Improving astaxanthin production in Escherichia coli by co-utilizing CrtZ enzymes with different substrate preference

Abstract

Background The bifunctional enzyme β-carotene hydroxylase (CrtZ) catalyzes the hydroxylation of carotenoid β-ionone rings at the 3, 3’ position regardless of the presence of keto group at 4, 4’ position, which is an important step in the synthesis of astaxanthin. The level and substrate preference of CrtZ may have great effect on the amount of astaxanthin and the accumulation of intermediates. Results In this study, the substrate preference of PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from Pantoea Agglomerans were certified and were combined utilization for increase astaxanthin production. Firstly, PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from P. Agglomerans were expressed in platform strains CAR032 (β-carotene producing strain) and Can004 (canthaxanthin producing strain) separately to identify their substrate preference for carotenoids with keto groups at 4,4’ position or not. The results showed that PCcrtZ led to a lower zeaxanthin yield in CAR032 compared to that of PAcrtZ. On the contrary, higher astaxanthin production was obtained in Can004 by PCcrtZ than that of PAcrtZ. This demonstrated that PCCrtZ has higher canthaxanthin to astaxanthin conversion ability than PACrtZ, while PACrtZ prefer using β-carotene as substrate. Finally, Ast010, which has two copies of PAcrtZ and one copy of PCcrtZ produced 1.82 g/L of astaxanthin after 70 h of fed-batch fermentation. Conclusions Combined utilization of crtZ genes, which have β-carotene and canthaxanthin substrate preference respectively, can greatly enhance the production of astaxanthin and increase the ratio of astaxanthin among total carotenoids.

Improved Astaxanthin Synthesis in Komagataella phaffii through β-Carotene Ketolase Screening and β-Carotene Hydroxylase Mutagenesis

“…(PsCrtZ), and Agrobacterium aurantiacum (AaCrtZ) showed a preference for keto-carotene, while those from Pantoea sp. (e.g., Pantoea ananatis or Pantoea agglomerans) exhibited a β-carotene substrate preference. , CrtZs from Brevundimonas sp. (BvCrtZ) can hydroxylate β-carotene, but its activity is lower than that of Pantoea sp .…”

“…ananatis or Pantoea agglomerans) exhibited a β-carotene substrate preference. 7,26 CrtZs from Brevundimonas sp. (BvCrtZ) can hydroxylate β-carotene, but its activity is lower than that of Pantoea sp.…”

“…The recent advancements in metabolic engineering have opened new avenues for the industrial production of astaxanthin, particularly through heterologous synthesis in model microorganisms. Significant research has been dedicated to astaxanthin synthesis in industrial microbes like Escherichia coli, − Saccharomyces cerevisiae, − Yarrowia lipolytica, − and Corynebacterium glutamicum , as detailed in Table S1. However, Komagataella phaffii, despite its widespread application in heterologous protein expression, is relatively unexplored as a chassis for astaxanthin synthesis, with existing studies indicating low astaxanthin yields. − …”

From Aquaculture to Aquaculture: Production of the Fish Feed Additive Astaxanthin by Corynebacterium glutamicum Using Aquaculture Sidestream

“…The total carotenoid concentrations of up to about 0.1 g L −1 observed in this study may indicate that more astaxanthin can be produced if all precursor carotenoids are converted to astaxanthin. This may be approached by adjusting the copy number and/or expression levels of crtZ and crtW as previously demonstrated for E. coli and S. cerevisiae [ 118 , 119 ].…”

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…In addition, due to the unique molecular structure of terpenoids, their chemical synthesis routes often involve tedious synthesis steps, leading to low final yields and environmental concerns . Attempts have been made to synthesize terpenoids by introducing the plant-origin pathway genes into model microbial chassis, such as Escherichia coli , and Saccharomyces cerevisiae. − But prokaryotes such as E. coli often have difficulty in expressing P450 enzymes properly, which play important roles in the synthesis of many terpenoids.…”