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外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探
杨智宇, 佟天奇, 刘磊, 平文祥, 葛菁萍
中国农学通报. 2020, 36(21): 104-112
外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探
Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae
为验证乙酸作为信号分子的作用,本研究分别将酿酒酵母(Saccharomyces cerevisiae)W141上清液(对照组)、W141-07 (△aldh6)上清液及1.5 g/L乙酸添加至对数生长期的W141发酵液中,检测2,3-BD产量、乙酰乳酸合成酶(ILV2)及2,3-丁二醇脱氢酶(BDH1)酶活。结果表明:当添加1.5 g/L乙酸时,2,3-BD产量、ILV2和BDH1酶活性均达到最高,分别为3.01±0.04 g/L、1.41±0.03 U/mg和0.12±0.002 U/mg,且较其余两组相比差异极显著(P<0.01)。同时,测定3组条件下ilv2(24 h)和bdh1(60 h)基因的表达情况时发现,添加W141-07上清液后,ilv2和bdh1基因的表达量分别下调了31.6%和25.0%;而添加1.5 g/L乙酸后,ilv2和bdh1的表达量均发生上调,分别是对照组的4.38及1.24倍。表明乙酸可作为信号分子驱动相关基因的表达,进而提高2,3-BD产量。
To verify the role of acetic acid as a signal molecule, the supernatant of Saccharomyces cerevisiae W141 (the control group) and W141-07 (△aldh6), as well as 1.5 g/L acetic acid were added into S. cerevisiae W141 fermentation broth at the logarithmic phase. The content of 2,3- butanediol(2,3-BD), the activity of acetolactate synthase (ILV2) and 2,3-BD dehydrogenase (BDH1) were detected. The results showed that under the condition of 1.5 g/L acetic acid, the content of 2,3-BD, the ILV2 and BDH1 enzyme activities reached the highest value, which were 3.01±0.04 g/L, 1.41±0.03 U/mg and 0.12±0.002 U/mg, respectively. The acetic acid addition group was significantly higher than those of supernatant addition groups (P<0.01). At the same time, the expressions of ilv2 (24 h) and bdh1 (60 h) genes under the three conditions were determined. After adding W141-07 supernatant, the expression levels of ilv2 and bdh1 genes were down-regulated by 31.6% and 25.0%, respectively. After adding acetic acid with a final concentration 1.5 g/L, the expression levels of ilv2 and bdh1 were up-regulated, which were 4.38 and 1.24 times that of the control, respectively. These results indicate that acetic acid can act as a signal molecule to drive the expression of related genes, thus to improve 2,3-BD production.
2,3-丁二醇 / 酿酒酵母 / 乙酸 / 荧光定量PCR / 2,3-丁二醇脱氢酶 / α-乙酰乳酸合成酶 {{custom_keyword}} /
2,3-butanediol / Saccharomyces cerevisiae / acetic acid / fluorescent quantitative polymerase chain reaction / 2,3-butanediol dehydrogenase / α-acetolactatesynthase {{custom_keyword}} /
表1 不同地膜覆盖对大棚草莓植株农艺性状的影响 cm |
处理 | 始花期 | 采收末期 | 株高增长量 | 冠幅增长量 | |||
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株高 | 冠幅 | 株高 | 冠幅 | ||||
黑膜(CK) | 20.8 | 40.9 | 35.1 | 46.4 | 14.3 | 5.5 | |
银黑双色膜 | 19.6 | 39.9 | 28.3 | 46.5 | 8.7 | 6.6 |
表2 不同地膜覆盖对大棚草莓品质的影响 |
处理 | 早期 | 后期 | |||||||
---|---|---|---|---|---|---|---|---|---|
可溶性固形物 SSC% | 酸度 | 糖酸比 | 硬度 | 可溶性固形物 SSC% | 酸度 | 糖酸比 | 硬度 | ||
黑膜(CK) | 11.4 | 0.9 | 12.6 | 2.94 | 9.6 | 0.8 | 11.7 | 1.84 | |
银黑双色膜 | 12.5* | 0.9 | 13.7* | 3.25 | 9.8 | 0.7* | 14.8* | 2.07* |
注*表示通过0.05信度的显著性检验。 |
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Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.
