Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5′ untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool.

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real‐time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell‐baculovirus expression vector system (IC‐BEVS), a well‐established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real‐time monitoring tool for recombinant adeno‐associated virus (rAAV) production in the IC‐BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm‐based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave‐one‐batch‐out cross‐validation. Our approach shows fluorescence spectroscopy allows real‐time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC‐BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.

Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible "tuning knob" for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution is performed. Adjacent triplets of the 200 bp long core promoter are mutated at a time by changing the wild-type sequence into cytosine or adenine triplets, resulting in 130 variants that are cloned upstream of an eGFP reporter gene providing a library for expression fine-tuning. Mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5'UTR had a significant effect on the eGFP fluorescence. Surprisingly, mutations in most other regions are tolerated, indicating that yeast core promoters can show a high tolerance toward small mutations, supporting regulatory models of degenerate motifs, or redundant design. The authors exploited these neutral core promoter positions, not affecting expression, to introduce extrinsic sequence elements such as cloning sites (allowing targeted core promoter/5'UTR modifications) and bacterial promoters (applicable in multi host vectors).