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

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中国农学通报 ›› 2020, Vol. 36 ›› Issue (21) : 104-112. DOI: 10.11924/j.issn.1000-6850.casb20190500171
生物科学

外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探

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Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae

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摘要

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,ilv2bdh1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,ilv2bdh1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。

Abstract

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.

关键词

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

Key words

2,3-butanediol / Saccharomyces cerevisiae / acetic acid / fluorescent quantitative polymerase chain reaction / 2,3-butanediol dehydrogenase / α-acetolactatesynthase

引用本文

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杨智宇 , 佟天奇 , 刘磊 , 平文祥 , 葛菁萍. 外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探. 中国农学通报. 2020, 36(21): 104-112 https://doi.org/10.11924/j.issn.1000-6850.casb20190500171
Yang Zhiyu , Tong Tianqi , Liu Lei , Ping Wenxiang , Ge Jingping. Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae. Chinese Agricultural Science Bulletin. 2020, 36(21): 104-112 https://doi.org/10.11924/j.issn.1000-6850.casb20190500171

0 引言

草莓是多年生常绿草本植物,在植物分类学上属于蔷薇科(Rosaceae)草莓属(Fragaria),在园艺学上属于浆果类果树。随着国内草莓产业的不断发展,逐步形成了集中性生产区域和专业化生产基地,种植规划逐渐趋向合理,草莓品种改良、繁育方式、栽培及管理技术逐步升级,成为优势农产品产业带、精品区的重要组成部分。据统计,2019年浙江省草莓种植面积6253.34 hm2、产量14.25万t、产值超20亿元[1]。草莓生长喜光,在光照充足的环境下,植株生长旺盛,叶片深绿色,发芽发育好,能够获得丰产。种植过密或遮荫时,由于光照不充足,将影响其正常生长[2-3]。同时,草莓属浅根系作物,叶面较大,叶、茎水分的蒸腾强,因此在整个生长期间都要求有比较充足的水分供应。范长娣等[4]分析闽北光、温、水等气象条件对草莓采收期、产量和单果重的影响,表明草莓移栽后一段时间的温度条件是决定其上市期的关键,温度和光温比是影响草莓产量的主导因子,月降水量、湿温比对产量影响次之;同时,温度低、降水少、湿温配合好,草莓月平均单果重较重。而浙北地区属亚热带季风气候,经常性遭遇低温连阴雨天气[5-7],此时正值大棚草莓开花坐果期和主要采收期,温室内光照不足,会使棚内作物受到损伤,严重时会导致作物停止生长。国内研究人员以番茄、黄瓜、菊花等蔬菜花卉等为试材,经过寡照处理后发现植株的株高、茎粗和叶面积生长速率减小[8-10]
塑料地膜是农业发展中重要的农业生产资料,具有提高地温、保持土壤水分、改善土壤理化性状、延长作物生育期、大幅提高作物产量等优势[11-13]。目前生产中常用的地膜类型为黑色PE膜和透明膜,虽然保水性比较好,可以防除杂草和降低棚内湿度,但也存在反光性差等缺点。围绕不同颜色的地膜应用,吕桂菊等[14]用透明地膜、银灰色反光膜和黑色膜在青椒上进行生产试验,结果显示,3种地膜对青椒均有增产作用,并以银灰色膜的增产效果最好,但Vc和可溶性固形物含量有所下;陈淑兰等[15]研究表明,银灰色地膜覆盖番茄前中后期产量都高于对照。因此,通过地膜覆盖来改善棚内小气候环境,提高作物产量,是生产实践中可以研究的方向。为了探究浙北地区冬春季连阴雨时空发生规律,摸清银黑双色地膜在大棚草莓生产中的应用效果,笔者利用2012—2020年11月—次年4月慈溪国家基本气象站逐日平均气温、24 h降水量观测资料以及日照时数观测资料对该地区低温连阴雨的发生特征进行分析,并于2020年9月在浙江省设施农业气象试验站大棚草莓生产中开展了不同种类地膜的应用效果对比试验,研究不同种类地膜覆盖对草莓植株生长指标及果实发育的影响,为大棚草莓低温寡照灾害防御及设施生产优化提供理论依据。

