High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

Mechanism of ethyl acetate synthesis by Kluyveromyces fragilis

Abstract The enzymes implicated in ethyl acetate synthesis and the catabolism of ethanol by Kluyveromyces fragilis were investigated under varying growth conditions. The culture was grown continuously to D = 0.25 h⁻¹ on diluted whey permeate. The results showed that ethyl acetate synthesis by Kluyveromyces fragilis is catalysed by both an esterase and an alcohol acetyltransferase. The esterase is a constitutive enzyme, while alcohol acetyltransferase is inducible. The catabolism of ethanol by Kluyveromyces fragilis resulted in production of ethyl acetate, acetate and acetaldehyde. The glyoxylic shunt is totally inactive in these conditions. The production of acetaldehyde is only governed by an alcohol dehydrogenase.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: studies of the ester stripping

Kluyveromyces marxianus is capable of converting lactose into ethyl acetate offering a chance for an economical reuse of whey. The microbial formation of ethyl acetate as a bulk product calls for an aerobic process and, thus, the highly volatile ethyl acetate is discharged from the aerated bioreactor. This stripping process was modeled and investigated experimentally. The stripping rate was proportional to the gas flow and nearly independent of the stirring rate since the stripping was governed by the absorption capacity of the exhaust gas rather than the phase transfer. Cooling the exhaust gas did not noticeably influence the stripping. One batch experiment is presented in detail to demonstrate the formation of ethyl acetate by K. maxianus DSM 5422 on whey. Further batch experiments showed that a substantial formation of ethyl acetate only occurred when the yeast growth was limited by a lack of trace elements. The highest product yield observed was 0.25 g ethyl acetate per g lactose which is nearly 50% of the theoretical maximum.     

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.     

Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxyglucose-resistant mutant of Kluyveromyces marxianus

Fourteen lactose-fermenting strains of Kluyveromyces marxianus , including its anamorph, Candida kefyr , were grown in two media containing 20% (w/v) sugar as either beet molasses or cheese whey. Strain NBRC 1963 of K. marxianus converted sucrose and lactose to ethanol in both media most efficiently. However, ethanol was produced from sucrose and not from lactose by strain NBRC 1963 in the medium containing equal amounts of sugar from beet molasses and cheese whey. The spontaneous mutants resistant to 2-deoxyglucose in the minimal medium composed of galactose as the sole carbon source were isolated from strain NBRC 1963. Among them, strain KD-15 vigorously produced ethanol in the media containing beet molasses, cheese whey, or both. The mutant strain KD-15 was insensitive to catabolite repression, as shown by the observation that β-galactosidase was not repressed in the presence of sucrose from beet molasses.     

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

马克斯克鲁维酵母的木糖和阿拉伯糖发酵

马克斯克鲁维酵母作为非常规酵母在燃料乙醇发酵中受到人们越来越多的关注。马克斯克鲁维具有天然的发酵戊糖的能力,但不同菌株的发酵能力存在较大差异。本研究比较了3株马克斯克鲁维菌株Kluyveromyces marxianus 9009/1911/1727(K.m 9009/1911/1727)在不同温度下的木糖和阿拉伯糖的发酵性能差异,结果发现不同发酵温度下,3株菌在耗糖速率、糖醇产率均表现出了显著的差异。菌株K.m 9009和K.m 1727在40℃下的发酵性能均优于30℃,这充分体现了马克斯克鲁维酵母的高温发酵优势。针对发酵差异,采用PCR方法获得3个不同菌株的戊糖代谢途径中的5种关键代谢酶(XR、XDH、XK、AR和LAD)的基因序列,并利用Clustalx 2.1进行了序列比对。结果显示3株菌的相关基因与文献中报道的1株克鲁维酵母的相应关键酶氨基酸编码序列相似性达98%以上,并且差异的氨基酸不在酶的关键位点处。在此基础上,通过Real-time实验,对木糖发酵差异最为明显的K.m 1727和K.m 1911的木糖代谢过程4个关键酶(XR、XDH、XK和ADH)的基因表达量进行测定,其结果显示对于耐热菌株K.m 1727,XDH和XK基因表达量低是导致木糖代谢过程中木糖醇积累、乙醇产量低的主要原因。最后,将所测得的马克斯克鲁维酵母的戊糖代谢关键酶序列与其他不同种属相比对,确定了其木糖和阿拉伯糖代谢途径,为进一步利用代谢工程方法提高戊糖发酵性能奠定了基础。     

