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Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In our previous study, a pathway for P3HP production was constructed in recombinant Esecherichia coli. Seven exogenous genes in P3HP synthesis pathway were carried by two plasmid vectors. However, the P3HP production was severely suppressed by strain instability du...
Citations
... Poly(3HP) is a well-characterized polyester, a biodegradable material with biocompatibility and high tensile strength (Catiker, 2015). Moreover, this material can synthesize prosthetics and heart valves, suggesting that Poly(3HP) has signi cant application prospects in medicine (Gao et al., 2014). Based on the excellent properties of 3-HP, the USA Department of Energy report in The glycerol pathway is currently the most studied pathway for the biological synthesis of 3-HP. ...
... In addition, the streptomycin resistance gene Str and the medium-copy-number replication origin p15A were selected to form the plasmid backbone. P TetR is an inducible promoter that, when tetracycline is present, can initiate the expression of downstream genes (Gossen and Bujard, 1992;Gossen et al.,1995). Plasmid construction was tested by colony PCR and enzyme digestion validation (see supplementary Fig.S1). ...
Background: 3-Hydroxypropionic acid (3-HP) is a platform compound that can produce many chemical commodities. This study focuses on establishing and optimizing the production of 3-HP in E. coli. We constructed a series of engineered E.coli strains which can produce 3-HP via the malonyl-CoA pathway. To increase the techniques the metabolic flux of precursor acetyl-CoA, CRISPR/Cas9-based DNA editing techniques were used to knock out the genes encoding pyruvate oxidase (poxB), lactate dehydrogenase (ldhA) and phosphate transacetylase (pta) reducing the by-products consumption. Simultaneously, to elevate the production of 3-HP and reduce the burden of the recombinant plasmid in Escherichia coli, the critical precursor of the malonyl-CoA pathway, acetyl-CoA carboxylase gene (accDABC), was overexpressed on the genome.
Results: We overexpressed the codon-optimized malonyl-CoA reductase gene (mcr) and increased 3-HP production also via adaptive laboratory evolution using the PpHpdR/PhpdH system to construct metabolite biosensors based on transcription factors. Combining the above metabolic engineering efforts with media and fermentation conditions optimization in a fermentor agitation resulted in the 3-HP titer of the engineered strain increasing about 63.5 times from the initial 0.34 g/L to 21.6 g/L.
Conclusions: This study encourages further bioprocess development to produce 3-HP from the malonyl-CoA pathway.
... In this study, the authors used the previously described enzymes DhaB and GdrAB for the glycerol dehydratase and reactivation step. For the second step, they used a propionaldehyde dehydrogenase from Salmonella typhimurium (PduP) [108]. The last step was performed by endogenous CoA transferases, and no gene was overexpressed. ...
Acrylic acid (AA) is a chemical with high market value used in industry to produce diapers, paints, adhesives and coatings, among others. AA available worldwide is chemically produced mostly from petroleum derivatives. Due to its economic relevance, there is presently a need for innovative and sustainable ways to synthesize AA. In the past decade, several semi-biological methods have been developed and consist in the bio-based synthesis of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. However, more recently, engineered Escherichia coli was demonstrated to be able to convert glucose or glycerol to AA. Several pathways have been developed that use as precursors glycerol, malonyl-CoA or β-alanine. Some of these pathways produce 3-HP as an intermediate. Nevertheless, the heterologous production of AA is still in its early stages compared, for example, to 3-HP production. So far, only up to 237 mg/L of AA have been produced from glucose using β-alanine as a precursor in fed-batch fermentation. In this review, the advances in the production of AA by engineered microbes, as well as the hurdles hindering high-level production, are discussed. In addition, synthetic biology and metabolic engineering approaches to improving the production of AA in industrial settings are presented.
... Various PASs have been successfully constructed. However encountering many problems such as invalidation during prolonged culture growth (Cooper and Heinemann 2000) , survival of plasmid-free cells by assimilating nutrition released from plasmid-containing cells (Gao et al. 2014) and so on. Another strategy of PAS is to knockout essential genes from the chromosomes and restore these genetic de ciencies on plasmids. ...
