The environmental burden of conventional plastics has sparked interest in sustainable alternatives such as polyhydroxyalkanoates (PHAs). However, despite ample research in bioprocess development and the use of inexpensive waste streams, production costs remain a barrier to widespread commercialization. Complementary to this, genetic engineering offers another avenue for improved productivity. Cupriavidus necator stands out as a model host for PHA production due to its substrate flexibility, high intracellular polymer accumulation, and tractability to genetic modification. This review delves into metabolic engineering strategies that have been developed to enhance the production of poly(3-hydroxybutyrate) (PHB) and related copolymers in C. necator. Strategies include the optimization of central carbon flux, redox and cofactor balancing, adaptation to oxygen-limiting conditions, and fine-tuning of granule-associated protein expression and the regulatory network. This is followed by outlining engineered pathways improving the synthesis of PHB copolymers, PHBV, PHBHHx, and other emerging variants, emphasizing genetic modifications enabling biosynthesis based on unrelated single-carbon sources. Among these, enzyme engineering strategies and the establishment of novel artificial pathways are widely discussed. In particular, this review offers a comprehensive overview of promising engineering strategies, serving as a resource for future strain development and positioning C. necator as a valuable microbial chassis for biopolymer production at an industrial scale.

Background: Not changing the native constitution of genes prior to their expression by a heterologous host can affect the amount of proteins synthesized as well as their folding, hampering their activity and even cell viability. Over the past decades, several strategies have been developed to optimize the translation of heterologous genes by accommodating the difference in codon usage between species. While there have been a handful of studies assessing various codon optimization strategies, to the best of our knowledge, no research has been performed towards the evaluation and comparison of codon harmonization algorithms. To highlight their importance and encourage meaningful discussion, we compared different open-source codon harmonization tools pertaining to their in silico performance, and we investigated the influence of different gene-specific factors. Results: In total, 27 genes were harmonized with four tools toward two different heterologous hosts. The difference in %MinMax values between the harmonized and the original sequences was calculated (ΔMinMax), and statistical analysis of the obtained results was carried out. It became clear that not all tools perform similarly, and the choice of tool should depend on the intended application. Almost all biological factors under investigation (GC content, RNA secondary structures and choice of heterologous host) had a significant influence on the harmonization results and thus must be taken into account. These findings were substantiated using a validation dataset consisting of 8 strategically chosen genes. Conclusions: Due to the size of the dataset, no complex models could be developed. However, this initial study showcases significant differences between the results of various codon harmonization tools. Although more elaborate investigation is needed, it is clear that biological factors such as GC content, RNA secondary structures and heterologous hosts must be taken into account when selecting the codon harmonization tool.

Tetrasomy 9p is a chromosomal disorder characterized by the presence of a supernumerary chromosome. This rare abnormality exhibits a broad phenotypic variability and is not clearly distinguishable from other more frequent aneuploidies in the prenatal setting. We present two prenatal cases with dissimilar phenotypes, one with solely increased fetal nuchal translucency and one with multiple congenital anomalies, and discuss prior studies. These cases illustrate the difficulty of prenatally diagnosing this condition based on phenotypic information alone. We conclude that invasive prenatal diagnosis with (molecular) karyotyping is the best choice for the prenatal detection of tetrasomy 9p.