Dataset Information

A cyanobacterial screening platform for Rubisco mutant variants

ABSTRACT: Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. Its relatively low catalytic constant as well as its tendency to also accept O2 as a substrate have made it a common target of enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library, without prior experimental knowledge, by coupling the phylogenetically-guided model EVmutation with “in silico evolution”. Starting with this targeted mutagenesis library, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. We identified an amino acid exchange which increased the thermostability of Gallionella Rubisco and conveyed resilience to detrimental exchanges. The establishment of this platform is a first step towards high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts. DIA proteomics was used to compare relative quantities of the native and heterologous RuBisCO, as well as the proteome at large at different gas conditions and with or without the heterologous enzyme.

INSTRUMENT(S):

ORGANISM(S): Synechocystis Sp. (strain Pcc 6803 / Kazusa)

SUBMITTER: Emil Sporre

LAB HEAD: Paul Hudson

PROVIDER: PXD059631 | Pride | 2025-08-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20231030_cbbM1_pcc6803.elib.peptides.txt	Txt
	20231030_cbbM1_pcc6803.elib.proteins.txt	Txt
	20231030_cbbM2_pcc6803.elib.peptides.txt	Txt
	20231030_cbbM2_pcc6803.elib.proteins.txt	Txt
	HF_20231030_DIA_350to1000_10mz_40min_1to32_700nLmin_60C_2kV_2uL_RA1.raw	Raw

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Publications

A Cyanobacterial Screening Platform for Rubisco Mutant Variants.

Hoffmann Ute A UA Schuppe Anna Z AZ Knave Axel A Sporre Emil E Brismar Hjalmar H Englund Elias E Syrén Per-Olof PO Hudson Elton P EP

ACS synthetic biology 20250707 7

Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. The relatively low specific activity and tendency to accept O2 as a substrate have made Rubisco an attractive but challenging target for enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first sh ...[more]

PMID: 40622942

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Project description:We have investigated the response of the cyanobacterium Synechocystis sp. PCC 6803 during growth at very low O2 concentration (bubbled with 99.9% N2/0.1% CO2). Significant transcriptional changes upon low O2 incubation included up-regulation of a cluster of genes that contained psbA1 and an operon that includes a gene encoding the two-component regulatory histidine kinase, Hik31. This regulatory cluster is of particular interest, since there are virtually identical copies on both the chromosome and on plasmid pSYSX. We used a knockout mutant lacking the chromosomal copy of hik31 and studied differential transcription during the aerobic- low O2 transition in this ΔHik31 strain and the wild type. We observed two distinct responses to this transition, one Hik31 dependent, the other Hik31 independent. The Hik31 independent responses included the psbA1 induction and genes involved in chlorophyll biosynthesis. In addition, there were changes in a number of genes that may be in involved in assembling or stabilizing Photosystem II (PSII), and the hox operon and the LexA-like protein (Sll1626) were up-regulated during low O2 growth. This family of responses mostly focused on PSII and overall redox control. There was also a large set of genes that responded differently in the absence of the chromosomal Hik31. In the vast majority of these cases, Hik31 functioned as a repressor and transcription was enhanced when Hik31 was deleted. Genes in this category encoded both core and peripheral proteins for Photosystem I and PS II, the main phycobilisome proteins, chaperones, the ATP synthase cluster and virtually all of the ribosomal proteins. These findings, coupled with the fact that ΔHik31 grew better than the wild-type under low O2 conditions, suggested that Hik31 helped to regulate growth and overall cellular homeostasis. We detected changes in the transcription of other regulatory genes that may compensate for the loss of Hik31. We conclude that Hik31 regulates an important series of genes that relate to energy production and growth and that helps to determine how Synechocystis responds to changes in O2 conditions.