1 材料与方法

1.1 试验地概况

试验于2020—2021年在慈溪市白沙路街道浙江省设施农业气象试验中心试验基地开展。试验田块地势平坦,土壤类型为壤土,肥力中等,排灌方便,前作为芹菜。试验大棚为标准钢管大棚,南北走向,棚宽8 m,顶高3.2 m,棚长40 m,面积320 m2

1.2 试验材料

供试草莓品种为本地主栽品种‘红颊’。供试地膜分别为银黑双色(上层为银灰色,下层为黑色)和黑色,材质均为聚乙烯,银黑双色地膜厚度为1.2丝,宽度为1 m,黑色地膜厚度为1丝,宽度为1 m,均购至慈溪市浒山创新薄膜经营部。

1.3 试验设计

试验采取大区对比法,在两个规格一致的塑料大棚内,分别做2个处理设计。其中处理1全棚覆盖常规黑膜,处理2全棚覆盖银黑双色膜(银色面对外)。每个处理设置3个小区,随机区组排列,每个小区种植草莓40株。供试草莓于2020年9月8日定植于塑料大棚中,2020年11月9日覆盖地膜,采用高垄栽培,垄高40 cm,垄底宽60 cm,每垄2行,行株距为15 cm×20 cm。两个大棚田间管理操作相同。

1.4 调查方法

1.4.1 气象数据来源

农田小气候观测设备位于两个塑料大棚中部,分别在0.5 m和1.5 m高度设置一个温湿度传感器和太阳总辐射仪,同时在地膜下10 cm处设置温、湿度探头1个,代表浅层土温。温湿度传感器型号DHC2型,太阳总辐射仪型号FS-S6A。数据采集频率为20 s/次,存储每30 min的平均值。棚外数据来源于距试验田100 m的慈溪市国家基本气象站2012—2021年所观测的气象资料,主要包括逐日气温、降水量和日照时数。

1.4.2 株高

在植株进入采收盛期后,分别在试验小区中随机选取10株,用直尺测量株高(植株基部到最高叶片的自然高度)、冠幅(植株冠丛最大幅度之间的距离),重复3次,取平均值。

1.4.3 叶片SPAD值

在植株进入采收盛期后,每隔7 d测定叶片SPAD值。每个小区随机选取5株健壮植株,选择中心展开叶往外数第三叶的中心小叶3个点位,用SPAD-502Plus叶绿素含量测定仪(日本柯尼卡)测量,取平均值作为各叶片的SPAD值[16]

1.4.4 产量和品质

果实成熟后,每个小区随机选取5株作为固定观测样本,对成熟果实进行随熟随采,记目测观察果型、果色、风味。用游标卡尺测量果实横径、纵径。用电子天平测量单果重。用PAL-1型数显糖度计(日本爱宕株式会社)测量可溶性固形物(SSC%)含量。用GMK-835F型水果酸度计测量果实酸度。用HLY-YD5数显果实硬度计测量果实硬度。

1.5 研究方法

1.5.1 连阴雨寡照指标

以国家基本气象站单个站点连续3 d或3 d以上降水量≥0.1mm,其中允许1 d无降水,且日照时数≤2 h,连续2 d无降水为过程结束标志,则定义该测站的1次连阴雨过程[17]

1.5.2 连阴雨发生频次

公式见式(1)。
F=20122020Vi9
(1)
式中F为2012—2020年11月—次年4月慈溪市大棚草莓花果期连阴雨发生频次;Vi为发生连阴雨的次数。

1.5.3 连阴雨强度

连阴雨强度即连阴雨过程降水量与过程日数之比,公式见式(2)。
K=1nAin
(2)
式中,K为连阴雨强度;Ai为日降水量;n为连阴雨过程日数。

1.6 数据处理

数据统计使用Excel 2010软件,数据分析使用SPSS Statistics 27统计分析软件,采用单因素ANOVA进行显著性检验,显著性水平为P<0.05。

