Modulating Microbial Materials - Engineering Bacterial Cellulose with Synthetic Biology.

The fusion of synthetic biology and materials science offers exciting opportunities to produce sustainable materials that can perform programmed biological functions such as sensing and responding or enhance material properties through biological means. Bacterial cellulose (BC) is a unique material for this challenge due to its high-performance material properties and ease of production from culturable microbes. Research in the past decade has focused on expanding the benefits and applications of BC through many approaches.

Microbial messengers: nucleic acid delivery by bacteria.

The demand for diverse nucleic acid delivery vectors, driven by recent biotechnological breakthroughs, offers opportunities for continuous improvements in efficiency, safety, and delivery capacity. With their enhanced safety and substantial cargo capacity, bacterial vectors offer significant potential across a variety of applications. In this review, we explore methods to engineer bacteria for nucleic acid delivery, including strategies such as engineering attenuated strains, lysis circuits, and conjugation machinery.

Self-pigmenting textiles grown from cellulose-producing bacteria with engineered tyrosinase expression.

Environmental concerns are driving interest in postpetroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose (BC) is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like BC to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods.

Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation.

Background: Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation.

Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics.

We describe construction of the synthetic yeast chromosome XI () and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence.

Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes.

A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of .

Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast , an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals.

Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology.

Naturally evolved organisms typically have large genomes that enable their survival and growth under various conditions. However, the complexity of genomes often precludes our complete understanding of them, and limits the success of biotechnological designs. In contrast, minimal genomes have reduced complexity and therefore improved engineerability, increased biosynthetic capacity through the removal of unnecessary genetic elements, and less recalcitrance to complete characterisation.

Screening microbially produced Δ-tetrahydrocannabinol using a yeast biosensor workflow.

Microbial production of cannabinoids promises to provide a consistent, cheaper, and more sustainable supply of these important therapeutic molecules. However, scaling production to compete with traditional plant-based sources is challenging. Our ability to make strain variants greatly exceeds our capacity to screen and identify high producers, creating a bottleneck in metabolic engineering efforts.

Inducible expression of large gRNA arrays for multiplexed CRISPRai applications.

CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter.

Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria.

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the tool kit (KTK), a standardized modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids.

Bacterial cellulose spheroids as building blocks for 3D and patterned living materials and for regeneration.

Engineered living materials (ELMs) based on bacterial cellulose (BC) offer a promising avenue for cheap-to-produce materials that can be programmed with genetically encoded functionalities. Here we explore how ELMs can be fabricated in a modular fashion from millimetre-scale biofilm spheroids grown from shaking cultures of Komagataeibacter rhaeticus. Here we define a reproducible protocol to produce BC spheroids with the high yield bacterial cellulose producer K.

Ten future challenges for synthetic biology.

After 2 decades of growth and success, synthetic biology has now become a mature field that is driving significant innovation in the bioeconomy and pushing the boundaries of the biomedical sciences and biotechnology. So what comes next? In this article, 10 technological advances are discussed that are expected and hoped to come from the next generation of research and investment in synthetic biology; from ambitious projects to make synthetic life, cell simulators and custom genomes, through to new methods of engineering biology that use automation, deep learning and control of evolution. The non-exhaustive list is meant to inspire those joining the field and looks forward to how synthetic biology may evolve over the coming decades.

Genetic Toolkits to Design and Build Mammalian Synthetic Systems.

Construction of DNA-encoded programs is central to synthetic biology and the chosen method often determines the time required to design and build constructs for testing. Here, we describe and summarise key features of the available toolkits for DNA construction for mammalian cells. We compare the different cloning strategies based on their complexity and the time needed to generate constructs of different sizes, and we reflect on why Golden Gate toolkits now dominate due to their modular design.

Living materials with programmable functionalities grown from engineered microbial co-cultures.

Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria.

Engineering Bacterial Cellulose by Synthetic Biology.

Synthetic biology is an advanced form of genetic manipulation that applies the principles of modularity and engineering design to reprogram cells by changing their DNA. Over the last decade, synthetic biology has begun to be applied to bacteria that naturally produce biomaterials, in order to boost material production, change material properties and to add new functionalities to the resulting material. Recent work has used synthetic biology to engineer several strains; bacteria that naturally secrete large amounts of the versatile and promising material bacterial cellulose (BC).

The second decade of synthetic biology: 2010-2020.

Synthetic biology is among the most hyped research topics this century, and in 2010 it entered its teenage years. But rather than these being a problematic time, we’ve seen synthetic biology blossom and deliver many new technologies and landmark achievements.

Entirely Off-Grid and Solar-Powered DNA Sequencing of Microbial Communities during an Ice Cap Traverse Expedition.

Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth's surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions.

Off-Colony Screening of Biosynthetic Libraries by Rapid Laser-Enabled Mass Spectrometry.

By leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering, the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction.