Thomas C. Williams

Synthetic Evolution of Metabolic Productivity Using Biosensors

Synthetic biology has progressed to the point where genes that encode whole metabolic pathways and even genomes can be manufactured and brought to life. This impressive ability to synthesise and assemble DNA is not yet matched by an ability to predictively engineer biology. These difficulties exist because biological systems are often overwhelmingly complex, having evolved to facilitate growth and survival rather than specific engineering objectives such as the optimisation of biochemical production. A promising and revolutionary solution to this problem is to harness the process of evolution to create microbial strains with desired properties. The tools of systems biology can then be applied to understand the principles of biological design, bringing synthetic biology closer to becoming a predictive engineering discipline.

Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities

AbstractBackgroundPredictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources (glucose, sucrose, galactose and ethanol) and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of “constitutive” and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter.ResultsThe “constitutive” promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels (PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources (sucrose, galactose and ethanol); in contrast to the “constitutive” promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several “constitutive” promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift.ConclusionsThe data provides a foundation for predictable and optimised control of gene expression levels on different carbon sources and throughout batch fermentation, including during and after the diauxic shift. This information can be applied for designing expression approaches to improve yields, rates and titres in yeast cell factories.

Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae

ABSTRACT Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p1-989), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p1-989 increased tolerance toward two other monoterpenes (β-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p1-989 is the first example of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C10 alkenes in yeast.

Yeast's balancing act between ethanol and glycerol production in low-alcohol wines

Alcohol is fundamental to the character of wine, yet too much can put a wine off‐balance. A wine is regarded to be well balanced if its alcoholic strength, acidity, sweetness, fruitiness and tannin structure complement each other so that no single component dominates on the palate. Balancing a wines positive fruit flavours with the optimal absolute and relative concentration of alcohol can be surprisingly difficult. Over the past three decades, consumers have increasingly demanded wine with richer and riper fruit flavour profiles. In response, grape and wine producers have extended harvest times to increase grape maturity and enhance the degree of fruit flavours and colour intensity. However, a higher degree of grape maturity results in increased grape sugar concentration, which in turn results in wines with elevated alcohol concentration. On average, the alcohol strength of red wines from many warm wine‐producing regions globally rose by about 2% (v/v) during this period. Notwithstanding that many of these ‘full‐bodied, fruit‐forward’ wines are well balanced and sought after, there is also a significant consumer market segment that seeks lighter styles with less ethanol‐derived ‘hotness’ on the palate. Consumer‐focussed wine producers are developing and implementing several strategies in the vineyard and winery to reduce the alcohol concentration in wines produced from well‐ripened grapes. In this context, Saccharomyces cerevisiae wine yeasts have proven to be a pivotal strategy to reduce ethanol formation during the fermentation of grape musts with high sugar content (> 240 g l−1). One of the approaches has been to develop ‘low‐alcohol’ yeast strains which work by redirecting their carbon metabolism away from ethanol production to other metabolites, such as glycerol. This article reviews the current challenges of producing glycerol at the expense of ethanol. It also casts new light on yeast strain development programmes which, bolstered by synthetic genomics, could potentially overcome these challenges.

Recent advances in synthetic biology for engineering isoprenoid production in yeast

Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, redirecting carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.

Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

BackgroundEngineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting.ResultsTo address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene.ConclusionsThe low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications.

The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production

The growth characteristics and underlying metabolism of microbial production hosts are critical to the productivity of metabolically engineered pathways. Production in parallel with growth often leads to biomass/bio-product competition for carbon. The growth arrest phenotype associated with the Saccharomyces cerevisiae pheromone-response is potentially an attractive production phase because it offers the possibility of decoupling production from population growth. However, little is known about the metabolic phenotype associated with the pheromone-response, which has not been tested for suitability as a production phase. Analysis of extracellular metabolite fluxes, available transcriptomic data, and heterologous compound production (para-hydroxybenzoic acid) demonstrate that a highly active and distinct metabolism underlies the pheromone-response. These results indicate that the pheromone-response is a suitable production phase, and that it may be useful for informing synthetic biology design principles for engineering productive stationary phase phenotypes.

Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.

The Multiplanetary Future of Plant Synthetic Biology

The interest in human space journeys to distant planets and moons has been re-ignited in recent times and there are ongoing plans for sending the first manned missions to Mars in the near future. In addition to generating oxygen, fixing carbon, and recycling waste and water, plants could play a critical role in producing food and biomass feedstock for the microbial manufacture of materials, chemicals, and medicines in long-term interplanetary outposts. However, because life on Earth evolved under the conditions of the terrestrial biosphere, plants will not perform optimally in different planetary habitats. The construction or transportation of plant growth facilities and the availability of resources, such as sunlight and liquid water, may also be limiting factors, and would thus impose additional challenges to efficient farming in an extraterrestrial destination. Using the framework of the forthcoming human missions to Mars, here we discuss a series of bioengineering endeavors that will enable us to take full advantage of plants in the context of a Martian greenhouse. We also propose a roadmap for research on adapting life to Mars and outline our opinion that synthetic biology efforts towards this goal will contribute to solving some of the main agricultural and industrial challenges here on Earth.

Photoresponsive endosomal escape enhances gene delivery using liposome–polycation–DNA (LPD) nanovectors

Lipid-based nanocarriers with stimuli responsiveness have been utilized as controlled release systems for gene/drug delivery applications. In our work, by taking advantage of the high complexation capability of polycations and the light triggered properties, we designed a novel photoresponsive liposome-polycation-DNA (LPD) platform. This LPD carrier incorporates verteporfin (VP) in lipid bilayers and the complex of polyethylenimine (PEI)/plasmid DNA (pDNA) encoding EGFP (polyplex) in the central cavities of the liposomes. The liposomes were formulated with cationic lipids, PEGylated neutral lipids and cholesterol molecules, which improve their stability and cellular uptake in the serum-containing media. We evaluated the nanocomplex stability by monitoring size changes over six days, and the cellular uptake of the nanocomplex by imaging the intracellular route. We also demonstrated that light triggered the cytoplasmic release of pDNA upon irradiation with a 690 nm LED light source. Furthermore, this light triggered mechanism has been studied at the subcellular level. The activated release is driven by the generation of reactive oxygen species (ROS) from VP after light illumination. These ROS oxidize and destabilize the liposomal and endolysosomal membranes, leading to the release of pDNA into the cytosol and subsequent gene transfer activities. Light-triggered endolysosomal escape of pDNA at different time points was confirmed by a quantitative analysis of colocalization between pDNA and endolysosomes. The increased expression of the reporter EGFP in human colorectal cancer cells was also quantified after light illumination at various time points. The efficiency of this photo-induced gene transfection was demonstrated to be more than double compared to non-irradiated controls. Additionally, we observed a reduced cytotoxicity of the LPDs compared with the polyplexes alone. This study has thus shown that light-triggered and biocompatible LPDs enable an improved control of efficient gene delivery, which will be beneficial for future gene therapies.