Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

过表达NADH激酶基因对酿酒酵母乙醇发酵的影响

甘油是酿酒酵母乙醇代谢途径中的主要副产物,降低甘油生成,可以提高乙醇的产率和原料的利用率。以工业酒精酵母单倍体S1(MATa)为研究对象,构建了一个4.5 kb左右的基因敲除突变盒gpd2Δ::PGK1PT-POS5-HyBR,利用醋酸锂转化法转入S1,得到重组菌S3(gpd2Δ::PGK1PT-POS5-HyBR),使得工业酒精酵母在敲除GPD2的同时整合过表达了NADH激酶基因POS5。结果表明,在150 g/L的葡萄糖摇瓶发酵实验中,重组菌S3在不影响菌株生理特性的条件下,乙醇得率(g ethanol/g glucose)比原始菌株S1提高了8%,甘油得率(g glycerol/g glucose)降低了33.64%。本研究证明过表达NADH激酶基因可降低乙醇发酵中副产物甘油的生成并提高乙醇得率。     

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source

Saccharomyces cerevisiae is a widely used host organism for the production of heterologous proteins, often cultivated in glucose-based fed-batch processes. This production system however has many factors limiting the productivity, mainly towards the end of the fermentation. For the optimised production of a Camelid antibody fragment this process was evaluated. In shake flask cultivations, it was found that ethanol has a strong effect on productivity increase and therefore glucose and ethanol fed-batch fermentations were compared. It appeared that specific heterologous protein production was up to five times higher in the ethanol cultivation and could be further optimised. Then the key characteristics of ethanol fed-batch fermentations such as growth rate and specific production were determined under ethanol limitation and accumulation and growth limiting conditions in the final phase of the process. It appeared that an optimal production process should have an ethanol accumulation throughout the feed phase of approximately 1% v/v in the broth and that production remains very efficient even in the last phase of the process. This productivity increase on ethanol versus glucose was also proven for several other Camelid antibody fragments some of which were heavily impaired in secretion on glucose, but very well produced on ethanol. This leads to the suggestion that the ethanol effect on improved heterologous protein production is linked to a stress response and folding and secretion efficiency.     

乙醇耐受性酿酒酵母菌株选育的研究进展

酿酒酵母是工业发酵生产乙醇的重要菌种,但是其发酵产物乙醇对酿酒酵母有明显的抑制作用.选育乙醇耐受性酿酒酵母是克服高浓度乙醇的抑制作用,提高乙醇产量的一条重要途径.本文对近年来国内外选育乙醇耐受性酵母的研究作一综述,旨在为乙醇耐受性酵母的选育提供参考.     

Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway

Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoyl-CoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.     

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.     

树干毕赤酵母和酿酒酵母混合糖发酵产乙醇

以树干毕赤酵母和酿酒酵母为发酵菌株,酸性蒸汽爆破玉米秸秆预水解液和纯糖模拟液为C源,采用固定化酵母细胞的方法,研究了酸爆玉米秸秆预水解液初始pH、N源种类及其浓度、3种发酵模式对树干毕赤酵母戊糖发酵的影响。结果表明:玉米秸秆预水解液适合发酵的初始pH范围为6.0～7.0;1.0 g/L的(NH4)2SO4作为N源,在40 g/L葡萄糖和25 g/L木糖培养基中发酵24 h,糖利用率达到99.47%,乙醇质量浓度为24.72 g/L,优于尿素和蛋白胨作为N源;3种模式的发酵体系中,以游离树干毕赤酵母和固定化酿酒酵母发酵性能最好,糖利用率和乙醇得率分别为99.43%和96.39%。     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

灭活原生质体融合选育木糖、葡萄糖共发酵酿酒酵母工程菌

为了选育高效利用木糖、葡萄糖共发酵,并使乙醇产量有所提高的酿酒酵母工程菌株。以酿酒酵母Saccharomyces cerevisiae W5和休哈塔假丝酵母Candida shehatae 20335为亲本株,确定了双亲株原生质体灭活剂量,并进行原生质体融合获得融合子,用高效液相色谱（HPLC）测定融合子以木糖、葡萄糖单碳源及混合碳源发酵时的乙醇得率。结果表明,获得一株发酵性能优良的融合子HDY2-14,其利用木糖和葡萄糖单碳源发酵的乙醇得率分别为0.213g/g和0.257g/g,混合碳源发酵的乙醇得率为0.310g/g,其中混合碳源乙醇得率比亲本株W5和20335的乙醇得率分别提高了20.2%和15.2%。     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.