# Innovative Solvent Production Through Genetic Engineering and Microbial consortium approaches

Cyrielle Debeir, Alexandre Delarouzée, Gwladys da Costa, François Wasels

► To cite this version:

Cyrielle Debeir, Alexandre Delarouzée, Gwladys da Costa, François Wasels. Innovative Solvent Pro-
duction Through Genetic Engineering and Microbial consortium approaches. FEMS MICRO, Jul
2025, Milan, Italy. FEMS MICRO Abstract book, 11, pp.324.17P, 2025. [hal-05323928](https://hal-05323928)

HAL Id: hal-05323928

https://ifp.hal.science/hal-05323928v1

Submitted on 21 Oct 2025

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## ABSTRACT

* Genetic engineering and cocultures are complementary approaches to improve fermentation yields

* The asaccharolytic C3 producing strain *Anaerotium propionicum* (*Apr*) was characterized in different conditions

* A Δhbd mutant of *Clostridium acetobutylicum* (*Cac*) was obtained using a CRISPR-Cas9 genetic tool

* Co-cultures designed to enhance propanol titer were conducted between the wild type or Δhbd mutant of *Cac* and *Apr*

<figure><img src="image_1.png" /></figure>

## RESULTS

### Characterization of *Anaerotium propionicum* and selection of the coculture medium

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Fermentation products of *A. propionicum*. One week batch cultures were performed in 10 mL working volume at 34°C, in its reference medium (A) or CGM (B), with different substrates and gas compositions.</figcaption></figure>

*CGM, Clostridium Growth Medium; **ACA, Anaerobic Cabinet Atmosphere, 93% N₂, 4% CO₂, 3% H₂

* Higher propionate titers are reached when **alanine** is present in the medium

* Gas composition of 100% CO₂ is toxic to the strain

* The highest propanol titer was **0.17 g/L** on CGM + ethanol with 100% N₂.
**No propionate** was produced in that condition

* **C3 titers are higher on CGM containing alanine**

### Cocultures with *Clostridium acetobutylicum*

<figure><img src="image_3.png" /><figcaption>**Figure 2:** (A) Fermentation products of cocultures of *Cac* and *Apr* and (B) substrate consumption for each condition. One week batch cultures were performed on CGM (containing 36 g/L glucose) + 3 g/L alanine at 34°C.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 3:** Metabolism of the Δhbd mutant of *Cac*. The mutant was constructed using a CRISPR/Anti-CRISPR tool. The only solvents produced are ethanol and acetone.</figcaption></figure>

Ack, acetate kinase; Adc, acetoacetate decarboxylase; Adh, alcohol dehydrogenase; Ald, aldehyde dehydrogenase; Aldc, acetolactate decarboxylase; Als, acetolactate synthase; Bcd, butyryl-CoA dehydrogenase; Buk, butyrate kinase; CtfA-CtfB, butyrate-acetoacetate CoA-transferase (A and B subunits); Crt, crotonase; EtfA-EtfB, electron transport flavoprotein (α and β subunits); Hbd, 3-hydroxybutyryl-CoA déshydrogénase; Ldh, lactate déshydrogénase; Pfor, pyruvate ferredoxine oxydoreductase; Pta, phosphate acetyltransferase; Ptb, phosphate butyryltransferase; Thl, thiolase.

* The Δhbd mutant was able to produce more than **19 g/L** of solvents, mostly ethanol

* The coculture between *Cac* and *Apr* had a **deleterious effect**:

→ Less glucose consumed by *Cac*

→ Less solvents produced

→ No propionate or propanol detected

* The coculture between Δhbd and Apr produced **1.39 g/L propanol** = 8 times the highest propanol titer obtained for Apr in monoculture

## CONCLUSIONS

* Apr was characterized on two different media in various conditions

* **Alanine** is necessary to reach high titers of propionate

* Cocultivating *Cac* and *Apr* seems **deleterious** for both strains

* A Δhbd mutant was successfully obtained using a CRISPR-Cas9 tool and characterized

* **1.39 g/L of propanol** (= 8 times the Apr monoculture highest titer) were produced in a coculture between this Δhbd mutant and Apr

## PERSPECTIVES

* Perform the Δhbd + Apr coculture on DSMZ 156 medium

* Construct an alanine hyperproducer mutant of *Cac* to increase syntrophic interactions between both strains

* Understand fundamental interactions between strains using a Fluorescence In Situ Hybridization (FISH) based tool