本发明涉及一种根皮素的生物合成方法，具体的说为一种酿酒酵母发酵生产根皮素的方法。背景技术：根皮素分子式：c15h14o5；英文名称：phloretin；cas：60-82-2；分子量：274.28；物理性质：易溶于甲醇、乙醇和丙酮，微溶于氯仿、难溶于水、石油醚和苯，方棱形或片状结晶(乙醇或甲醇)。根皮素是国外新近研究开发出来的一种新型天然皮肤美白剂，继熊果苷又一天然美容产品，主要分布于苹果、梨等多汁水果的果皮及根皮枝叶中。其主要用作皮肤天然美白剂，它能抑制酪氨酸酶活性、黑色素活性，对各种皮肤色斑有淡化作用。据最新研究发现其还有抗炎、免疫抑制，心血管保护等作用。全球每年用量不少于20吨，需求每年以20％速率增加，具有较大市场需求。目前，现有提取根皮素的工艺，有酸水解、酶解和直接提取。实际生产中多以柚皮苷为原料，兰尼镍为催化剂，在氢氧化钠溶液中，催化氢化合成柚皮苷二氢查耳酮，然后在盐酸溶液中，柚皮苷二氢查耳酮水解得到根皮素。该法存在化学废液的排放，化学污染严重。还有是以苹果皮为原材料，通过乙醇溶液为提取剂、浓缩后酸水解再萃取干燥，得到根皮素，该法采用酸水解，污染严重。本发明旨在解决这一技术问题，提供一种条件反应温和、合成效率高的根皮素生物合成方法。技术实现要素：本发明的目的在于解决现有技术的不足，提供一种酿酒酵母发酵生产根皮素的方法，该方法产量高，无污染，生产周期短。本发明解决其技术问题所采用的技术方案是：一种酿酒酵母发酵生产根皮素的方法，所述合成方法以对羟基苯丙酸为原料，酿酒酵母菌为宿主菌，经过宿主菌体内若干酶的催化而完成；所述宿主菌按下述方法构建获得：1)根据酿酒酵母密码子偏好性优化基因ha4cl和ebchs的密码子，然后利用双向启动子，将两个基因构建于酿酒酵母表达载体ycplac22，得到载体1，表达载体ycplac22带有大肠杆菌的氨苄霉素抗性和酿酒酵母的筛选标记trp基因；2)将丙二酸转化为丙二酰辅酶a的两个基因rlmatb和rlmatc依据酿酒酵母密码子偏好性进行优化，然后将两个基因同时构建于酿酒酵母表达载体ycplac33，得到载体2，表达载体ycplac33带有大肠杆菌的氨苄霉素抗性基因和酿酒酵母的筛选标记ura基因；3)将上述载体1和2同时转入野生型酿酒酵母w303中，根据酿酒酵母的筛选标记trp和ura基因，筛选阳性克隆，然后提取酿酒酵母基因组，进一步pcr验证；4)将强化乙醇到丙二酰辅酶a的途径关键基因seacsl641p，scadh2，scald6，scacc1s659a，s1157a整合到酿酒酵母第16条染色体的yprcdelta15整合位点，同时整合leu筛选标记基因，得到酿酒酵母宿主菌。本发明的酿酒酵母发酵生产根皮素的合成路线如下，如图1所示：优选的，步骤1)中ha4cl和ebchs基因根据向日葵和灯盏花中基因的氨基酸序列来合成。优选的，步骤2)中两个基因rlmatb和rlmatc源于豆科植物根瘤菌。优选的，步骤4)中seacsl641p基因源于沙门氏菌，scadh2、scacc1s659a，s1157a和scald6基因源于酿酒酵母。酿酒酵母发酵生产根皮素的方法，所述合成方法步骤如下：1)根据酿酒酵母密码子偏好性优化基因ha4cl和ebchs的密码子，然后利用双向启动子，将两个基因构建于酿酒酵母表达载体ycplac22，得到载体1，表达载体ycplac22带有大肠杆菌的氨苄霉素抗性和酿酒酵母的筛选标记trp基因；2)将丙二酸转化为丙二酰辅酶a的两个基因rlmatb和rlmatc依据酿酒酵母密码子偏好性进行优化，然后将两个基因同时构建于酿酒酵母表达载体ycplac33，得到载体2，表达载体ycplac33带有大肠杆菌的氨苄霉素抗性基因和酿酒酵母的筛选标记ura基因；3)将上述载体1和2同时转入野生型酿酒酵母w303中，根据酿酒酵母的筛选标记trp和ura基因，筛选阳性克隆，然后提取酿酒酵母基因组，进一步pcr验证；4)将强化乙醇到丙二酰辅酶a的途径关键基因seacsl641p，scadh2，scald6，scacc1s659a，s1157a整合到酿酒酵母第16条染色体的yprcdelta15整合位点，同时整合leu筛选标记基因，得到酿酒酵母宿主菌；5)将酿酒酵母宿主菌培养，诱导表达；4)在表达后的菌液中添加对羟基苯丙酸底物，继续反应20-26h。5)将反应液中的根皮素提取、分离纯化。优选的，利用甲醇将根皮素从发酵液中萃取出来，甲醇与发酵液的体积比1∶1。本专利涉及到的基因为at4cl、ebchs、mddbr、hapal和ccc4h(见表1)。表1专利中用到的物种和基因简介本发明的有益效果是：本发明制备根皮素与现有技术不同之处在于：1)以低成本化合物为原料，工艺原材料成本低。2)以改造微生物为技术基础，在微生物体内进行生物酶的催化合成，避免大规模的提取、分离、纯化过程，从生产成本和对环境友好方面容易控制。3)本工艺在生产设备的规模和数量上比其他方法要要少，操作简单，易于产业转化。4)本工艺仅在生产的最后步骤有分离纯化工序，产品纯化工艺简单，产品质量比一般方法纯度更好、含量更高。5)合成过程没有废液排放，环境友好，无污染，可持续生产，本发明工艺从经济、环境和职业健康角度均为优良的工业化生产指路线。附图说明图1为本发明的根皮素生物合成路线图；图2(a)为本发明中的ycplac22-ha4cl-ebchs质粒图谱；图2(b)为本发明中的ycplac33-rlmatb-rlmatc质粒图谱；图3为本发明合成样品的液相结果图；图4为本发明合成样品的质谱结果图。具体实施方式下面通过具体实施例，并结合附图，对本发明的技术方案作进一步的具体说明。以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例1质粒构建。