Improving Reproducibility in Synthetic Biology

Synthetic biology holds great promise to deliver transformative technologies to the world in the coming years. However, several challenges still remain to be addressed before it can deliver on its promises. One of the most important issues to address is the lack of reproducibility within research of the life sciences. This problem is beginning to be recognised by the community and solutions are being developed to tackle the problem. The recent emergence of automated facilities that are open for use by researchers (such as biofoundries and cloud labs) may be one of the ways that synthetic biologists can improve the quality and reproducibility of their work. In this perspective article, we outline these and some of the other technologies that are currently being developed which we believe may help to transform how synthetic biologists approach their research activities

Bioengineering horizon scan 2020.

Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017). Here we report the results of a new horizon scan that is based on inputs from a larger and more international group of 38 participants. The final list of 20 issues includes topics spanning from the political (the regulation of genomic data, increased philanthropic funding and malicious uses of neurochemicals) to the environmental (crops for changing climates and agricultural gene drives). The early identification of such issues is relevant to researchers, policy-makers and the wider public

Neither 1 G nor 2 G fuel ethanol: setting the ground for a sugarcane-based biorefinery using an iSUCCELL yeast platform

First-generation (1 G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2 G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating, and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here we propose an alternative biorefinery, where the sucrose-rich stream from the 1 G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are threefold: 1) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; 2) decreased cooling times after enzymatic hydrolysis prior to fermentation; 3) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1 G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2 G technologies

EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available

Emergence of Phenotypically Distinct Subpopulations Is a Factor in Adaptation of Recombinant <i>Saccharomyces cerevisiae</i> under Glucose-Limited Conditions

Cells cultured in a nutrient-limited environment can undergo adaptation, which confers improved fitness under long-term energy limitation. We have shown previously how a recombinant Saccharomyces cerevisiae strain, producing a heterologous insulin product, under glucose-limited conditions adapts over time at the average population level. Here, we investigated this adaptation at the single-cell level by application of fluorescence-activated cell sorting (FACS) and showed that the following three apparent phenotypes underlie the adaptive response observed at the bulk level: (i) cells that drastically reduced insulin production (23%), (ii) cells with reduced enzymatic capacity in central carbon metabolism (46%), and (iii) cells that exhibited pseudohyphal growth (31%). We speculate that the phenotypic heterogeneity is a result of different mechanisms to increase fitness. Cells with reduced insulin productivity have increased fitness by reducing the burden of the heterologous insulin production, and the populations with reduced enzymatic capacity of the central carbon metabolism and pseudohyphal growth have increased fitness toward the glucose-limited conditions. The results highlight the importance of considering population heterogeneity when studying adaptation and evolution. IMPORTANCE The yeast Saccharomyces cerevisiae is an attractive microbial host for industrial production and is used widely for manufacturing, e.g., pharmaceuticals. Chemostat cultivation mode is an efficient cultivation strategy for industrial production processes as it ensures a constant, well-controlled cultivation environment. Nevertheless, both the production of a heterologous product and the constant cultivation environment in the chemostat impose a selective pressure on the production organism, which may result in adaptation and loss of productivity. The exact mechanisms behind the observed adaptation and loss of performance are often unidentified. We used a recombinant S. cerevisiae strain producing heterologous insulin and investigated the adaptation occurring during chemostat growth at the single-cell level. We showed that three apparent phenotypes underlie the adaptive response observed at the bulk level in the chemostat. These findings highlight the importance of considering population heterogeneity when studying adaptation in industrial bioprocesses

The transcriptome and flux profiling of Crabtree‐negative hydroxy acid producing strains of Saccharomyces cerevisiae reveals changes in the central carbon metabolism

Saccharomyces cerevisiae is a yeast cell factory of choice for the production of many bio‐based chemicals. However, it is also a Crabtree‐positive yeast and so it shuttles a large portion of carbon into ethanol, even under aerobic conditions. To minimise the carbon loss, ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. Deletion of PDC genes has a profound impact on S. cerevisiae physiology, and it is not yet well understood how PDC‐negative yeasts are affected when engineered to produce other products than ethanol. In this study, we introduced pathways for the production of three hydroxy acids (lactic, malic, or 3‐hydroxypropionic acid) into an evolved PDC‐negative strain. We characterised these strains via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. The expression of lactic acid and malic acid biosynthesis pathways improved the maximum specific growth rate (μmax) of the strain by 64 and 20% respectively, presumably due to NAD+ regeneration. On the contrary, the 3HP pathways expression decreased the μmax. All strains showed a very high flux (>90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The transcriptional profile was least affected by the production of lactic acid and more by malic or 3‐hydroxypropionic acids. The study, for the first time, directly compares the flux and transcriptome profiles of several different hydroxy acid producing strains of an evolved PDC‐negative S. cerevisiae and suggests directions for future metabolic engineering