches to improve terpenoid production. This review focuses on the biosynthesis and biodiversity of terpenoids, understanding the terpene synthase enzyme family involved in their synthesis and the engineering efforts to create microbial cell factories for terpenoid production.</p>

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Updating genome annotation for the microbial cell factory Aspergillus niger using gene co-expression networks

10.1101/398842 ◽

2018 ◽

Author(s):

p Schäp ◽

MJ Kwon ◽

B Baumann ◽

B Gutschmann ◽

S Jung ◽

...

Keyword(s):

Aspergillus Niger ◽

Fungal Pathogens ◽

Meta Analysis ◽

Microbial Cell ◽

Model Systems ◽

Model Organisms ◽

Cell Factory ◽

Microbial Cell Factory ◽

Cell Factories ◽

Promising Source

AbstractA significant challenge in our understanding of biological systems is the high number of genes with unknown function in many genomes. The fungal genus Aspergillus contains important pathogens of humans, model organisms, and microbial cell factories. Aspergillus niger is used to produce organic acids, proteins, and is a promising source of new bioactive secondary metabolites. Out of the 14,165 open reading frames predicted in the A. niger genome of only 2% have been experimentally verified and over 6,000 are hypothetical. Here we show that gene co-expression network analysis can be used to overcome this limitation. A meta-analysis of 155 transcriptomics experiments generated co-expression networks for 9,579 genes (∼65%) of the A. niger genome. By populating this dataset with over 1,200 gene functional experiments from the genus Aspergillus and performing gene ontology enrichment, we could infer biological processes for 9,263 of A. niger genes, including 2,970 hypothetical genes. Experimental validation of selected co-expression sub-networks uncovered four transcription factors involved in secondary metabolite synthesis, which were used to activate production of multiple natural products. This study constitutes a significant step towards systems-level understanding of A. niger, and the datasets can be used to fuel discoveries of model systems, fungal pathogens, and biotechnology.

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