Sustainable Production of Novel Biomaterials in Escherichia coli

Journal of biological engineering, 2013

Polyhydroxyalkanoates (PHAs) are a group of biodegradable plastics that are produced by a wide variety of microorganisms, mainly as a storage intermediate for energy and carbon. Polyhydroxybutyrate (PHB) is a short-chain-length PHA with interesting chemical and physical properties. Large scale production of PHB is not wide-spread mainly due to the downstream processing of bacterial cultures to extract the PHB. Secretion of PHB from Escherichia coli could reduce downstream processing costs. PHB are non-proteinaceous polymers, hence cannot be directly targeted for secretion. Phasin, PhaP1, is a low molecular weight protein that binds to PHB, reducing PHB granule size. In this study PHB is indirectly secreted with PhaP1 from E. coli via type I secretion using HlyA signal peptides. This study demonstrated the successful secretion of phasin and phasin bound PHB outside of the cell and into the culture medium. The secretion of PHB was initiated between 24 and 48 h after induction. After 4...

PLOS ONE, 2015

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB). 4 / 11 0.5 g of either purified P(3HB) or commercially sourced P(3HB) (Sigma-Aldrich, #363502-10G) were treated with 20μl PhaZ1 (0.36 μg/μl; BBa_K1149010) in 800 μl potassium phosphate buffer Forward-Design of Engineered phaCAB Operons PLOS ONE |

Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2 % (w/v) starch in M9 media containing 0.15 % (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4 % of cell dry mass.

New Biotechnology, 2018

Major ampullate spider silk represents a promising protein-based biomaterial with diverse commercial potential ranging from textiles to medical devices due to its excellent physical and thermal properties. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli), alfalfa and goats. This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative downstream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants of $761 kg-1 and $23 kg-1 with greenhouse gas emissions of 572 kg CO2-eq. kg-1 and 55 kg CO2-eq. kg-1 , respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plants include improved protein yield, process optimization, and an N th plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and the impact of alternative technologies on the system.

Synthetic and Systems Biotechnology

Society is on the cusp of harnessing recent advances in synthetic biology to discover new bio-based products and routes to their affordable and sustainable manufacture. This is no more evident than in the discovery and manufacture of Synthetic Biological Materials, where synthetic biology has the capacity to usher in a new Materials from Biology era that will revolutionise the discovery and manufacture of innovative synthetic biological materials. These will encompass novel, smart, functionalised and hybrid materials for diverse applications whose discovery and routes to bio-production will be stimulated by the fusion of new technologies positioned across physical, digital and biological spheres. This article, which developed from an international workshop held in Manchester, United Kingdom, in 2017 [1], sets out to identify opportunities in the new materials from biology era. It considers requirements, early understanding and foresight of the challenges faced in delivering a Discovery to Manufacturing Pipeline for synthetic biological materials using synthetic biology approaches. This challenge spans the complete production cycle from intelligent and predictive design, fabrication, evaluation and production of synthetic biological materials to new ways of bringing these products to market. Pathway opportunities are identified that will help foster expertise sharing and infrastructure development to accelerate the delivery of a new generation of synthetic biological materials and the leveraging of existing investments in synthetic biology and advanced materials research to achieve this goal.

Applied and Environmental Microbiology, 2012

Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production. Volume 78 Number 9 aem.asm.org 3179 on April 9, 2012 by guest http://aem.asm.org/ Downloaded from FIG 3 Molecular weight distributions of P(3HB) synthesized by recombinant E. coli DH5␣ harboring pGET109-pha series plasmids. The cultivation time was 12 h (dashed lines) or 72 h (solid lines). Determination of molecular weight was carried out in triplicate, and typical distributions for each polymer are shown.

Environmental Microbiology, 2001

Journal of Biotechnology, 1998

The poly(b-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria -Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

Biopolymers, 1994

The morphology and primary crystal structure of SLPF, a protein polymer produced by genetically engineered Escherichia coli bacteria, were characterized. SLPF is a segmented copolymer consisting of amino acid sequence blocks modeled on the crystalline segments of silk fibroin and the cell attachment domain of human fibronectin. Wide angle x-ray scattering (WAXS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and molecular simulations were used to analyze the primary crystal structure of SLPF. TEM experiments conducted on SLPF droplets cast from formic acid on amorphous carbon film demonstrated that these protein films have a microstructure formed of woven sheaves. The sheaves are composed of well-defined whisker crystallites. The width of the whiskers, 11.8 +/- 2.2 nm, may be correlated to the length of the silk-like segment in SLPF as predicted by molecular simulations. WAXS data, TEM images, SAED, patterns, molecular simulations, and theoretical diffraction patterns all were consistent with the crankshaft model proposed for Silk I by Lotz and Keith.

Polymers

Bacteria are considered as the major cell factories, which can effectively convert nitrogen and carbon sources to a wide variety of extracellular and intracellular biopolymers like polyamides, polysaccharides, polyphosphates, polyesters, proteinaceous compounds, and extracellular DNA. Bacterial biopolymers find applications in pathogenicity, and their diverse materialistic and chemical properties make them suitable to be used in medicinal industries. When these biopolymer compounds are obtained from pathogenic bacteria, they serve as important virulence factors, but when they are produced by non-pathogenic bacteria, they act as food components or biomaterials. There have been interdisciplinary studies going on to focus on the molecular mechanism of synthesis of bacterial biopolymers and identification of new targets for antimicrobial drugs, utilizing synthetic biology for designing and production of innovative biomaterials. This review sheds light on the mechanism of synthesis of ba...