Production of High Levels of 3S,3′S-Astaxanthin in Yarrowia lipolytica via Iterative Metabolic Engineering

Abstract

Astaxanthin is a highly value-added keto-carotenoid compound. The astaxanthin 3S,3′S-isomer is more desirable for food additives, cosmetics, and pharmaceuticals due to health concerns about chemically synthesized counterparts with a mixture of three isomers. Biosynthesis of 3S,3′S-astaxanthin suffers from limited content and productivity. We engineered Yarrowia lipolytica to produce high levels of 3S,3′S-astaxanthin. We first assessed various β-carotene ketolases (CrtW) and β-carotene hydroxylases (CrtZ) from two algae and a plant. HpCrtW and HpCrtZ from Haematococcus pluvialis exhibited the strongest activity in converting β-carotene into astaxanthin in Y. lipolytica. We then fine-tuned the HpCrtW and HpCrtZ transcriptional expression by increasing the rounds of gene integration into the genome and applied a modular enzyme assembly of HpCrtW and HpCrtZ simultaneously. Next, we rescued leucine biosynthesis in the engineered Y. lipolytica, leading to a five-fold increase in biomass. The astaxanthin production achieved from these strategies was 3.3 g/L or 41.3 mg/g dry cell weight under fed-batch conditions, which is the highest level reported in microbial chassis to date. This study provides the potential for industrial production of 3S,3′S-astaxanthin, and this strategy empowers us to build a sustainable biorefinery platform for generating other value-added carotenoids in the future.

Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica

“…In previous studies, Ma et al (2021) constructed a Y. lipolytica strain that could produce 453 mg/L of astaxanthin in 5-L fermenter through targeting the enzyme fusions (CrtW-Z) to subcellular compartment [ 14 ]. Zhu et al (2022) adjusted the expression levels of CrtW and CrtZ, restored leucine biosynthesis, and performed fed-batch fermentation in the synthetic CSM-Ura medium supplemented with YAG concentrated medium, resulting in the production of 3.3 g/L astaxanthin [ 9 ]. Above studies enhanced the production of astaxanthin by promoting the cell growth and substrate conversion.…”

“…Therefore, using microorganisms as efficient platforms for astaxanthin production has attracted considerable attentions [ [4] , [5] , [6] ]. In relevant studies, astaxanthin titers in engineered Escherichia coli , Saccharomyces cerevisiae , and Y. lipolytica strains varied from 404.78 mg/L to 3.3 g/L in different culture scales [ [7] , [8] , [9] ].…”

“…Although lots of efforts have been made for astaxanthin production in engineered microbes, a higher astaxanthin titer is urgently required for cost-effective commercialization, which is mainly due to the poor conversion efficiency of the precursor β-carotene to astaxanthin. Therefore, many strategies have been developed to improve the conversion efficiency of β-carotene to astaxanthin [ [9] , [10] , [11] , [12] , [13] ]. One of the most significant strategies was focused on optimizing the catalytic efficiency of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ).…”

The Role of Astaxanthin as a Nutraceutical in Health and Age-Related Conditions

“…Several methods are being developed for this purpose. Thus, using the genetically modified organisms (GMO) Yarrowia lipolytica, Escherichia coli, and Saccharomyces cerevisiae, an enhancement in the synthesis of ASX to 8–10 mg/g DCW occurred [ 38 , 62 , 63 ]. However, the commercial application of these genetically modified microorganisms and the use of their products in food or feed will need to undergo special regulations to ensure their safety.…”

Modulating DHA-Producing Schizochytrium sp. toward Astaxanthin Biosynthesis via a Seamless Genome Editing System

“…Previous work suggested that only HpCrtW initiated the biosynthesis of astaxanthin in H. pluvialis 48 and Y. lipolytica. 49 Thus, we ligated two copies of HpcrtW with one copy of HpcrtZ and then fused them with three different combinations of upstream genes: idi/hmgr, idi/ hmgr/hmgs, and idi/hmgr/crtE. All the above-mentioned vectors were transformed into strain M326 to generate engineered strains designated IRW2Z, IRSW2Z, and IREW2Z.…”