Microbial synthesis of commodity chemicals often be conducted in recombinant plasmid-based expression systems, in which plasmids play pivotal roles on productivity. The recombinant plasmids always encounter instability, leading to losses in product recovery of entire process. To maintain the stability of plasmids, several mechanisms have been evolved. Plasmid addition system, selectively killing plasmid-free cells, is regard as a useful strategy to improve the proportion of plasmid-containing cells. In this study, a novel plasmid addition system using an essential gene grpE that encodes a molecular cochaperone as selection marker, avoiding use of antibiotics, was constructed in Escherichia coli . The solo copy of grpE gene on the chromosome was knocked out and relocated on multicopy plasmids. The generated strains can maintain high ratio of plasmid-harboring cells without antibiotics supplementation in mineral salts media and exhibit improved cell growth and increased tolerance to phloroglucinol. Using this system in phloroglucinol synthesis, it could significantly increase the phloroglucinol titer from 0.75 g/L to 1.26 g/L, which was further increased to 1.78 g/L when biotin-[acetyl-CoA-carboxylase] ligase BirA was overexpressed. It can be expected that this system will be a powerful tool for microbial manufacture of important chemicals in E . coli .
... Consequently, it would be a requirement to generate genetically stable strains where expression cassettes are inserted into the genome. In addition, plasmid removal may increase performance through a decreased metabolic liability related with plasmid preservation and replication [54]. To build a strain-free of plasmids, the E. Coli aldH has been incorporated into the K. pneumonia. ...
In this paper, we have described the production of 3-Hydroxypropanoic acid using glycerol as a key intermediate in the presence of engineered microbes. 3-hydroxypropanoic acid (3-HP) is an important interface chemical that is in great need around the world. 3-HP can be generated from a variety of renewable energy sources. Acrylic acid and its several derivatives are made from it as a starting material 3-hydroxypropanoic acid (3-HP) is a highly sought-after platform chemical, used in bioplastics in their polymerized state. Many microbes naturally have biosynthetic pathways for producing 3-HP, and some of these pathways have been inserted into commonly used cell factories like Saccharomyces cerevisiae and Escherichia coli. Recent advances in synthetic biology and metabolic engineering have resulted in more effective methods for bio-production of 3-HP. Among them are novel methods for implementing heterologous pathways, rational enzyme engineering, precise gene expression control, carbon flux redirection based on in silico predictions from genome-scale metabolic models, and fermentation state optimization. Despite extensive research into 3-HP production in existing industrially applicable cell factories, modern production methods have not yet achieved the standards required for commercialization. In this paper, we compare yields and titers obtained in various microbial cell factories, as well as possible methods for achieving industrially important cell factories, to assess the current state of 3-HP bio-production.
... Consequently, it would be a requirement to generate genetically stable strains where expression cassettes are inserted into the genome. In addition, plasmid removal may increase performance through a decreased metabolic liability related with plasmid preservation and replication [54]. To build a strain-free of plasmids, the E. Coli aldH has been incorporated into the K. pneumonia. ...
In this paper, we have described the production of 3-Hydroxypropanoic acid using glycerol as a key intermediate in the presence of engineered microbes. 3-hydroxypropanoic acid (3-HP) is an important interface chemical that is in great need around the world. 3-HP can be generated from a variety of renewable energy sources. Acrylic acid and its several derivatives are made from it as a starting material 3-hydroxypropanoic acid (3-HP) is a highly sought-after platform chemical, used in bioplastics in their polymerized state. Many microbes naturally have biosynthetic pathways for producing 3-HP, and some of these pathways have been inserted into commonly used cell factories like Saccharomyces cerevisiae and Escherichia coli. Recent advances in synthetic biology and metabolic engineering have resulted in more effective methods for bio-production of 3-HP. Among them are novel methods for implementing heterologous pathways, rational enzyme engineering, precise gene expression control, carbon flux redirection based on in silico predictions from genome- scale metabolic models, and fermentation state optimization. Despite extensive research into 3-HP production in existing industrially applicable cell factories, modern production methods have not yet achieved the standards required for commercialization. In this paper, we compare yields and titers obtained in various microbial cell factories, as well as possible methods for achieving industrially important cell factories, to assess the current state of 3-HP bio-production.
