Biology subjects

Claassens, N. J.

Publications and source records attributed to Claassens, N. J..

22 records · Page 2Linked to original sources

bioRxiv2022

Revealing determinants of translation efficiency via whole-gene codon randomisation and machine learning

Codon usage refers to the occurrence of synonymous codons in protein-coding genes. It is known for decades that codon usage contributes to translation efficiency and hence to protein production levels. However, its role in protein synthesis is still only partly understood. This lack of understanding hampers the design of synthetic genes for efficient protein production. In this study, we developed a method to generate a large, synonymous codon library of the gene encoding the red fluorescent protein (RFP). After expression in Escherichia coli, 1459 clones of this library were selected of which we measured protein production levels and determined the full coding sequences. Using different machine learning approaches, this data was used to reveal correlations between codon usage and protein production. Interestingly, protein production levels can be relatively accurately predicted (Pearson correlation of 0.762) by a Random Forest model, which only relies on the sequence information for the first 8 codons. This study clearly demonstrated the key role of codons at the start of the coding sequence. As such, it provides not only important fundamental insights on the influence of codon usage on protein production but also relevant clues on optimising the design of efficiently translated synthetic genes.

molecular biology↗

bioRxiv2021

Design, construction and optimization of a synthetic RNA polymerase operon in Escherichia coli.

Prokaryotic genes encoding functionally related proteins are often clustered in operons. The compact structure of operons allows for co-transcription of the genes, and for co-translation of the polycistronic messenger RNA to the corresponding proteins. This leads to reduced regulatory complexity and enhanced gene expression efficiency, and as such to an overall metabolic benefit for the protein production process in bacteria and archaea. Interestingly, the genes encoding the subunits of one of the most conserved and ubiquitous protein complexes, the RNA polymerase, are not clustered in a single operon. Rather, its genes are scattered in all known prokaryotic genomes, generally integrated in different ribosomal operons. To analyze the impact of this genetic organization on the fitness of Escherichia coli, we constructed a bacterial artificial chromosome harboring the genes encoding the RNA polymerase complex in a single operon. Subsequent deletion of the native chromosomal genes led to a reduced growth on minimal medium. However, by using adaptive laboratory evolution the growth rate was restored to wild-type level. Hence, we show that a highly conserved genetic organization of core genes in a bacterium can be reorganized by a combination of design, construction and optimization, yielding a well-functioning synthetic genetic architecture.

synthetic biology↗

bioRxiv2020

Photorespiration pathways in a chemolithoautotroph

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of 2-phosphoglycolate, an essential process termed photorespiration, was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, but remains uncharacterized in chemolithoautotrophic bacteria. Here, we study photorespiration in the model chemolithoautotroph Cupriavidus necator (Ralstonia eutropha) by characterizing the proxy-process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium and instead the bacterial-like glycerate pathway is the main photorespiratory pathway. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports photorespiration. In this cycle, glyoxylate is condensed with acetyl-CoA to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support photorespiration in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so-far unknown photorespiration pathway, highlighting important diversity in microbial carbon fixation metabolism.

microbiology↗

bioRxiv2020

Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator

Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necators Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing the formate bioeconomy.

synthetic biology↗