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. To address this, we genetically engineer Komagataeibacter rhaeticus to create a bacterial strain that grows self-pigmenting BC. Melanin biosynthesis in the bacteria from recombinant tyrosinase expression achieves dark black coloration robust to material use. Melanated BC production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-pigmentation with tools from synthetic biology, through the optogenetic patterning of gene expression in cellulose-producing bacteria. With this study, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

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. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization. RESULTS: K. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation. CONCLUSIONS: The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.

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. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

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 (synXI) 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. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae.

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 Saccharomyces cerevisiae, 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. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.

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 Saccharomyces cerevisiae 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. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

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. Here, we review the past and current genome minimisation and re-functionalisation efforts, with an emphasis on the latest advances facilitated by synthetic genomics, and provide a critical appraisal of their potential for industrial applications.

Screening microbially produced Δ9-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. Here, we present a yeast-based biosensor for detecting microbially produced Δ9-tetrahydrocannabinol (THC) to increase throughput and lower the cost of screening. We port five human cannabinoid G protein-coupled receptors (GPCRs) into yeast, showing the cannabinoid type 2 receptor, CB2R, can couple to the yeast pheromone response pathway and report on the concentration of a variety of cannabinoids over a wide dynamic and operational range. We demonstrate that our cannabinoid biosensor can detect THC from microbial cell culture and use this as a tool for measuring relative production yields from a library of Δ9-tetrahydrocannabinol acid synthase (THCAS) mutants.

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. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.

Komagataeibacter 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 Komagataeibacter 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 Komagataeibacter 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. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags, and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fiber system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

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. rhaeticus and demonstrate for the first time their potential for their use as building blocks to grow ELMs in 3D shapes. Using genetically engineered K. rhaeticus, we produce functionalized BC spheroids and use these to make and grow patterned BC-based ELMs that signal within a material and can sense and report on chemical inputs. We also investigate the use of BC spheroids as a method to regenerate damaged BC materials and as a way to fuse together smaller material sections of cellulose and synthetic materials into a larger piece. This work improves our understanding of BC spheroid formation and showcases their great potential for fabricating, patterning and repairing ELMs based on the promising biomaterial of bacterial cellulose.

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. We look forward to future advances, including accessory packs for cloning toolkits that can facilitate editing, orthogonality, advanced regulation, and integration into synthetic chromosome construction.

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. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.

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.

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 Komagataeibacter strains; bacteria that naturally secrete large amounts of the versatile and promising material bacterial cellulose (BC). In this review, we summarize how genetic engineering, metabolic engineering and now synthetic biology have been used in Komagataeibacter strains to alter BC, improve its production and begin to add new functionalities into this easy-to-grow material. As well as describing the milestone advances, we also look forward to what will come next from engineering bacterial cellulose by synthetic biology.

Third-party vessel allografts in kidney and pancreas transplantation: Utilization, de novo DSAs, and outcomes.

Third-party vascular allografts (VAs) are an invaluable resource in kidney and pancreas transplantation when vascular reconstruction is needed and additional vessels from the organ donor are not available. We report the largest single-center experience to date on VA use, at a high-volume U.S. transplant center. Over a 7-year period, VAs were used for vascular reconstruction of 65 kidneys and 5 pancreases, in 69 recipients. The renal vein required reconstruction more often with right kidney transplantation (72.5% vs 27.5%, P < .001), and the renal artery required reconstruction more often with left kidney transplantation (67.6% vs 32.4%, P = .003). Eleven patients (15.9%) developed anti-VA de novo HLA donor-specific antibodies (dnDSAs) at a median time after transplantation of 19.0 months. Higher number of HLA mismatches between the VA donor and the recipient, and development of anti-organ allograft dnDSAs were significant predictors of anti-VA dnDSA development. Those with anti-VA dnDSAs had a higher rate of organ allograft rejection (45.4% vs 13.8%, P = .03) compared to those without, but there was no significant difference in incidence of vascular complications or graft outcomes. VAs can help circumvent challenging surgical situations. Anti-VA dnDSAs do not adversely affect organ allograft outcomes; however, they can contribute to HLA sensitization in the recipients.