Avoiding overflow metabolite formation in Komagataella phaffii fermentations to enhance recombinant protein production.

BACKGROUND: Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. RESULTS: High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. CONCLUSIONS: These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Benzalkonium chloride antagonises aminoglycoside antibiotics and promotes evolution of resistance.

BACKGROUND: Biocide disinfectants are essential tools in infection control, but their use can inadvertently contribute to emergence of antibiotic-resistant bacteria. In this study we systematically examine the effect of the biocide benzalkonium chloride, which is primarily used for surface disinfection but is also present as a preservative in many consumer products, on the activity of aminoglycoside antibiotics in Acinetobacter baumannii. METHODS: The effect of subinhibitory BAC on aminoglycoside treatment of A. baumannii ATCC17978 was investigated using time-to-kill assays, MIC determination, directed evolution experiments, fluctuation tests and labelled gentamicin accumulation assays. Further MIC determinations and directed evolution experiments were performed with additional Gram-negative ESKAPE pathogens. FINDINGS: In A. baumannii ATCC17978, BAC prevents gentamicin killing and drastically increases the frequency at which resistant mutants emerge, through reducing intracellular antibiotic accumulation. BAC also increases the MIC of multiple aminoglycoside antibiotics (kanamycin, tobramycin, streptomycin, gentamicin and amikacin). BAC promotes the emergence of mutants with reduced gentamicin susceptibility in other Gram-negative ESKAPE pathogens but does not always alter the MIC. These effects occur at BAC concentrations which are similar to residual levels in high-use environments, and just below the concentration range for BAC when used as a preservative in eye drops and ear drops. INTERPRETATION: Our results suggest that subinhibitory BAC has the potential to antagonise aminoglycoside activity and promote the emergence of bacterial mutants with reduced susceptibility. We suggest that the extremely widespread use of BAC in clinical and home settings and its long half-life mean there is potential for these interactions to occur in the environment, or in patients who use BAC-containing products while taking aminoglycosides to treat skin, eye or ear infections, although such co-exposure is likely to be rare. We suggest that biocide stewardship is needed to prevent the types of exposure that can contribute to antibiotic resistance. FUNDING: This work was funded by the National Health and Medical Research Council of Australia. The funders had no role in study design, interpretation or decision to publish.

Adaptive laboratory evolution of native methanol assimilation in Saccharomyces cerevisiae.

Utilising one-carbon substrates such as carbon dioxide, methane, and methanol is vital to address the current climate crisis. Methylotrophic metabolism enables growth and energy generation from methanol, providing an alternative to sugar fermentation. Saccharomyces cerevisiae is an important industrial microorganism for which growth on one-carbon substrates would be relevant. However, its ability to metabolize methanol has been poorly characterised. Here, using adaptive laboratory evolution and 13C-tracer analysis, we discover that S. cerevisiae has a native capacity for methylotrophy. A systems biology approach reveals that global rearrangements in central carbon metabolism fluxes, gene expression changes, and a truncation of the uncharacterized transcriptional regulator Ygr067cp supports improved methylotrophy in laboratory evolved S. cerevisiae. This research paves the way for further biotechnological development and fundamental understanding of methylotrophy in the preeminent eukaryotic model organism and industrial workhorse, S. cerevisiae.

Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies.

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.

Benchmarking two Saccharomyces cerevisiae laboratory strains for growth and transcriptional response to methanol.

One-carbon compounds, such as methanol, are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals, fuels, foods, and pharmaceuticals. Efficient biological production often requires extensive genetic manipulation of a microbial host strain, making well-characterised and genetically-tractable model organisms like the yeast Saccharomyces cerevisiae attractive targets for the engineering of methylotrophic metabolism. S. cerevisiae strains S288C and CEN.PK are the two best-characterised and most widely used hosts for yeast synthetic biology and metabolic engineering, yet they have unpredictable metabolic phenotypes related to their many genomic differences. We therefore sought to benchmark these two strains as potential hosts for engineered methylotrophic metabolism by comparing their growth and transcriptomic responses to methanol. CEN.PK had improved growth in the presence of methanol relative to the S288C derivative BY4741. The CEN.PK transcriptome also had a specific and relevant response to methanol that was either absent or less pronounced in the BY4741 strain. This response included up-regulation of genes associated with mitochondrial and peroxisomal metabolism, alcohol and formate dehydrogenation, glutathione metabolism, and the global transcriptional regulator of metabolism MIG3. Over-expression of MIG3 enabled improved growth in the presence of methanol, suggesting that MIG3 is a mediator of the superior CEN.PK strain growth. CEN.PK was therefore identified as a superior strain for the future development of synthetic methylotrophy in S. cerevisiae.

Convergent microevolution of Cryptococcus neoformans hypervirulence in the laboratory and the clinic.

Reference strains are a key component of laboratory research, providing a common background allowing for comparisons across a community of researchers. However, laboratory passage of these strains has been shown to lead to reduced fitness and the attenuation of virulence in some species. In this study we show the opposite in the fungal pathogen Cryptococcus neoformans, with analysis of a collection of type strain H99 subcultures revealing that the most commonly used laboratory subcultures belong to a mutant lineage of the type strain that is hypervirulent. The pleiotropic mutant phenotypes in this H99L (for "Laboratory") lineage are the result of a deletion in the gene encoding the SAGA Associated Factor Sgf29, a mutation that is also present in the widely-used H99L-derived KN99a/α congenic pair. At a molecular level, loss of this gene results in a reduction in histone H3K9 acetylation. Remarkably, analysis of clinical isolates identified loss of function SGF29 mutations in C. neoformans strains infecting two of fourteen patients, demonstrating not only the first example of hypervirulence in clinical C. neoformans samples, but also parallels between in vitro and in vivo microevolution for hypervirulence in this important pathogen.

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

BACKGROUND: Engineering 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. RESULTS: To 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. CONCLUSIONS: The 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.