Exponential growth rates of species of the yeast genus Kluyveromyces

Doubling times were measured during exponential growth of 19 strains belonging to 10 of the 17 species of the yeast genus Kluyveromyces. Growth was in shaken aerobic batch culture at 25°C, in a chemically defined medium with d-glucose as sole carbon source. Doubling times were strikingly uniform, being mainly between 2 and 3.5 h.     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation

The ability of Kluyveromyces marxianus to convert lactose into ethyl acetate offers a chance for an economic reuse of whey. Former experiments with K. marxianus DSM 5422 proved limitation of growth by iron (Fe) or copper as a precondition for significant ester synthesis. Several aerobic batch and chemostat cultivations were done with whey-borne media of a variable Fe content for exploring the effect of Fe on growth, the Fe content of biomass, and metabolite synthesis. At low Fe doses, Fe was the growth-limiting factor, the available Fe was completely absorbed by the yeasts, and the biomass formation linearly depended on the Fe dose governed by a minimum Fe content in the yeasts, x _Fe,min. At batch conditions, x _Fe,min was 8.8???g/g, while during chemostat cultivation at D?=?0.15?h^?1, it was 23???g/g. At high Fe doses, sugar was the growth-limiting factor, Fe was more or less absorbed, and the formed biomass became constant. Significant amounts of ethyl acetate were only formed at Fe limitation while high Fe doses suppressed ester formation. Analysis of formed metabolites such as glycerol, pyruvate, acetate, ethanol, ethyl acetate, isocitrate, 2-oxoglutarate, succinate, and malate during chemostat cultivation allowed some interpretation of the Fe-dependent mechanism of ester synthesis; formation of ethyl acetate from acetyl-SCoA and ethanol is obviously initiated by a diminished metabolic flux of acetyl-SCoA into the citrate cycle and by a limited oxidation of NADH in the respiratory chain since Fe is required for the function of aconitase, succinate dehydrogenase, and the electron-transferring proteins.     

一个外切菊粉酶基因的克隆表达及酶学性质

从马克斯克鲁维酵母（Kluyveromycesmarxianus）DSM5418中克隆出外切菊粉酶（INU）的成熟肽编码区域,在毕赤酵母（Pichiapastoris）GS115中实现了高效分泌表达,体积酶活力达到15．27U／mL,进一步对重组酶进行了纯化与表征。经过（NH4）2SO4沉淀、透析和分子筛过滤后,得到了纯度大于95％的纯化重组酶,SDS-PAGE分析发现INU的表观相对分子质量为9．0×10^4,大于理论预测值6．0×10^4。纯化酶液的表征结果表明,INU的最适温度和最适pH分别为55℃和5．0,在此条件下INU对菊粉的K。值和比酶活分别为1．90mmol／L和433．86U／mg,对蔗糖的K。值和比酶活分别为27．81mmol/L和1249．49U／mg,I/S值为0．34;HPLC分析表明,INU酶解菊粉的产物由果糖和葡萄糖组成;金属离子Mn2＋、Fe3｜、K｜和Co2＋对酶有促进作用,而Zn2＋、Cu2＋、Ni2＋、SDS和EDTA对酶活力有不同程度的抑制作用。     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation

Kluyveromyces marxianus is able to transform lactose into ethyl acetate as a bulk product which offers a chance for an economical reuse of whey-borne sugar. Ethyl acetate is highly volatile and allows its process-integrated recovery by stripping from the aerated bioreactor. Extensive formation of ethyl acetate by K. marxianus DSM 5422 required restriction of yeast growth by a lack of trace elements. Several aerobic batch processes were done in a 1-L stirred reactor using whey-borne culture medium supplemented with an individual trace element solution excluding Mn, Mo, Fe, Cu, or Zn for identifying the trace element(s) crucial for the observed ester synthesis. Only a lack of Fe, Cu, or Zn restricted yeast growth while exclusion of Mn and Mo did not exhibit any effect due to a higher amount of the latter in the used whey. Limitation of growth by Fe or Cu caused significant production of ethyl acetate while limitation by Zn resulted in formation of ethanol. A lack of Fe or Cu obviously makes the respiratory chain inefficient resulting in an increased mitochondrial NADH level followed by a reduced metabolic flux of acetyl-SCoA into the citrate cycle. Synthesis of ethyl acetate from acetyl-SCoA and ethanol by alcoholysis is thus interpreted as an overflow metabolism.     

