Validated Growth Rate-Dependent Regulation of Lipid Metabolism in Yarrowia lipolytica

Given the strong potential of Yarrowia lipolytica to produce lipids for use as renewable fuels and oleochemicals, it is important to gain in-depth understanding of the molecular mechanism underlying its lipid accumulation. As cellular growth rate affects biomass lipid content, we performed a comparative proteomic analysis of Y. lipolytica grown in nitrogen-limited chemostat cultures at different dilution rates. After confirming the correlation between growth rate and lipid accumulation, we were able to identify various cellular functions and biological mechanisms involved in oleaginousness. Inspection of significantly up- and downregulated proteins revealed nonintuitive processes associated with lipid accumulation in this yeast. This included proteins related to endoplasmic reticulum (ER) stress, ER–plasma membrane tether proteins, and arginase. Genetic engineering of selected targets validated that some genes indeed affected lipid accumulation. They were able to increase lipid content and were complementary to other genetic engineering strategies to optimize lipid yield.     

田口设计优化解脂耶罗维亚酵母产油发酵培养基

本研究以粗甘油为碳源通过田口设计对解脂耶罗维亚酵母产油脂的培养基进行优化。首先通过单因素实验得到对结果影响最显著的因素,在单因素实验的基础上利用Minitab 17设计田口式正交实验,对实验中各因素水平的均值和信噪比进行分析。结果表明培养基中的C/N和MgSO₄·7H₂O的浓度对油脂产量有极显著影响,CaCl₂的浓度有显著影响。最佳培养基组成为C/N为100∶1（粗甘油50 g/L,硫酸铵1.08 g/L）,MgSO₄·7H₂O 3 g/L,CaCl₂ 0.2 g/L,KH₂PO₄ 4 g/L。使用最佳培养基配方在28℃180 r/min条件下培养4 d,油脂产量达到1.859 g/L,与田口设计的预测结果 1.782 g/L接近,相对于未优化时油脂产量提高了48.5%。      

Utilization of Whey and Grape Must for Citric Acid Production by Two Yarrowia lipolytica Strains

In this study, utilization of whey and grape must were investigated for citric acid production using Yarrowia lipolytica NBRC 1658 and a domestic strain Y. lipolytica 57. In addition to its use as a sole nutrient source, whey was also fortified with glucose or fructose as well as other nutrients. The best results for citric acid production were obtained in the medium containing whey supplemented with fructose. Maximum citric acid concentrations in this medium were 49.23 and 32.65 g/L for the domestic and NBRC 1658 strains, respectively. In grape must, maximum citric acid concentrations obtained using domestic and NBRC 1658 strains were 32.09 and 10.39 g/L, respectively. Both of the natural nutrient sources were found to be promising for utilization in citric acid production process. A domestic Turkish yeast strain was confirmed to be superior for citric acid production for the first time. This can be targeted for enhancing citric acid production efficiencies from locally available substrates such as whey or grape must.     

Distribution of Mixing Efficiency in a Split-Cylinder Gas-Lift Bioreactor with Immobilized Yarrowia Lipolytica Cells Used for Olive Oil Mill Wastewater Treatment

The distribution of mixing in a split-cylinder gas-lift bioreactor has been investigated for suspensions of immobilized Yarrowia lipolytica cells with particles diameter varying between 3 and 4.2 mm. The results indicated important variation of mixing time on the height of riser or downcomer, as well as different behavior of suspensions flows in these two regions. Therefore, for the riser, the mixing efficiency increases from its bottom to the top, allowing the biocatalyst particles with intermediate size (3.6-mm diameter) reaching the most intense circulation of suspension. The analysis of the suspension flow in the downcomer region revealed that the intermediate positions are associated with the highest mixing mainly for the largest immobilized yeast particles (4.2-mm diameter). In both cases, the influence of aeration on turbulence extent is positive only for air superficial velocity up to 1.05–1.35 × 10^?3 m/s, the magnitude of this effect being correlated with the biocatalyst size and position on the riser or downcomer. By means of the experimental data, mathematical correlations for mixing time have been proposed for each circulation region, taking into consideration both the operational parameters and the distance from the bioreactor bottom. These equations offer a good concordance with the experiment, the average deviation being of 5.82% for the riser and 6.06% for the downcomer zone.     

