Integrated Optimization of Hemoprotein Biomanufacturing in Yeast: From Upstream Engineering of Heme Synthesis to Downstream Purification of Human Hemoglobin

Blood transfusions are a vital part of modern healthcare systems, providing a life-saving treatment in a wide range of medical emergencies. However, despite its significance, this common procedure comes with inherent limitations, including the risk of blood-borne diseases, limited shelf life, demanding storage requirements, need for cross-matching, logistical hurdles, and reliance on altruistic donors. Hemoglobin-based Oxygen Carriers (HBOCs) offer a promising alternative to overcome these constraints. Nevertheless, cost-effective biomanufacturing of HBOCs remains a significant hurdle. This thesis aims to address this challenge by exploring the genetic optimization of microbial cell factories, particularly Saccharomyces cerevisiae and Komagataella phaffii, to enhance HBOC production metrics.

The initial study examines how iron supplementation, in conjunction with the deletion of either the HAP1 or ROX1 gene, influences heme biosynthesis in S. cerevisiae, a crucial aspect of HBOC production. These genes encode essential transcription factors involved in oxygen homeostasis and heme biosynthesis. Using transcriptomics and proteomics analysis, the study reveals the complex interplay between oxygen, iron, and heme regulatory networks, and provide vital insights into the potential of targeted genetic modifications combined with iron supplementation to enhance heme production, which acts as the foundation for the next studies in this thesis.

Building on these insights, the second study explores the potential of a multi-dimensional genetic engineering strategy to optimize the production of recombinant human hemoglobin (rHbA) in S. cerevisiae. The approach targeted multiple genes related to heme formation, including overexpressing the rate-limiting enzyme in the pathway (HEM3), decoupling the negative feedback loop on heme biosynthesis (Δhap1 or Δrox1), constitutively activating the iron regulon for increased flux towards incorporation into heme (Δgrx3/4 or AFT1-1^𝑢𝑝), and facilitating full iron utilization for heme formation (Δcth1/2). The successful combination of these modifications revealed a positive synergetic “push-pull” effect, substantially enhancing rHbA production. Additionally, the study reveals a unique red colony phenotype in certain strains, warranting further research to explore their full potential.

The third study expands the application space of these findings by applying the robust genetic engineering strategy to produce other industrially relevant hemoproteins. These include fetal hemoglobin (HbF), fusion fetal hemoglobin (F-HbF), horseradish peroxidase (HRP), and human cytochrome P450 (CYP2C9). The previously identified best genetic modifications led to significant enhancements in both heme synthesis and the formation of these hemoproteins, offering promising advances in the development of novel therapies, environmental remediation strategies, and industrial processes.

The fourth study built on this success by fine-tuning the genetic engineering strategy, specifically aiming to minimize downstream degradation of the synthesized co-factor heme and the translated protein globin chains. This was achieved by deleting the HMX1 and/or PEP4 genes. The principle of diminishing returns epistasis was evident in the results, with the additional modifications yielding smaller improvements than earlier optimizations. Nevertheless, these modifications significantly increased the total activity of formed rHbA, representing an incremental advancement towards the large-scale, cost-effective biomanufacturing of HBOCs.

The fifth study transitioned from S. cerevisiae to Komagataella phaffii, another yeast offering several advantages such as higher cell densities, protein secretion, and stress resistance. By adapting the proven engineering strategy from S. cerevisiae, significant improvements in bound and total heme levels were observed. Moreover, by expressing hemoglobins for secretion, this study set the stage for the subsequent study on purification.

The final study focused on an essential step in biomanufacturing HBOCs - the downstream purification process. It explores the potential of a novel, high-capacity, salt-tolerant multimodal cation exchange nonwoven membrane (MMC-MPCA) for efficient purification of human hemoglobin from clarified K. phaffii cell culture supernatant, a critical step in HBOC biomanufacturing. This membrane demonstrated excellent productivity, superior dynamic binding capacity, remarkable elution purity levels, and robust reusability, offering a pathway toward more efficient, scalable, and cost-effective downstream processing for HBOC biomanufacturing.

In conclusion, these studies collectively provide extensive insights into the genetic optimization of microbial cell factories for the production of HBOCs. The findings underscore the significant potential of microbial biomanufacturing in addressing global blood supply shortages, marking a incremental stride forward in the field of biotechnology.