Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

Abstract High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora . Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from β-carotene is canthaxanthin (4,4'-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt . We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.     

Differential stimulation of an arid-environment winter ephemeral Dimorphotheca pluvialis (L.) Moench by ultraviolet-B radiation under nutrient limitation

A South African winter ephemeral D. pluvialis was exposed, under low and high nutrient conditions, to four different daily doses of biologically effective UV-B radiation. These simulated different depletions (range 0–30%) in the ozone layer at the southerly distribution limit (33° 56′S) of this species, and included daily UV-B doses received at the northerly distribution limit (26° 38′S) without ozone depletion. Growth inhibition by increased UV-B radiation was observed during early vegetative stages, but only under low nutrient conditions. Thereafter, net CO₂ assimilation rate, growth and reproduction were stimulated by an increase in UV-B radiation, though doses above those approximating a 20% ozone depletion appeared to be inhibitory. Differential stimulation occurred in the two nutrient treatments. Under high nutrient conditions, photosynthesis (specifically carboxylation efficiency), and numbers of leaves, inflorescences and diaspores per plant, and leaf areas increased, but leaf thickness decreased with increased UV-B radiation. Under low nutrient conditions, dry masses of leaves, stems, inflorescences and diaspores, and total above-ground dry masses increased with increased UV-B radiation. Foliar organic carbon and nitrogen concentrations and foliar concentrations of UV-B absorbing compounds were unaffected by increased UV-B radiation, but foliar P concentrations declined. Diaspore viability declined with increased UV-B radiation. The net effect was a 35 to 43% reduction in viable diaspore production under high nutrient conditions at UV-B doses equivalent to those currently received at the northerly distribution limit during the reproductive phase. It is concluded that anticipated increases in UV-B radiation could reduce regeneration success, and seedling survival in areas of low soil fertility, particularly at lower latitudes, and consequently increase the risk of localized population extinctions from stochastic causes.     

Cell fragility — The key problem of microalgae mass production in closed photobioreactors

The gap between the theoretical biological potential of microalgae and the biomass productivity obtained with algal culture in tubular biophotoreactors is due to a reduced growth rate related to hydrodynamic stress of pumping. High levels of mixing are necessary to reach a turbulent flow of the culture, in order to optimize the light regime. The optimal conditions of pumping to produce this significant liquid mixing may produce some cell damage. Factors affecting this hydrodynamic stress (geometry of the bioreactor involved, type of pump utilized, morphology of algal cells, physiological conditions of microalgae, etc.) are discussed.     

Astaxanthin preparation by fermentation of esters from Haematococcus pluvialis algal extracts with Stenotrophomonas species

Natural astaxanthin (Ax) is an additive that is widely used because of its beneficial biochemical functions. However, the methods used to produce free Ax have drawbacks. Chemical saponification methods produce several by‐products, and lipase‐catalyzed hydrolysis methods are not cost effective. In this study, a bacterial strain of Stenotrophomonas sp. was selected to enzymatically catalyze the saponification of Ax esters to produce free all‐trans‐Ax. Through single‐factor experiments and a Box–Behnken design, the optimal fermentation conditions were determined as follows: a seed culture age of 37.79 h, an inoculum concentration of 5.92%, and an initial broth pH of 6.80. Under these conditions, a fermentation curve was drawn, and the optimal fermentation time was shown to be 60 h. At 60 h, the degradation rate of the Ax esters was 98.08%, and the yield of free all‐trans‐Ax was 50.130 μg/mL. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:649–656, 2016     

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L^?1 day^?1) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L^?1 day^?1 by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

雨生红球藻虾青素合成研究进展

虾青素是一种重要的次级类胡萝卜素,具有高活性的抗氧化功能,广泛应用于食品保健、医药、水产养殖等领域。雨生红球藻是一种在胁迫条件下能够大量积累虾青素的微藻。文中回顾了雨生红球藻虾青素的生物合成研究的进展,包括虾青素生物合成的诱导与调控、虾青素合成与光合作用及脂类代谢的关系等研究现状。     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis： Effects of Cultivation Parameters

The green alga, Haematococcus pluvlalis Flotow is used as a source of the ketocarotenoid astaxanthin for application in fish aquaculture, pharmaceutical and cosmetic industries. Ceils of the green alga were induced by the application of different light and starvation conditions to evaluate the effect in astaxanthin accumulate. The conditions used for the Induction were high light intensity （170 μmol·m^-2·s^-1）, iron starvation, sulfur starvation and phosphate starvation. The results show that stresses applied in culture, which interfere with cell division, trigger the accumulation of astaxanthin. Notably, sulfur starvation results in a massive accumulation of this commercially important carotenoid.     

不同理化因子对雨生红球藻CG-11碳酸酐酶活性的影响

以雨生红球藻CG-11为实验藻株,探讨在不同CO2、HCO3-、Zn2＋浓度以及pH和氮磷比例条件下,藻细胞的碳酸酐酶活性对这些理化因子的响应。结果表明,通入空气实验组的碳酸酐酶活性最高,为（75.20±1.53）U·mg-1（Chla）,通入5%CO2条件下的碳酸酐酶活性为（9.96±1.43）U·mg-1（Chla）;高浓度HCO3-对碳酸酐酶活性亦具有明显抑制作用,培养液中可溶性无机碳的浓度与碳酸酐酶活性呈负相关;在实验设置的pH范围内,pH9.0时碳酸酐酶活性最高,为（62.32±3.25）U·mg-1（Chla）;适当的氮磷比与Zn2＋浓度显著提高了雨生红球藻CG-11的生长速率,碳酸酐酶的活性亦有明显提高。     

Time- and media-dependent secondary carotenoid accumulation in Haematococcus pluvialis

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.     

雨生红球藻的紫外、激光复合诱变育种

用紫外线和激光复合诱变生产虾青素的雨生红球藻,以适宜条件下的生长速率和亚适宜条件下的虾青素累积能力为筛选指标。结果表明,与原始出发株比,紫外线诱变后,色青累积速率提高37.8％,但生长速率有所下降,紫外线,激光复合诱变结果,生长速率提高11.1%,培养1个月时虾青素累积量提高52.2%。电镜观察结果表明,激光可刺激叶绿体发育,从而改善了紫外线诱变后的生长抑制状况,展示复合诱变是筛选高产虾青素藻株的有效方法。