Dataset Information

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

ABSTRACT: Type I polyketide synthases (T1PKSs) hold an enormous potential as a rational production platform for the biosynthesis of speciality chemicals. However, despite the great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enables the production of an unnatural polyketide in each of the hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. Here, we not only highlight the significance of codon optimization but also establish the groundwork for high-throughput assembly and characterization of PKS pathways in alternative hosts.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli Corynebacterium Glutamicum Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD042749 | Pride | 2023-09-01

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 142

Similar Datasets

Project description:Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).

			Action	DRS
	10_G-R1.raw	Raw
	10_G-R2.raw	Raw
	10_G-R3.raw	Raw
	11_G-R1.raw	Raw
	11_G-R2.raw	Raw

Dataset Information

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets