Evaluation of heterologous α‐amylase production in two expression platforms dedicated for Yarrowia lipolytica: commercial Po1g–pYLSC (php4d) and custom‐made A18–pYLTEF (pTEF)

In view of the constantly increasing demand for cost‐effective, low‐energy and environmentally friendly industrial processes and household care products, enzyme production occupies an essential place in the field of biotechnology. Along with increasing demand for industrial and household care enzymes, the demand for heterologous expression platforms has also increased. Apart from the conventional hosts, e.g. Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, routinely used in heterologous protein expression, the non‐conventional ones have become more and more exploited in this field. Among the available yeast host systems, Yarrowia lipolytica appears to be an attractive alternative. The aim of this study was to compare efficiency of two Yarrowia‐based expression platforms, commercial Po1g–pYLSC and custom‐made A18‐pYLTEF, in expression of an insect‐derived, raw‐starch‐digesting α‐amylase, to select the ‘champion’ system for further studies on this valuable enzyme. Both expression platforms were compared with respect to copy number of the integrated expression cassette/transformed genome, and the recombinant strains performance (Po1g–pYLSC‐derived 4.29 strain, and A18‐pYLTEF‐derived B9 strain) during batch bioreactor cultures. Our results demonstrate that the average number of integration events into the recipient's genome was comparable for both expression systems under investigation, but with varying distribution of the multicopy integrants; and the number of the recombinant gene copies was highly correlated with the acquired amylolytic activity of the strains. Due to severe susceptibility of the recombinant AMY1 polypeptide to native proteases of the custom‐made expression system, the final yield of the enzyme was substantially lower when compared to the commercial Po1g–pYLSC (reaching a maximum level of 142.84 AU/l). Copyright © 2015 John Wiley & Sons, Ltd.     

Cell surface characterization of Yarrowia lipolytica IMUFRJ 50682

In the present work, the surface characteristics of a wild-type strain of Yarrowia lipolytica (IMUFRJ50682) were investigated. Six different methods to characterize cell surfaces--adhesion to polystyrene; hydrophobic interaction chromatography (HIC); microbial adhesion to solvents (MATS) test; zeta potential; microbial adhesion to hydrocarbons (MATH) test; and contact angle measurement (CAM)--were employed to explain the cell surface behaviour of Y. lipolytica (IMUFRJ50682). This Y. lipolytica strain presents significant differences at the cell surface compared with another Y. lipolytica strain (W29) previously reported in the literature. The main difference is related to the higher cell adhesion to non-polar solvents. The proteins present on the cell wall of Y. lipolytica IMUFRJ50682 seem to play an important role in these particular surface characteristics because of the consistent reduction of this yeast hydrophobic character after the action of pronase on its cell wall.     

共轭亚油酸合成相关基因在耶氏解脂酵母中异源表达及活性研究

为了探究植物乳杆菌(Lactobacillus plantarum)中与共轭亚油酸(CLA)生物合成相关的3个基因:(亚)油酸水合酶基因(mcra)、短链脱氢酶/氧化还原酶基因(dh)、乙酰乙酸脱羧酶基因(dc)在耶氏解脂酵母(Yarrowia lipolytica,Y.lipolytica)中异源表达后能否具有活性,利用两个耶氏解脂酵母整合表达质粒(p INA 1269和p INA 1312),将3个基因分别导入耶氏解脂酵母营养缺陷型宿主菌Polf(Ura~-,Leu~-)中,构建了重组菌株。在不同重组菌中添加相应的底物:亚油酸(LA)和10-羟基-顺12-十八碳烯酸(10-HOE),然后对反应体系进行脂肪酸检测,得到基因对应的不同产物:10-HOE和10-氧代-反11-十八碳烯酸(10-oxo-trans 11-octadecenoic acid),证明mcra、dh、dc在耶氏解脂酵母中进行了异源表达并且具有活性。     

Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 Delta strain

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.     

The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1

Cytochromes P450 constitute a superfamily of haem-thiolate mono-oxygenases that are involved in the oxidative metabolism of lipophilic subtrates. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. We have expressed human cytochrome P450 CYP1A1 under the POX2 promoter (pPOX2-CYP1A1) in Y. lipolytica, with or without overproduction of Y. lipolytica CPR expressed under the ICL promoter (pICL-CPR) or the POX2 promoter (pPOX2-CPR). Activity of cytochrome CYP1A1 was analysed by conversion of hydroxyresorufin to resorufin. Strain JMY330 and JMY330-CPR present no activity, the monocopy cytochrome CYP1A1 integrant JMY331 and JMY331-CPR derivatives present an average activity of 32.0 pM/min/dw and 48.3 and 64.6 pM/min/dw for pICL-CPR and pPOX2-CPR, respectively. Increase of CPR expression resulted in about two-fold higher activity. The multicopy 1A1 integrant JMY339 and JMY339-CPR derivatives present an activity of 129 pM/min/dw and 815-1845 pM/min/dw, respectively. Increase of CPR expression resulted in 6.3-12.8-fold higher activity, depending on the CPR transformant. We observed a 50-fold increase of activity between the monocopy integrant JMY331 as compared to the multicopies integrant JMY339-CPR in which CPR was overexpressed.     

重组解脂亚罗酵母合成菜油甾醇

菜油甾醇是一种具有重要生理活性的植物甾醇。为构建合成菜油甾醇的工程菌株,通过敲除解脂亚罗酵母polf菌株中麦角固醇合成基因ERG5,促进麦角-5,7,24-三烯醇的体内积累,进而表达经密码子优化的非洲爪蟾DHCR7基因,使麦角-5,7,24-三烯醇在DHCR7和polf胞内ERG4的共同催化作用下,转化成菜油甾醇,最后利用GC-MS对重组菌株的发酵产物进行检测分析。结果表明,重组菌株polf-ERG5--DHCR7+(PED)合成菜油甾醇的产量达0.485 mg/g(以细胞干重计)。重组解脂亚罗酵母合成菜油甾醇菌株的成功构建,为生物合成法合成植物甾醇提供了新思路。     

Candida rugosa脂肪酶交联酶晶体稳定性研究

交联酶晶体（Ｃｒｏｓｓ－Ｌｉｎｋｅｄ Ｅｎｚｙｍｅ Ｃｒｙｓｔａｌ,ＣＬＥＣｓ）是一种新型的生物催化剂。本研究对Ｃａｎｄｉｄａ ｒｕｇｏｓａ脂肪酶交联酶晶体的制备以及其稳定性进行了探索,制备得到了交联酶晶体不仅保持酶原有的特性,而且提高了酶在极端环境条件（高酸碱度、高温）下的稳定性,并初步研究了交联酶晶体在有机溶液中的稳定性。     

1,13-十三碳二羧酸发酵工艺的研究

以热带假丝酵母(Candidatropicalis)为菌种发酵生产1,13 十三碳二羧酸(DCl3)是现阶段最经济的方法。通过对接种量、补碱工艺、发酵级数和空气流量的优化以及采取中间补加正十三碳烷烃(C13)、延长发酵周期等措施,确定了最佳的DC13发酵工艺,15m3发酵DC13的产量达到850kg,主要原材料正十三碳烷烃的消耗下降了11 6%。     

溶氧对耶氏解脂酵母油脂积累和柠檬酸分泌的影响

产油耶氏解脂酵母能将培养基中过量的碳源转化为油脂储存于细胞内并分泌大量的柠檬酸。在耶氏解脂酵母发酵过程中通过关联搅拌转速和通气量调控培养基中的溶氧含量处于5%、10%、20%、30%和不控制5种水平,来研究溶氧对其油脂积累和柠檬酸分泌的影响。结果表明,随着发酵培养基中溶氧的增加,耶氏解脂酵母细胞内油脂含量和柠檬酸分泌量均有所增加,且不控制培养基中溶氧时,细胞内油脂含量和柠檬酸产量均最高,油脂含量达到细胞干重的11.62%(w/w),柠檬酸产量达到21.0 g/L。培养基中不同的溶氧含量还会影响油脂的脂肪酸组成,高溶氧能够促进油酸含量的增加,溶氧不控制时油酸的含量最高,达到48.62%。本研究为耶氏解脂酵母产脂发酵培养过程中溶氧的控制提供了重要的实验依据。