解脂假丝酵母脂肪酶的纯化及性质研究

通过采用盐析和透析等提纯方法,对解脂假丝酵母(Candidalipolytica)脂肪酶进行了纯化,比活提高了2.69倍,回收率45%.该酶反应的最适pH值为7.5,最适温度为38℃,pH稳定范围为6.0～7.5,温度稳定性较差,以2mol/L的蔗糖作保护剂效果较好.该酶为金属酶,5mmol/L的钙离子有较好的活化效果.     

Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane

Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. Gamma-decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl-L-alpha-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This effect was more important than that of benzyl alcohol, which is known as a fluidizing agent in living cells, and may explain the toxic action of gamma-decalactone in microorganisms.     

Biosynthesis of lipids and organic acids by Yarrowia lipolytica strains cultivated on glucose

The biochemical behavior of wild‐type or genetically modified (presenting decreased expression of intracellular acyl‐CoA oxidases) Yarrowia lipolytica strains cultivated on commercial glucose was studied. Flask nitrogen‐limited cultures were performed favoring the production of organic acids (and potentially the accumulation of lipid). Nitrogen depletion induced secretion of citric acid, while intracellular lipid was not produced in high quantities. Maximum total citric acid up to 49 g/L (yield 0.85 g/g glucose) was produced. In some of the wild‐type strains, an increase of glucose in the medium also induced noticeable production of acetic acid. Increasing the amount of added glucose led to an increase in the total lipid quantity (%) produced, although in the stationary growth phase the concentration of lipid declined, indicating lipid degradation even for the genetically modified strains. Total lipid amount did not exceed the value of 14%, while neutral fractions increased with increase in glucose concentration. In all cases, the total microbial lipids and major lipid fractions were composed of C₁₆ and C₁₈ (principally unsaturated) fatty acids. Finally, in several of the strains cultured in media containing a low glucose concentration, unicellular morphology was observed, while at high glucose concentrations mycelia were predominant.     

食品检验用解脂耶氏酵母菌标准物质制备研究

目的 为满足实验室酵母菌日常检验质量控制需求及实验室间比对,制备解脂耶氏酵母菌检验用标准物质。方法 通过生化、基质辅助激光解吸电离飞行时间质谱及ITS位点测序对研究用菌株进行菌种鉴定确认后,刮取适宜菌苔于保护剂中混匀后冻干,制备10_⁴ CFU/样品浓度的标准物质。参照CNAS-GL003等标准要求进行均匀性检验后,采用单因素方差分析对结果进行评价。将样品放于25 ℃和37 ℃及-20 ℃、4 ℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据国家标准GB 4789.15,将研制的本标准物质加入7类食品基质共20件样品中进行检验,验证真实食品样品中适用性。组织3家实验室,对本标准物质进行协作标定。结果CMCC98025经生化鉴定为解脂耶氏酵母菌,准确度为93%;MALDI-TOF MS鉴定结果为解脂耶氏酵母,分数为2.012;ITS位点测序结果与NCBI Genbank中已有序列比对,匹配最优结果为解脂耶氏酵母Yarrowia lipolytica(Accession number ：CP061015.1;Query Cover：95%;Ident：100%)。均匀性测试结果符合正态分布,通过单因子方差分析计算F_0.05(19,20)=2.137,F值为1.697,F＜F_0.05,符合均匀性要求。25 ℃及37 ℃培养箱中放置7天,标准物质中菌含量仍保持在10_⁴ CFU/样品,可保持稳定。标准物质于4 ℃储存28天及-20 ℃储存90天,菌含量为10_⁴ CFU/样品,复苏率均在91%以上,说明4 ℃放置较短时间及-20 ℃放置较长时间样品稳定。7类食品样品基质中加入本标准物质后进行检验,均可检出,回收率为81.3%。经三家实验室协作标定,本标准物质平均浓度为2.0～3.0×10_⁴ CFU/样品。结论 本研究制备的解脂耶氏酵母菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于食品中解脂耶氏酵母菌定性检验,今后可作为实验室日常检验工作的阳性对照质控样品,也可作为实验室间比对样品发放,以保障日常检验工作结果可靠性,进一步提升检验机构人员的检验水平。