# Eliciting Clavulanic Acid Biosynthesis: The Impact of Bacillus velezensis FZB42 on the Metabolism of Streptoyces clavuligerus ATCC 27064

## TL;DR

This study shows how adding Bacillus velezensis cells can boost clavulanic acid production in Streptomyces clavuligerus by altering gene expression.

## Contribution

The novel use of B. velezensis to enhance CA production and the detailed transcriptomic analysis of the interaction.

## Key findings

- Live Bacillus velezensis cells increased clavulanic acid production by 2.0-fold in Streptomyces clavuligerus.
- Transcriptomic analysis revealed 410 up-regulated and 594 down-regulated genes in S. clavuligerus under B. velezensis influence.
- Up-regulated genes included those in the clavulanic acid biosynthesis cluster and oxidative stress defense pathways.

## Abstract

Background/Objectives: Clavulanic acid (CA) is produced by cell suspension cultures of Streptomyces clavuligerus ATCC 27064, and is widely used as a beta-lactamase inhibitor to combat antibiotic resistance. CA titers are moderate due to bioprocess complexity, prompting ongoing efforts to overcome these limitations. Methods: In this study, we aimed to evaluate the effect of live and inactivated Bacillus velezensis FZB42 cells on CA production in S. clavuligerus, and to explore the transcriptional response underlying this interaction using RNA-seq technology. Results: The addition of dead and live cells of B. velezensis improved CA production by 1.4 and 2.0-fold, respectively. Furthermore, the transcriptome of S. clavuligerus, obtained with live cells of B. velezensis FZB42 at the peak of maximum CA production, revealed that 410 genes were up-regulated and 594 were down-regulated under these conditions, with a padj < 0.05. Most of the genes from the cephamycin C and CA clusters were up-regulated, which correlates well with the increase in CA production. Likewise, S. clavuligerus ATCC 27064 enhanced the expression of genes encoding enzymes that scavenge endogenous H2O2, as well as other genes related to oxidative stress defense. Regarding downregulated genes, we found that S. clavuligerus decreased the expression of genes involved in the biosynthesis of terpenoids, polyketides, and lantibiotics, as well as the expression of the operon involved in the synthesis of the pyrroloquinoline quinone (PQQ) cofactor. Conclusions: These findings contribute to the understanding of S. clavuligerus metabolism and pave the way for future metabolic engineering efforts aimed at obtaining CA-overproducing strains.