根据本发明中提供的氨基酸序列和酿酒酵母密码子偏好性设计相应基因的核酸序列，将基因ha4cl和ebchs送商业公司合成，设计20bp左右大小的同源臂，利用一步克隆试剂盒，将两个基因构建于载体ycplac22上，得载体1，如图2(a)所示，表达载体ycplac22带有大肠杆菌的氨苄霉素抗性和酿酒酵母的筛选标记trp基因；将源于豆科植物根瘤菌的丙二酸转化为丙二酰辅酶a的两个基因rlmatb和rlmatc依据酿酒酵母密码子偏好性进行优化，然后将两个基因同时构建于酿酒酵母表达载体ycplac33，得到载体2，如图2(b)所示，表达载体ycplac33带有大肠杆菌的氨苄霉素抗性基因和酿酒酵母的筛选标记ura基因；将上述载体1和2同时转入野生型酿酒酵母w303中。实施例2转化过程。1)挑取单菌落在对应的培养基中过夜摇培12h；2)用分光光度计测培养的菌液的od600的值；3)以初od600值为0.2od转接到50ml的新鲜ypad培养基中；4)活化4-5小时让酿酒酵母增值两代菌液od600值在0.8-0.9；3600rpm离心5min收集菌体，25mlddh2o洗两次(所用的ddh2o最好是当天灭菌的)；5)1ml水重悬到无菌1.5ml离心管中；(超净台内用)6)13000rpm离心30s收集菌体；7)1ml水重悬分装每管100μl用于转化，将分装的菌液在掌上离心机上离心20s弃上清加转化体系；(以上操作必须在超净台中完成，保证无菌环境)8)转化体系配比：peg3350(50％(w/v过滤除菌))240ulliac1.0m(过滤除菌)36ulssdna(2.0mg/ml)50ul共转化dna片段和水34ultotal360ul；9)共转化所需的片段，按每个片段加入400ng混合混匀即可；10)用混合好的转化体系重悬菌体，于30℃培养箱中保温20min；11)42℃热激40min涂相应的筛选板，倒置放于30℃培养箱中培养2-3天，待转化子长出来，挑选单克隆，提基因组验证；实施例3整合过程1)提取酿酒酵母基因组，将酿酒酵母16号染色体的yprcdelta15整合位点上下游同源臂通过pcr提取出片段，上游片段1，下游片段2。2)将上游片段和筛选标签leu基因、基因(包含启动子和终止子)seacsl641p，scadh2，scald6，scaccls659a，s1157a通过同源臂一起转入酿酒酵母w303中，在酿酒酵母体内，利用同源重组，酿酒酵母自身会将导入的基因片段整合到酿酒酵母的基因组中。实施例4合成根皮素1)将酿酒酵母宿主菌培养，在培养后的菌液中添加对羟基苯丙酸和丙二酸底物，反应120-160h；2)将反应液中的根皮素提取、分离纯化，利用甲醇将根皮素从发酵液中萃取出来，甲醇与发酵液的体积比1∶1。实施例4合成的样品的液相和质谱结果如图3和图4所示。所述仅为本发明的较佳实施例，并不用以限制本发明，凡是在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应在本发明的保护范围之列。序列表<110>嘉兴欣贝莱生物科技有限公司<120>一种酿酒酵母发酵生产根皮素的方法<160>6<170>siposequencelisting1.0<210>1<211>540<212>prt<213>酿酒酵母(saccharomycescerevisiae)<400>1metalaserglyleuglyileileproalaserleuleuproalaile151015thrileproleuhisleuproleuhisserthrcysproglyalaile202530serleuproleualaalaprocysleuilealaglyalathrglygly354045valhisthrthralaalavalglyleuthrseralaleuvalalaser505560alaleuhisglyglyglyileserleuglyalavalilemetileleu65707580leuproalaserproglyprovalthrserproileglyalaserthr859095leuglyalaileserthrmetalaalaproproprothralaalagly100105110ileileleuglyvalleualaseralaserleuileileilethrgly115120125seralahisileproleuvalleualathralaseralaalaserile130135140leuleuvalcysilealaseralaproleuglycysleuhisproser145150155160glyleuthrseralaalaglythrleuleuproglyileglyvalser165170175seralaalavalvalalaleuprothrserserglythrthrglyleu180185190proleuglyvalmetleuthrhisleuglyleuvalthrservalala195200205glyglyvalalaglyglyalaproalaleuthrilehisserglyala210215220valleumetcysserleuproleuprohisilethrserleualaser225230235240ileleuleucysglyleualaalaglyalaalaileleuleumetser245250255leuproalailevalproproleuglyleuileglyleuthrleuval260265270thrileglyproprovalproproilevalleuthrilealaalaala275280285glyglyleuvalalaleuthralametserserilealathrvalmet290295300serglyalaalaproleuglyleualaleuglyalathrvalalamet305310315320leuproproalaalaleuleuglyglyglythrglymetthrglyala32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