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2,3-butanediol is a promising bulk chemical due to its extensive industry applications. The state-of-the-art nature of microbial 2,3-butanediol production is reviewed in this paper. Various strategies for efficient and economical microbial 2,3-butanediol production, including strain improvement, substrate alternation, and process development, are reviewed and compared with regard to their pros and cons. This review also summarizes value added derivatives of biologically produced 2,3-butanediol and different strategies for downstream processing. The future prospects of microbial 2,3-butanediol production are discussed in light of the current progress, challenges, and trends in this field. Guidelines for future studies are also proposed.
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2,3-丁二醇是生物制造产品体系中一种重要的精细化工原料和潜在平台化合物,广泛应用于材料、医药、食品及航空航天等领域。利用生物质可再生资源为原料生产2,3-丁二醇符合当前发展低碳经济的国家需求。本文回顾了生物制造2,3-丁二醇的研究历史,分析了微生物合成2,3-丁二醇的代谢机理,总结了提高生物制造2,3-丁二醇经济性的有效途径,包括廉价原料的替代、菌株选育与遗传改造和发酵过程控制等,并对2,3-丁二醇的各种下游分离过程进行了对比分析;指出今后研究重点应着眼于努力提高生物质的利用效率,同时实现高效的2,3-丁二醇生物转化两方面,并在此基础上开发2,3-丁二醇的系列高值衍生物,以进一步拓展其应用领域。
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总结了不同 2,3-丁二醇立体异构体的生物合成机制, 以及有利于这些立体异构体高效合成的一些策略, 包括构建全细胞催化剂及利用合成生物学技术重建代谢途径等先进方法; 同时指出, 未来的研究重点是进一步利用合成生物学的方法, 以提高不同立体构型 2,3-丁二醇的生物合成能力, 并建立这些异构体高效可行的分离方法.
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Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH(4))(2)HPO(4) were identified as the most significant factors by the two-level Plackett-Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation.
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Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.
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In order to produce 2,3-butanediol (2,3-BD) with a high titer, it is necessary to engineer Saccharomyces cerevisiae by deleting the competing pathway and overexpressing the 2,3-BD biosynthetic pathway. A pyruvate decarboxylase (Pdc)-deficient mutant was constructed and evolved for rapid glucose consumption without ethanol production. Genome re-sequencing of the evolved strain (SOS4) revealed a point mutation (A81P) in MTH1 coding for a transcriptional regulator involved in glucose sensing, unlike the previously reported Pdc-deficient mutant which had internal deletion in MTH1. When alsS and alsD genes from Bacillus subtilis, and endogenous BDH1 gene were overexpressed in SOS4, the resulting strain (BD4) not only produced 2,3-BD efficiently, but also consumed glucose faster than the parental strain. In fed-batch fermentation with optimum aeration, 2,3-BD concentration increased up to 96.2 g/L. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD for industrial applications.
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2,3-丁二醇是克雷伯氏菌发酵产1,3-丙二醇的主要副产物,为减少2,3-丁二醇的产生,利用Red重组技术对克雷伯氏菌2,3-丁二醇合成途径关键酶基因budC和budA进行了敲除。突变株发酵性能实验结果表明,所获得的两株突变株生长性能受到不同程度的影响;budC基因的缺失使菌株1,3-丙二醇产量提高了10%,2,3-丁二醇降低为原来的70%,而budA基因缺失则使菌株无2,3-丁二醇和1,3-丙二醇的产生,但乳酸、琥珀酸、乙醇和乙酸的产量较出发菌株都有明显增长。通过进一步对budC基因缺失菌株主要产物分析,推测在该菌中存在2,3-丁二醇回补途径,这一结果为低副产物克雷伯氏菌的改造提供了新依据。
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【目的】乙酰乳酸合成酶(ALS)是异丁醇生物合成中的关键酶, 实现ALS的高效表达对调控异丁醇代谢途径有重要意义。【方法】根据GenBank中ALS的基因序列(alsS)设计引物, 以枯草芽孢杆菌168基因组DNA为模板通过PCR扩增技术得到目标酶基因, 目的片段全长为1 713 bp。将alsS连接到pET-30a(+)上, 得到重组质粒pET-30a(+)-alsS, 并在Escherichia coli BL2l(DE3)中实现表达。【结果】对表达条件进行了优化, 获得最佳表达条件为: 诱导温度30 °C, 诱导起始菌体OD600为0.6?0.8, 诱导剂IPTG浓度为1?mmol/L, 诱导时间为6 h。表达的乙酰乳酸合成酶大部分以可溶性形式存在于菌体内, 优化后酶活可达到24.4 U/mL, 比优化前提高了7.13倍。经HisTrapTMFF亲和层析后获得电泳纯的ALS, 比活为95.2 U/mg。【结论】ALS的有效表达为在大肠杆菌体内构建异丁醇代谢途径打下了基础。
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童颖佳, 邬文嘉, 彭辉, 等. 微生物合成2,3-丁二醇的代谢工程[J]. 化工学报, 2016,67(7):2656-2671.