2 结果与分析

2.1 2012—2020年慈溪市连阴雨寡照发生频率分布特征

根据1.5.1对连阴雨寡照的定义,得到2012—2020年11月—次年4月慈溪市连阴雨寡照发生频率统计(图1)。由此可知,2012—2020年慈溪市大棚草莓开花坐果期(11月—次年4月)连阴雨共出现86次,概率为17.7%(图1)。连阴雨发生次数最多为11月,最少为4月。连阴雨≥7 d累计出现27次,持续10 d以上的长连阴雨出现11次,其中7 d以上的连阴雨出现最多的是11月,其次是1月。2019年12月—1月出现了长达29 d的连阴雨过程。整体来看,慈溪大棚草莓花果期连阴雨发生次数呈微下降趋势,线性趋势系数为 -0.005。
图1 2012—2020年11月—次年4月逐月连阴雨发生次数

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2.2 2012—2020年慈溪市连阴雨寡照发生强度分布特征

图2给出了2012—2020年11月—次年4月慈溪市连阴雨寡照发生强度分布特征,由此可知,在此期间慈溪市大棚草莓花果期连阴雨强度平均为6.3。2018—2020年大棚草莓花果期连阴雨强度整体处在高位区,其中2018年11月—2019年4月连阴雨强度值最大,为8.33。整体来看,慈溪大棚草莓花果期连阴雨发生强度呈增强趋势,线性趋势系数为0.0264。
图2 2012—2020年11月—次年4月逐月累计降水量和连阴雨强度

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2.3 不同地膜覆盖温室内光温环境的变化

图34给出了不同地膜覆盖平均浅层地温和棚内太阳总辐射随日期变化情况。由此可知,在草莓全生育期,大棚内地温呈现先降后升的趋势,定植后,在未覆盖地膜的阶段,两个处理地温基本保持一致,2020年11月9日覆盖地膜后,银黑双色膜处理组浅层地温整体低于黑膜覆盖处理组,平均气温低0.4℃;随着入春外界日平均气温上升到10℃以上,2021年3月10日起,银黑双色膜处理组浅层地温则高于黑膜覆盖处理组0.5℃;整体来看,在气温较低的冬春季,两种地膜均能有效提升和保持浅层土温至10℃以上。从棚内太阳总辐射变化趋势来看,覆盖地膜后,银黑双色膜处理组太阳总辐射整体高于黑膜覆盖组,平均值高9.1 W/m2。由此说明在气温较低的冬季,银黑双色膜的增温效果不及黑膜,但是由于银色涂层能强烈反射太阳辐射,提高棚内太阳总辐射值,可以起到增加光效的作用。
图3 不同地膜覆盖平均浅层地温随日期的变化

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图4 不同地膜覆盖棚内太阳总辐射随日期的变化

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2.4 不同地膜覆盖对大棚草莓植株农艺性状的影响

表1给出了不同地膜覆盖草莓植株农艺性状对比,由此可知,使用黑色地膜植株在始花期和采收末期的株高均高于使用银黑双色膜。整个生育期,使用黑色地膜株高增长量14.3 cm,而使用银黑双色地膜,植株株高增长量8.7 cm。从冠幅指标来看,使用黑膜和银黑双色膜区别不大。
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
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

2.5 不同地膜覆盖对大棚草莓叶片SPAD值的影响

图5给出了进入采收期后,不同地膜覆盖大棚草莓叶片SPAD值变化趋势。由图可知,随着生育进程的发展,不同地膜覆盖草莓叶片SPAD值变化趋势较为一致,呈现明显下降后稳步提升的过程。2021年1月22日—2月2日、2021年2月25日—3月8日出现2次明显的连阴雨过程,通过对叶片SPAD值连续测定显示,在此期间,银黑双色膜覆盖处理的叶片叶绿素值要高于黑膜处理组,特别是2月下旬出现的连阴雨过程,在覆盖银黑双色膜棚内的植株叶片SPAD值较为稳定,而在覆盖黑膜棚内,植株叶片SPAD值有明显下降的趋势。说明在低温寡照环境下,银黑双色膜对植株叶片叶绿素的合成有促进作用。
图5 不同地膜覆盖对大棚草莓叶片SPAD值的影响

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2.6 不同地膜覆盖对大棚草莓产量的影响

图6~7给出了不同地膜覆盖对大棚草莓产量以及果实大小、单果重等影响产量形成的指标统计,由此可知,在生产早期(12月—次年2月),银黑双色膜处理草莓平均产量3629.4 kg/hm2,黑膜覆盖处理草莓平均产量2951.1 kg/hm2,增产可达20%;到了生产后期,两种处理产量水平较为一致。从全期来看,银黑双色膜处理可以提高大棚草莓的产量和产值。而从草莓果型来看,不同地膜覆盖对果实横径有一定影响,银黑双色膜处理对横径的增长有一定的促进作用,而对果实纵径则没有明显影响。
图6 不同地膜覆盖对大棚草莓小区产量的影响

