Bridging Nature and Engineering: Protein-Derived Materials for Bio-Inspired Applications.

The sophisticated, elegant protein-polymers designed by nature can serve as inspiration to redesign and biomanufacture protein-based materials using synthetic biology. Historically, petro-based polymeric materials have dominated industrial activities, consequently transforming our way of living. While this benefits humans, the fabrication and disposal of these materials causes environmental sustainability challenges. Fortunately, protein-based biopolymers can compete with and potentially surpass the performance of petro-based polymers because they can be biologically produced and degraded in an environmentally friendly fashion. This paper reviews four groups of protein-based polymers, including fibrous proteins (collagen, silk fibroin, fibrillin, and keratin), elastomeric proteins (elastin, resilin, and wheat glutenin), adhesive/matrix proteins (spongin and conchiolin), and cyanophycin. We discuss the connection between protein sequence, structure, function, and biomimetic applications. Protein engineering techniques, such as directed evolution and rational design, can be used to improve the functionality of natural protein-based materials. For example, the inclusion of specific protein domains, particularly those observed in structural proteins, such as silk and collagen, enables the creation of novel biomimetic materials with exceptional mechanical properties and adaptability. This review also discusses recent advancements in the production and application of new protein-based materials through the approach of synthetic biology combined biomimetics, providing insight for future research and development of cutting-edge bio-inspired products. Protein-based polymers that utilize nature's designs as a base, then modified by advancements at the intersection of biology and engineering, may provide mankind with more sustainable products.

Single Crossover to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC 7120 (hereafter Anabaena 7120) is a model cyanobacterium for studying pathways such as photosynthesis and nitrogen fixation along with many other metabolic pathways common to plants. In addition, since Anabaena 7120 forms specialized N2-fixing cells, called heterocysts, to perform uniquely solar-powered, oxic nitrogen fixation under fixed-nitrogen depleted conditions, this cyanobacterium provides the unique opportunity to study cellular differentiation in bacteria. Since more than 155,810 sequenced prokaryotic genomes are currently available (Zhang et al., Microbiome 8(1):134, 2020), target gene inactivation, combined with analyses of the corresponding mutant's phenotype, has become a powerful tool to assess gene function through detecting a loss-of-function in the knockout mutant. In the method described here, a single crossover approach is used to knockout a target gene in Anabaena 7120. The method requires inserting an internal fragment of the target gene into the cyanobacterial integration vector pZR606 to create a knockout plasmid, and then is introduced to Anabaena 7120 via conjugative transformation. A single crossover, occurring via homologous recombination, disrupts the target gene, creating 3'- and 5'-deleted fragments (Fig. 1). The mutant containing the inactivated gene can then be studied to determine any loss of function, thereby defining the gene's function. This gene inactivation approach is based on an integrative vector pZR606 (Chen et al., Appl Microbiol Biotechnol 99:1779-1793, 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Double Crossover Approach to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC7120 (hereafter Anabaena 7120) is a nitrogen-fixing, filamentous cyanobacterium. Given its diverse metabolism, it serves as an excellent model organism, particularly for studying cell differentiation, nitrogen fixation, photosynthesis, production of high-value chemicals, and synthetic biology. Gene knockout is a common approach to assess the function of gene products through assessing phenotypic loss of function. In the method described here, a double crossover approach is used to inactivate a target gene or target genes in Anabaena 7120. This method involves replicating the gene(s) from the wild-type genomic DNA and inserting them into an integrative plasmid vector. An internal portion of the genes may be removed and replaced with a GFP-Spectinomycin (gfp-sp) cassette. The plasmid is then introduced into Anabaena 7120 where a double crossover event occurs between the wild-type chromosome and the cargo plasmid, effectively replacing the wild-type gene with the disrupted gene from the plasmid. The gfp-sp cassette combined with the sacB gene serve as positive selection to identify double crossover mutants (Cai and Wolk (1990), 172(6):3138-3145, J. Bacteriol). Finally, the functional genes are cloned into another replicating plasmid vector to produce a cargo plasmid, which is conjugatively introduced into the mutant for a complementation test. By comparing the phenotypes among the wild-type, mutant, and complement, one should see a loss of function in the mutant which is recovered in the complement, thereby defining the function of the target gene. The double crossover approach described here for Anabaena PCC 7120 may be broadly applicable to the study of gene function in cyanobacteria and other prokaryotic organisms.

Identification of two genes required for heptadecane production in a N2-fixing cyanobacterium Anabaena sp. strain PCC 7120.

Cyanobacteria photosynthetically produce long-chain hydrocarbons, which are considered as infrastructure-compatible biofuels. However, native cyanobacteria do not produce these hydrocarbons at sufficient rates or yields to warrant commercial deployment. This research sought to identify specific genes required for photosynthetic production of alkanes to enable future metabolic engineering for commercially viable production of alkanes. The two putative genes (alr5283 and alr5284) required for long-chain hydrocarbon production in Anabaena sp. PCC 7120 were knocked out through a double crossover approach. The knockout mutant abolished the production of heptadecane (C17H36). The mutant is able to be complemented by a plasmid bearing the two genes along with their native promoters only. The complemented mutant restored photosynthetic production of heptadecane. This combined genetic and metabolite (alkanes) profiling approach may be broadly applicable to characterization of knockout mutants, using N2-fixing cyanobacteria as a cellular factory driven by solar energy to produce a wide range of commodity chemicals and drop-in-fuels from atmospheric gases (CO2 and N2 gas) and mineralized water.

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review.

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO2 , H2 O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357-1371, 2016.

Evaluation of UV-C mutagenized Scheffersomyces stipitis strains for ethanol production.

We evaluated fermentation capabilities of five strains of Scheffersomyces stipitis (WT-2-1, WT-1-11, 14-2-6, 22-1-1, and 22-1-12) that had been produced by UV-C mutagenesis and selection for improved xylose fermentation to ethanol using an integrated automated robotic work cell. They were incubated under both facultative and anaerobic conditions to evaluate ethanol production on glucose, xylose, cellobiose, and a combination of all three sugars. The medium contained 50 g/L total sugar and 5 g/L yeast extract. The strains performed significantly better under facultative compared with anaerobic conditions. As expected, glucose was the most readily fermented sugar with ~100% fermentation efficiency (FE) under facultative conditions but only 5% to 16% FE anaerobically. Xylose utilization was 20% to 40% FE under facultative conditions but 9% to 25% FE anaerobically. Cellobiose was the least fermented sugar, at 18% to 27% FE facultatively and 8% to 11% anaerobically. Similar trends occurred in the sugar mixture. Under facultative conditions, strain 22-1-12 produced 19.6 g/L ethanol on glucose, but strain 14-2-6 performed best on xylose (4.5 g/L ethanol) and the sugar combination (8.0 g/L ethanol). Ethanol titers from glucose under anaerobic conditions were again highest with strain 22-1-12, but none of the strains produced ethanol from xylose. Future trials will evaluate nutrient addition to boost microaerophilic xylose fermentation.