2,3-丁二醇(2,3-BD)是一种重要的微生物代谢产物,广泛应用于食品、医药、化工等多个领域。微生物合成2,3-BD的效率不高一直制约着其生物制造工业化进程,应用代谢工程的理论和方法优化微生物的代谢途径有望解决这一问题。本文全面总结了近年来微生物合成2,3-BD研究过程中的菌株改造和构建技术,包括过表达合成途径中的关键酶编码基因、敲除旁路代谢途径关键酶编码基因、应用辅因子工程手段对天然菌株代谢网络进行重新设计和合理改造,以及利用合成生物学技术在模式菌株中构建全新的代谢途径,实现2,3-BD的高效生物合成。最后,本文对未来的研究方向进行了展望,提出了进一步利用先进的合成生物学方法构建高效细胞工厂的指导性建议。
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为了解产酸克雷伯氏菌对木质纤维素水解液中主要抑制物的耐受和代谢,考察了产酸克雷伯氏菌发酵生产2,3-丁二醇 (2,3-butanediol,2,3-BDO) 过程中对3种发酵抑制物乙酸、糠醛和5-羟甲基糠醛 (5-hydroxymethylfurfural HMF) 的耐受以及抑制物浓度的变化,检测了糠醛和HMF的代谢产物。结果表明:产酸克雷伯氏菌对乙酸、糠醛和HMF的耐受浓度分别为30 g/L、4 g/L和5 g/L。并且部分乙酸可作为生产2,3-丁二醇的底物,在0~30 g/L浓度范围内可提高2,3-丁二醇的产量。发酵过程中产酸克雷伯氏菌可将HMF和糠醛全部转化,其中约70% HMF被转化为2,5-呋喃二甲醇,30% HMF和全部糠醛被菌体代谢。研究表明在木质纤维素水解液生产2,3-丁二醇的脱毒过程中可优先考虑脱除糠醛,一定浓度的乙酸可以不用脱除。
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The effective use of xylose may significantly enhance the feasibility of using lignocellulosic hydrolysate to produce 2,3-butanediol (2,3-BD). Previous difficulties in 2,3-BD production include that the high-concentration xylose cannot be converted completely and the fermentation rate is slow. This study investigated the effects of yeast extract, ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and acetic acid on 2,3-BD production from xylose. The central composite design approach was used to optimize the concentrations of these components. It was found that simultaneous addition of yeast extract, Na2EDTA, and acetic acid could significantly improve 2,3-BD production. The optimal concentrations of yeast extract, Na2EDTA, and acetic acid were 35.2, 1.2, and 4.5 g/L, respectively. The 2,3-BD concentration in the optimized medium reached 39.7 g/L after 48 hours of shake flask fermentation, the highest value ever reported in such a short period. The xylose utilization ratio and the 2,3-BD concentration increased to 99.0% and 42.7 g/L, respectively, after 48 hours of stirred batch fermentation. Furthermore, the 2,3-BD yield was 0.475 g/g, 95.0% of the theoretical maximum value. As the major components of lignocellulosic hydrolysate are glucose, xylose, and acetic acid, the results of this study indicate the possibility of directly using the hydrolysate to effectively produce 2,3-BD.
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The efficient fermentation of lignocellulosic hydrolysates in the presence of inhibitors is highly desirable for bioethanol production. Among the inhibitors, acetic acid released during the pretreatment of lignocellulose negatively affects the fermentation performance of biofuel producing organisms. In this study, we evaluated the inhibitory effects of acetic acid on glucose and xylose fermentation by a high performance engineered strain of xylose utilizing Saccharomyces cerevisiae, SXA-R2P-E, harboring a xylose isomerase based pathway. The presence of acetic acid severely decreased the xylose fermentation performance of this strain. However, the acetic acid stress was alleviated by metal ion supplementation resulting in a 52% increased ethanol production rate under 2g/L of acetic acid stress. This study shows the inhibitory effect of acetic acid on an engineered isomerase-based xylose utilizing strain and suggests a simple but effective method to improve the co-fermentation performance under acetic acid stress for efficient bioethanol production.
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