... In a pH-stat fed-batch culture, the resultant E. coli strain produced 141.6 g l −1 of poly(3HB) with the productivity of 4.63 g l −1 h −1 , which was comparable to the high records obtained by native producers [87]. Other SCL-PHAs such as poly(3HP) and poly(4HB) have also been produced in recombinant E. coli by constructing their synthetic pathways [88,89,[230][231][232]. For poly(3HP), several synthetic pathways have been developed (Figure 11.5, gray highlight), such as the C3 substrate (glycerol or 1,3-propanediol)-based pathways and the malonyl-CoA pathway [231]. ...
Metabolic engineering has become an essential technology for developing microbial cell factories for producing chemicals and materials from sustainable and renewable feedstocks. Equipped with the ever‐advancing tools and strategies of modern systems biology and synthetic biology, traditional metabolic engineering has been upgraded to a next and systems level, which enables more rapid and sophisticated engineering of microbes for producing desired bioproducts. Escherichia coli has been playing a key role as a workhorse in the production of various chemicals and materials and also as a model bacterium in the development of advanced engineering tools and strategies. In this chapter, we review the achievements in metabolic engineering of E. coli for the biobased production of chemicals and materials together with accompanying tools and strategies.
... Poly(hydroxypropionate) (P3HP) is a short chain-length (scl) PHA ( Fig. 5) with interesting properties, such as high rigidity, ductility and tensile strength [86]. In addition, P3HP has a great potential to be used as a flexible and stable biomaterial, with an approximate melting temperature (Tm) of 77°C and an approximate glass transition temperature (Tg) of −22°C [87]. ...
In recent years, growing attention has been devoted to naturally occurring biological macromolecules and their ensuing application in agriculture, cosmetics, food and pharmaceutical industries. They inherently have antigenicity, low immunogenicity, excellent biocompatibility and cytocompatibility, which are ideal properties for the design of biomedical devices, especially for the controlled delivery of active ingredients in the most diverse contexts. Furthermore, these properties can be modulated by chemical modification via the incorporation of other (macro)molecules in a random or controlled way, aiming at improving their functionality for each specific application. Among the wide variety of natural polymers, microbial polyhydroxyalkanoates (PHAs) and exopolysaccharides (EPS) are often considered for the development of original biomaterials due to their unique physicochemical and biological features. Here, we aim to fullfil a gap on the present associated literature, bringing an up-to-date overview of ongoing research strategies that make use of PHAs (poly (3-hydroxybutyrate), poly (3-hydroxybutyrate-co-3-hydroxyvalerate), poly (3-hydroxyoctanoate), poly(3-hydroxypropionate), poly (3-hydroxyhexanoate-co-3-hydroxyoctanoate), and poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)) and EPS (bacterial cellulose, alginates, curdlan, pullulan, xanthan gum, dextran, hyaluronan, and schizophyllan) as sources of interesting and versatile biomaterials. For the first time, a monograph addressing the properties, pros and cons, status, challenges, and recent progresses regarding the application of these two important classes of biopolymers in biomedicine is presented.
... To mitigate metabolic burden, the heterologous genes were stably integrated via homologous recombination into the E. coli chromosome. Previous studies have demonstrated that genome integration of foreign genes is superior to plasmid-based expression, in which the heterologous protein is expressed more stably and with less impact on growth [34][35][36]. In concordance with previous studies showing the benefit of gene integration, the growth of iRJ1 with integrated RNaseJ1 gene transformed with different reporter plasmids was more consistent compared with plasmid co-transformants, with only minor retardation in the induced condition (Fig. 4b). ...
