Increased ethanol production from glycerol by Saccharomyces cerevisiae strains with enhanced stress tolerance from the overexpression of SAGA complex components

During the industrial production of ethanol using yeast, the cells are exposed to stresses that affect their growth and productivity; therefore, stress-tolerant yeast strains are highly desirable. To increase ethanol production from glycerol, a greater tolerance to osmotic and ethanol stress was engineered in yeast strains that were impaired in endogenous glycerol production by the overexpression of both SPT3 and SPT15, components of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex. The engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) formed significantly more biomass compared to the strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), and both engineered strains displayed increased biomass when compared to the control YPH499 fps1Δgpd2Δ (pESC-TRP) strain. The trehalose accumulation and ergosterol content of these strains were 2.3-fold and 1.6-fold higher, respectively, than the parent strains, suggesting that levels of cellular membrane components were correlated with the enhanced stress tolerance of the engineered strains. Consequently, the ethanol production of the engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) was 1.8-fold more than that of strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), with about 8.1g/L ethanol produced. In conclusion, we successfully established that the co-expression of SPT3 and SPT15 that improved the fermentation performance of the engineered yeast strains which produced higher ethanol yields than stress-sensitive yeast strains.     

Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol‐3‐phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4‐fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC‐TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4‐fold compared with 3.6% for the wild‐type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3‐fold improvement, compared with 3.6% for the wild‐type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343–347. © 2012 Wiley Periodicals, Inc.     

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38?±?0.01 g g^?1 xylose consumed) was improved from that of the control strain (0.31?±?0.01 g g^?1 xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1–MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1–MDH2–PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.     

高效合成番茄红素酿酒酵母菌株的构建

番茄红素作为一种高附加价值的萜类化合物已受到国内外研究者的广泛关注。首先对酿酒酵母Saccharomyces cerevisiae模式菌株S288c和YPH499合成番茄红素的能力进行分析比较,结果表明YPH499更适合作为底盘细胞用于番茄红素的合成。随后比较组成型启动子GPDpr、TEF1pr和诱导型启动子GAL1pr、GAL10pr对番茄红素合成的影响,结果发现以GPDpr、TEF1pr作为番茄红素合成途径基因crtE、crt B和crtI的启动子,摇瓶发酵60 h后,番茄红素产量为15.31 mg/L;以GAL1pr和GAL10pr为启动子时,其产量为123.89 mg/L,提高8.09倍。继续改造甲羟戊酸(MVA)途径,过量表达N-末端截短的关键酶基因t HMG1(3-羟基-3-甲基戊二酸单酰辅酶A还原酶),番茄红素产量为265.68 mg/L,单位菌体产量72.79 mg/g。文中所设计构建的异源表达番茄红素合成途径的酿酒酵母菌株单位细胞产量高,可以进一步改造和优化后用于番茄红素的工业化生产。     

Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ?GPD1?GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.     

Metabolic Engineering of a Glycerol-Oxidative Pathway in Lactobacillus panis PM1 for Utilization of Bioethanol Thin Stillage: Potential To Produce Platform Chemicals from Glycerol

Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production.     

Fatty acid ethyl esters, nonoxidative ethanol metabolites, synthesis, uptake, and hydrolysis by human platelets

The consumption of alcohol is known to have both positive and negative effects on the functioning of the cardiovascular system in general, and on platelet function in particular. Fatty acid ethyl esters (FAEEs) are non-oxidative metabolite of ethanol that may mediate the ethanol effect on platelet function leading to either bleeding or clotting. The aim of the current study was to investigate the synthesis, uptake, and hydrolysis of FAEEs by human platelets. Isolated platelets were incubated with ethanol for various times, and FAEE synthesis were measured by gas chromatography mass-spectrometry (GC-MS). In addition, platelets were incubated with (14)C-ethyl oleate, and FAEE uptake and hydrolysis were measured. There was significant synthesis of FAEEs by human platelets within 30 min of exposure to ethanol. The major FAEE species formed by human platelets exposed to ethanol were ethyl palmitate and ethyl stearate. FAEE uptake by human platelets showed maximum uptake by 60 s. The majority of FAEEs (50-80%) incorporated into platelets remained intact for up to 10 min. FAEE hydrolysis led to an increase in free fatty acids, with minimal subsequent esterification of the free fatty acids into phospholipids, triglycerides, and cholesterol esters. These studies show that FAEEs, non-oxidative metabolite of ethanol, can be incorporated into, synthesized, and hydrolyzed by human platelets.     

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.     

Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae

Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost of reduced glycerol yield have most often been based on deletion of either one or two isogenes (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1Δ gpd2Δ double deletion strain produced zero glycerol but showed an osmosensitive phenotype and abolished anaerobic growth. Our current approach has sought to generate "intermediate" phenotypes by reducing both isoenzyme activities without abolishing them. To this end, the GPD1 promoter was replaced in a gpd2Δ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1Δ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1Δ gpd2Δ double-deletion strain, these strains were able to completely ferment the sugars under quasi-anaerobic conditions in both minimal medium and during simultaneous saccharification and fermentation (SSF) of liquefied wheat mash (wheat liquefact). This result implies that our strains can tolerate the ethanol concentration at the end of the wheat liquefact SSF (up to 90 g liter(-1)). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type.     

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.     

spt15突变基因对酿酒酵母木糖利用的影响

利用基因工程手段得到重组菌YPH499-3中的spt15有效突变基因,通过表达载体pYX212转化入酿酒酵母原始菌株YPH499中,重新获得酿酒酵母重组菌株。对其性状进行研究,结果表明该菌株能有效利用木糖并共发酵木糖和葡萄糖。在30oC、200r/min,发酵72h时,50g/L木糖的利用率为82.0%,乙醇产率为28.4%;当木糖和葡萄糖以质量比1:1混合发酵时,木糖和葡萄糖的利用率分别为80.4%和100%,乙醇产率为31.4%;同时发现木糖醇的含量极低。从而验证了有效突变基因spt15-10对酿酒酵母共发酵木糖和葡萄糖产酒精的影响。     

Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase

An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose.     

Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.     

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 h when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 h. We also engineered a E. coli strain to produce 24 mg/L of prenol in an ethanol-containing medium, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

Overproduction of free fatty acids in E. coli: implications for biodiesel production

Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。