# CRISPR/Cas9-Mediated Metabolic Engineering of Endophytic Pseudomonas loganensis sp. nov. for the Production of Nutritionally Valuable Carotenoids

## TL;DR

Scientists engineered a new type of bacteria to produce valuable carotenoids, offering a sustainable alternative to traditional methods.

## Contribution

A novel endophytic bacterial chassis was developed for carotenoid production using CRISPR-Cas9.

## Key findings

- CRISPR-Cas9 was used to create strains producing zeaxanthin, lycopene, β-carotene, and astaxanthin.
- Optimized conditions increased carotenoid titers up to 12-fold compared to unoptimized LB medium.
- The engineered bacteria show potential for sustainable, large-scale carotenoid bioproduction.

## Abstract

Carotenoids
with significant nutritional and antioxidant
properties
have been widely utilized in the food, feed, pharmaceutical, and cosmetic
industries. They improve the nutritional value of foodstuffs and have
been used as natural food colorants. However, their current supply
chain is mainly dependent on extraction from plants and chemical synthesis,
both of which have bottlenecks, including environmental concerns,
toxicity, and allergenicity. To address global demand for sustainable
and environmentally friendly production of nutrients, we engineered
the endophytic Pseudomonas loganensis sp. nov. as a niche microbial chassis for nutritionally valuable
carotenoid production. Using CRISPR-Cas9, we knocked out key carotenogenic
genes to construct strains capable of producing zeaxanthin, lycopene,
and β-carotene. Additionally, an overexpression plasmid was
introduced to produce astaxanthin. HPLC analysis confirmed the successful
production of four target carotenoids. The culture conditions and
media compositions were optimized using response surface methodology,
resulting in a ∼5-fold increase in the titers of zeaxanthin
(13.4 mg/L), lycopene (9.67 mg/L), and β-carotene (23.53 mg/L),
and a ∼12-fold increase in astaxanthin titer (1 mg/L) compared
to LB medium without optimization. Our results indicate the potential
of endophytic bacteria as a microbial chassis for carotenoid bioproduction,
underscoring the potential of synthetic biology to contribute to global
efforts toward nutritional security and sustainable food systems.