耐镉马克思克鲁维酵母重金属镉吸附特性的研究

菌株YS-K1是从镉污染土壤中筛选获得的一株耐镉马克思克鲁维酵母Kluyveromyces marxianus YS-K1,对其镉吸附特性研究结果表明,该菌对镉具有良好的耐受性和吸附性,即使在镉浓度高达140mg/L的培养基中也能生长,当镉浓度在30mg/L以下时,24h后能吸附溶液中90%以上的镉,且最佳吸附条件为pH6.0,温度30℃。该菌所吸附的镉大部分集中在细胞壁上,占总吸附镉的76%,细胞膜上的占9%,细胞质中的占15%;能量抑制剂2,4-二硝基苯酚(DNP)和二环己基碳二亚胺(DCC),分别能降低菌株15%和22%的镉吸附量。在Kluyveromyces marxianus YS-K1细胞中,锌离子与镉离子有共用的结合位点,锌离子不仅会与镉离子竞争细胞表面的结合位点,减少其对镉的吸附量,而且也会与镉离子竞争进入细胞的通道位点,减少其对镉的积累量,同时能部分恢复镉离子对菌株生长的抑制作用。     

Growth and beta-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure

AIMS: To investigate the effect of total air pressure raise on cell growth and intracellular beta-galactosidase activity in batch cultures of Kluyveromyces marxianus CBS 7894. METHODS AND RESULTS: A pressurized bioreactor was used for K. marxianus batch cultivation under increased air pressure from 1.2 to 6 bar. Under these conditions no inhibition of cell growth was observed. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization led to an enhancement of the cell growth rate obtained at atmospheric pressure without aeration. The specific beta-galactosidase productivity increased from 5.8 to 17.0 U gCD-1 h-1 using a 6-bar air pressure instead of air at atmospheric pressure. The antioxidant enzyme superoxide dismutase (SOD) was slightly induced by the air pressure raise, which indicates that the defensive mechanisms of the cells can cope with an air pressure up to 6 bar. CONCLUSIONS: These experiments showed that the increase of air pressure up to 6 bar is an alternative to other methods of preventing the oxygen limitation and can be applied in the beta-galactosidase production by K. marxianus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results here reported proved that, in what biological aspects are concerned, it is possible to use the air pressure increase as an optimization parameter of beta-galactosidase production in high-density cell cultures of K. marxianus strains.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

Metals sorption from aqueous solutions by Kluyveromyces marxianus: Process optimization,equilibrium modeling and chemical characterization

The dead Kluyveromyces marxianus biomass, a fermentation industry waste, was used to explore its sorption potential for lead, mercury, arsenic, cobalt, and cadmium as a function of pH, biosorbent dosage, contact time, agitation speed, and initial metal concentration. The equilibrium data fitted the Langmuir model better for cobalt and cadmium, but Freundlich isotherm for all metals tested. At equilibrium, the maximum uptake capacity (Qmax) was highest for lead followed by mercury, arsenic, cobalt, and cadmium. The RL values ranged between 0–1, indicating favorable sorption of all test metals by the biosorbent. The maximum Kf value of Pb showed its efficient removal from the solution. However, multi-metal analysis depicted that sorption of all metals decreased except Pb. The potentiometric titration of biosorbent revealed the presence of functional groups viz. amines, carboxylic acids, phosphates, and sulfhydryl group involved in heavy metal sorption. The extent of contribution of functional groups and lipids to biosorption was in the order: carboxylic>lipids>amines>phosphates. Blocking of sulfhydryl group did not have any significant effect on metal sorption.     

菊粉酶基因的异源表达、分离纯化及酶学性质

为进一步提高菊粉酶在生物技术领域的应用,研究了来源于马克斯克鲁维酵母Kluyveromyces marxianus YX01的菊粉酶性质。通过在毕赤酵母GS115宿主细胞中异源表达该菊粉酶基因(inu),获得了一种外切型菊粉酶,经聚丙烯酰胺凝胶电泳(SDS-PAGE)验证其分子量为86.0 k Da。进一步在该菊粉酶上增加6个His标签,采用聚乙二醇(PEG)20 000透析浓缩和Ni-NTA Agarose静态亲和吸附作用的方法,完成菊粉酶的分离纯化,纯化倍数和酶回收率分别为3.6和33.1%。比较发现粗酶液与纯酶的酶学性质相似,且菊粉酶的最适反应温度为60℃,最适p H值为4.62,并测得该酶的Km和Vmax值,以菊粉为底物时,Km和Vmax值分别为80.53 g/L和4.49 g/(L·min);以蔗糖底物时,Km和Vmax值分别为183.10 g/L和20.20 g/(L·min)。金属离子Mn2+、Ca2+、Cu2+、Zn2+和Fe2+对酶活力具有不同程度的抑制作用,其中Cu2+、Zn2+和Fe2+的抑制作用最为显著。这些研究为进一步提高菊粉酶在工业化的应用奠定了基础。     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000