Deregulation of phytoene-β-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19

Abstract

Background: A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels.

Results: Total carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24-72 h was only 14-17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose.

Conclusion: We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

Keywords: Astaxanthin; Gene expression; Glucose metabolism; Phaffia rhodozyma; Phytoene-β-carotene synthase.

Fig. 1

Carotenoid profiles of WT JCM9042 at 40 g/L glucose (a), JCM9042 at 110 g/L glucose (b), mutant MK19 at 40 g/L glucose (c), and MK19 at 110 g/L glucose (d)

Fig. 2

Relative expression of crtI (a), ast (b), crtE (c), and pbs (d) genes at glucose concentrations of 40, 110, and 160 g/L

Fig. 3

Relative expression of creA in MK19 and JCM9042 at glucose induction times 0, 30, 60, and 120 min as indicated in the X-axis captions

Fig. 4

Relative expression of creA, alcA, and carotenoid synthesis genes in JCM9042 (a) and MK19 (b) at glucose induction times 0 and 30 min. Expression at 48 h was defined as 1. alcA, encoding the alcohol dehydrogenase I and repressed by creA, was used as control

Fig. 5

(a) Relative expression of alcA and carotenoid synthesis genes in MK19 and MK19-CreA9 (mutant 9) at 24 h at 40 g/L glucose. Expression in MK19 was defined as 1. (b) Astaxanthin content of MK19 and mutant 9 as a function of fermentation time

Fig. 6

Relative expression of glucose signaling pathway genes in MK19 and JCM9042 at glucose induction times 0 and 30 min. Expression in wild type JCM9042 at 48 h was defined as 1

Fig. 7

Regulatory pattern of glucose signaling pathway on pbs in JCM9042 (a) and MK19 (b)