Biology subjects

Shaikh, K. M.

Publications and source records attributed to Shaikh, K. M..

5 recordsLinked to original sources

bioRxiv2026

Targeted Deletions Uncover Previously Uncharacterised C1-fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum

Recycling carbon oxides from industrial off-gases and gasified organic waste using gas fermentation offers a sustainable route for biomanufacturing of fuels and chemicals. Acetogens are attractive biocatalysts here and fix CO and CO2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C1-fixing gene cluster limits rational engineering of acetogen cell factories. Here, we investigated five highly conserved but previously uncharacterised C1-fixing cluster genes to quantify their contributions to the growth of the model-acetogen Clostridium autoethanogenum using targeted CRISPR/Cas9-mediated single-gene deletions, physiological characterisation, comparative computational structural analyses, CRISPRi, and functional genomics. Importantly, we identified acsV, lpdA, cooC, and cooC2 as essential for autotrophy but not heterotrophy, whereas deletion of gcvH reduced autotrophic growth and shifted carbon flux from acetate towards 2,3-butanediol. Amino acid supplementation experiments and computational analyses indicate that lpdA and gcvH do not primarily function in glycine or serine biosynthesis but instead fulfil specialised roles in WLP-associated C1 and redox metabolism. Furthermore, genetic and physiological analyses showed that the two CooC-family ATPases, CooC and CooC2, have non-redundant roles in growth, with only cooC2 deletion impairing heterotrophy. Acetate supplementation tests revealed that {Delta}acsV cells retained metabolic activity despite abolished autotrophy, and computational analyses suggest a likely role for acsV in ATP-dependent corrinoid activation. Surprisingly, CRISPRi-mediated repression of acsV was translationally buffered, resulting in altered carbon partitioning without impairing autotrophy. Together, our findings provide insights into the physiological importance of the C1-fixing cluster genes in acetogens and support their metabolic engineering as gas-fermenting cell factories.

synthetic biology↗

bioRxiv2026

Genetic engineering of carbon monoxide dehydrogenases produces distinct autotrophic phenotypes in Clostridium autoethanogenum

Acetogens are promising microbes for sustainable biomanufacturing but improving acetogen gas fermentation requires efficient conversion of CO and CO2 into fuels and chemicals. Carbon monoxide dehydrogenase (CODH) enzymes couple carbon fixation to energy conservation in acetogens and serve as potential regulatory modules for tuning autotrophic metabolism. Intriguingly, the model-acetogen Clostridium autoethanogenum lost its unique truncation in the bifunctional CODH (acsA), essential for autotrophy, during autotrophic adaptive laboratory evolution while obtaining superior phenotypes. Additionally, protein expression of the monofunctional CODH cooS1 is high and conditionally-regulated in C. autoethanogenum. Here, we genetically engineered CODHs in C. autoethanogenum by replacing the stop codon in acsA with leucine (strain Leu_SNP) or serine (Ser_SNP), and deleting cooS1 ({Delta}cooS1). Phenotyping in autotrophic batch and chemostat cultures revealed altered growth profiles and significant redistribution of carbon and redox flows in SNP strains, whereas {Delta}cooS1 showed moderate and condition-dependent effects. Surprisingly, structural modelling identified no conformational differences between wild-type and mutant AcsA proteins. While transcriptomics showed limited transcriptional changes in {Delta}cooS1, it suggested potential transcriptional adjustments linked to reduced robustness and altered product profile of Leu_SNP. Our results demonstrate the impact of CODHs on autotrophy and offer targets for rational engineering of acetogen cell factories.

bioengineering↗

bioRxiv2025

Unprecedented exoelectrogenic activity in Parachlorella kessleri MACC-38: The interplay of photosynthetic electron transport and the oxidative pentose phosphate pathway

Exoelectrogenesis is the ability of living cells to export electrons. In photosynthetic organisms, exoelectrogenesis is of particular interest because it can be used for transduction of solar energy into electric current in biophotovoltaics or reducing power for biocatalysis. Previously, we identified and characterized the green microalga Parachlorella kessleri MACC-38 producing unprecedented current largely dependent on the photosynthetic electron transport chain (PETC). In this study, our comparative photosynthetic characterization demonstrates that chlororespiration is a crucial factor maintaining the PETC redox balance in MACC-38. Our data points that the oxidative pentose phosphate pathway (OPPP) is activated during exoelectrogenesis to meet the increased demand of reducing power. We hypothesize that chlororespitration prevents oversaturation of PETC during OPPP activation. These findings provide valuable insights into the fundamental mechanisms of exoelectrogenesis in green algae. By elucidating the complex interconnection between PETC, OPPP and exoelectrogenesis, our results pave the way towards improved bioelectrochemical and biocatalysis technologies.

plant biology↗

bioRxiv2023

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2- concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules. One-sentence summaryIn Chlamydomonas, the pyrenoid-localized bestrophin-like protein BST4 is a putative ion channel involved in pH regulation of the thylakoid lumen, possibly by mediating bicarbonate transport.

plant biology↗

bioRxiv2023

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both {Delta}hp and {Delta}nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

synthetic biology↗