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图7 不同地膜覆盖对大棚草莓果实横、纵径的影响

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2.7 不同地膜覆盖对大棚草莓品质的影响

表2给出了不同地膜覆盖对大棚草莓品质指标的统计。由表可知,生产早期银黑双色膜处理草莓可溶性固形物显著高于CK处理,果实酸度与CK无明显差别,生产后期银色双色膜处理组草莓酸度显著低于CK处理。在整个生产期,银黑膜处理组草莓糖酸比均显著高于CK,说明银黑双色膜处理有助于增加糖酸比,提高果实风味。从果实硬度来看,银黑双色膜处理果实硬度更高,更有助于商品化运输。整体来看,大棚覆盖银黑双色膜可以起到促进果实品质提高的作用。
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。

3 结论与讨论

通过统计分析,2012—2020年慈溪市大棚草莓开花坐果期(11月—次年4月)连阴雨发生概率为17.7%,发生次数呈微下降趋势,线性趋势系数为 -0.005,连阴雨强度平均为6.3,发生强度呈增强趋势,线性趋势系数为0.0264。连阴雨发生次数最多为11月,12月次之,最少为4月;7 d以上的连阴雨出现最多的是11月,其次是1月。而11—12月为草莓头茬果关键的开花坐果期,连阴雨寡照直接影响着草莓的早期产量和品质,会造成在草莓实际生产中植株生长减缓、产量品质下降、发病率提高等情况。
此次实验表明,银黑双色地膜与传统黑色地膜具有相似的增温效果,能够有效提升大棚内浅层地温,保证草莓在冬春季的正常生长。虽然银黑双色地膜对地温的提升作用不及黑色地膜,但是表面的银色涂层能强烈反射太阳辐射,提高棚内太阳总辐射值,可以起到增加光效的作用。而棚内太阳总辐射的增加,可以帮助叶片叶绿素的合成,特别是出现连阴雨天气过程时,低温寡照的环境会减缓植株生长,银膜相较于黑膜能有效促进植株叶片叶绿素的合成,提高叶片SPAD值。叶片是植物进行光合作用的重要场所,光温条件将直接影响叶绿体色素的合成以及光合速率的快慢[18-19],从而对植物的生长发育造成重要影响。有研究表明,低温寡照胁迫下黄瓜叶片光合作用的减弱,是由于黄瓜叶片光合色素含量下降、气孔导度下降、光系统受损,致使叶片捕获的光能减少、气体交换受阻、用于光化学反应的能量减少而引起的[20],这与此次试验结果相一致。
从草莓产量来看,在关键的12月—2月,银黑双色膜处理草莓平均产量3629.4 kg/hm2,黑膜覆盖处理草莓平均产量2951.1 kg/hm2,增产可达20%。而入春后,随着外界气温升高,光照逐渐充足,银黑膜对产量的促进影响不再明显。另外,从草莓品质指标来看,覆盖银黑双色膜,有助于提高草莓果实糖酸比和果实硬度,果实风味更佳,也有助于商品化运输。
因此,在生产上,可以采用覆盖银黑双色地膜,来改善大棚内小气候环境,达到草莓增产增收的作用。目前,慈溪市坎墩街道农创园已开展银黑双色地膜在大棚草莓生产中的应用,得到农户的较好反响。银黑双色地膜的应用,可以推广到大棚番茄、西瓜、葡萄等其他作物,积极应对浙北地区较为频发的连阴雨寡照天气,以达到进一步提升农作物产量和品质的作用。同时,如何应用数值预报产品定量预测连阴雨天气以及草莓如何根据气象条件科学指导果农进行栽培技术管理,以减轻不利气象因素对草莓产量和品质的影响,是可以进一步深入研究的方向。

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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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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.

基金

国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492)
黑龙江省教育厅重点项目“利用肺炎克雷伯氏菌发酵生产2,3-丁二醇及机理探讨”(HDJCCX-2016Z05)

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