Background
Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA.
Results
We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell.
Conclusions
The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.
... 3-hydroxypropoic acid, acrylate, and 1,3-propanediol (1,3-PDO) were previously utilized as dependent precursors for the biosynthesis of poly(3HP) and 3-HP-containing copolymer (Gao et al., 2014). However, these expensive precursors and Vitamin B 12 increased poly(3HP) production cost making it economically not feasible. ...
... The metabolic pathway synthesis of poly(3HP) yields nearly a twofold increase while at the same time obtaining high value-added by-products. To stabilize poly(3HP) production, Gao et al. (2014) constructed a genetically stable recombinant strain of E. coli. Based on amino acid synthesis and metabolism, chromosomal gene integration and plasmid addiction system technology were used to obtain high-yield poly(3HP) engineering strain E. coli Q1738 (Gao et al., 2014). ...
... To stabilize poly(3HP) production, Gao et al. (2014) constructed a genetically stable recombinant strain of E. coli. Based on amino acid synthesis and metabolism, chromosomal gene integration and plasmid addiction system technology were used to obtain high-yield poly(3HP) engineering strain E. coli Q1738 (Gao et al., 2014). ...
Many single-use non-degradable plastics are a threat to life today, and several polyhydroxyalkanoates (PHAs) biopolymers have been developed in the bioplastic industry to place petrochemical-based plastics. One of such is the novel biomaterial poly(3-hydroxypropionate) [poly(3HP)] because of its biocompatibility, biodegradability, and high yield synthesis using engineered strains. To date, many bio-polymer-based functional composites have been developed to increase the value of raw microbial-biopolymers obtained from cheap sources. This review article broadly covers poly(3HP), a comprehensive summary of critical biosynthetic production pathways comparing the yields and titers achieved in different Microbial cell Factories. This article also provides extensive knowledge and highlights recent progress on biosensors’ use to optimize poly(3HP) production, some bacteria host adopted for production, chemical and physical properties, life cycle assessment for poly(3HP) production using corn oil as carbon source, and some essential medical applications of poly(3HP).
... Characterization of synthetic promoters has been performed using plasmid-based expression systems or genomically integrated probes (Jensen and Hammer, 1998;Hammer et al., 2006;Zobel et al., 2015). However, varying plasmid copy numbers and high fitness costs for the host makes plasmid-based expression systems less suitable for promoter characterization in particular, and for metabolic engineering in general (Gao et al., 2014;Jahn et al., 2014;Lindmeyer et al., 2015;San Millan and MacLean, 2017). Genomic integration of the probe is preferred for characterization procedures (Zobel et al., 2015). ...
... In addition, an often-overlooked disadvantage of using multicopy plasmids for synthetic promoter screening is that they favor the selection of relatively weak promoters, as the combined effect of a strong constitutive promoter at high copy number may pose a too high burden. Genomic integration abolishes these copy number effects, as well as clonal variations, which have also been observed for different Pseudomonas strains (Friehs, 2004;Gao et al., 2014;Zobel et al., 2015). Nevertheless, the integration site in the genome must be chosen wisely and must be the same for all promoters. ...
Understanding the composability of genetic elements is central to synthetic biology. Even for seemingly well-known elements such as a sigma 70 promoter the genetic context-dependent variability of promoter activity remains poorly understood. The lack of understanding of sequence to function results in highly limited de novo design of novel genetic element combinations. To address this issue, we characterized in detail concatenated “stacked” synthetic promoters including varying spacer sequence lengths and compared the transcription strength to the output of the individual promoters. The proxy for promoter activity, the msfGFP synthesis from stacked promoters was consistently lower than expected from the sum of the activities of the single promoters. While the spacer sequence itself had no activity, it drastically affected promoter activities when placed up- or downstream of a promoter. Single promoter-spacer combinations revealed a bivalent effect on msfGFP synthesis. By systematic analysis of promoter and spacer combinations, a semi-empirical correlation was developed to determine the combined activity of stacked promoters.
