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液滴微流控助力链霉菌高通量筛选技术突破

阅读量：102

发布时间：2021-08-25

来源：酯香香化学采购网

　　【化工仪器网项目成果】链霉菌是重要的工业微生物，可以生产蛋白、小分子药物等高附加值产品。工业生产中，常用随机诱变手段产生大量的链霉菌突变库，但缺乏与之相适配的高通量筛选手段用以获得目标突变株。已报道的基于流式细胞分选的方法只能对链霉菌的原生质体或孢子进行筛选，由于抗生素等次级代谢产物多产生于菌丝发酵的平台期，因而原生质体或孢子均无法代表链霉菌的真实发酵状态，并不适合次级代谢产物的筛选；流式细胞仪也无法筛选胞外产物信号，因此开发链霉菌高效培养、检测和分选的高通量筛选技术具有重要意义。

　　中国科学院天津工业生物技术研究所研究员王猛带领的高通量编辑与筛选平台实验室，开发了基于液滴微流控的链霉菌高通量筛选技术。研究调整与优化链霉菌孢子液滴包埋参数及菌株液滴分选参数，使链霉菌高通量筛选技术的检测分选速度达到约每小时1万个菌株，人工混库的单轮分选富集率超过330倍。团队运用该技术在启动子强度分析、启动子改造、分泌蛋白表达等方面进行了测试应用，对数十种内源和异源启动子的分析表明，液滴微流控技术测定的启动子表达强度信号与传统的酶标板和显微镜荧光测定结果一致；从弱启动子ermE*p和强启动子gapdh(EL)p出发的启动子改造结果表明，采用液滴微流控筛选技术可以梯度改变启动子强度，有效获得启动子增强或减弱的突变体，充实元件库用于后续的代谢工程改造，基于弱启动子ermE*p改造及筛选，获得相比野生型强度为133.5%、343.2%和347.9%的突变体；基于强启动子gapdh(EL)p改造及筛选，获得强度为野生型10.1%、24.7%和94.6%的突变体；针对链霉菌胞外产物纤维素酶，应用液滴微流控技术快速从随机突变库中筛选获得纤维素酶产量分别为出发菌株169.2%至211.4%的多个高产菌株。

　　此外，研究表明，链霉菌从孢子出发可以在液滴中稳定培养7天以上，完成萌发、分化、进行各种生物合成直至走向衰亡的完整生命周期。这一特性使开发的基于液滴微流控的链霉菌高通量筛选平台可以用于菌体生长早期对于启动子等调控元件的筛选，也可以用于生长中期对于胞内、胞外目标蛋白的筛选，且在次级代谢产物如抗生素等筛选方面具有应用前景。

　　科研团队撰写的博客发表在Nature Portfolio Bioengineering Community的专栏Behind the paper上，从形态学角度探讨了液滴微流控筛选方法与丝状微生物的适配性，为该平台在更多丝状微生物改造和筛选方面的应用提供了新思路。

　　相关研究结果发表在Communications Biology上。研究工作得到国家重点研发计划、国家自然科学基金、中科院重点项目及天津市合成生物技术创新能力提升行动的支持。

基于液滴微流的链霉菌高通量筛选技术示意图

不同启动子的强度分析比较图：a、酶标仪，b、液滴微流控，c、荧光显微镜

内容来源于酯香香化学采购网

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[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000161s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000171s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` IN (34,92) AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000170s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` IN (95,38) AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000200s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 1 AND `status` = 1 [ RunTime:0.000208s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 1 [ RunTime:0.000217s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,1,%' ) [ RunTime:0.000221s ]
[ SQL ] SHOW COLUMNS FROM `yzn_goods` [ RunTime:0.000651s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (25,26,29,30,48,1) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.001015s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 2 AND `status` = 1 [ RunTime:0.000239s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 2 [ RunTime:0.000201s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,2,%' ) [ RunTime:0.000195s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (42,43,2) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000962s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 7 AND `status` = 1 [ RunTime:0.000215s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 7 [ RunTime:0.000213s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,7,%' ) [ RunTime:0.000202s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (10,14,32,33,49,50,51,52,53,54,55,56,57,58,96,97,7) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000982s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 [ RunTime:0.000206s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 16 [ RunTime:0.000196s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,16,%' ) [ RunTime:0.000190s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (59,60,61,62,63,16) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000939s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 [ RunTime:0.000216s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 17 [ RunTime:0.000194s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,17,%' ) [ RunTime:0.000212s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (40,64,65,66,67,68,100,17) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000934s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 34 AND `status` = 1 [ RunTime:0.000221s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 92 AND `status` = 1 [ RunTime:0.000184s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (34,92) [ RunTime:0.000245s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,34,%' or arrparentid like '0,92,%' ) [ RunTime:0.000193s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (35,70,71,93,94,34,92) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000905s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 95 AND `status` = 1 [ RunTime:0.000169s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 38 AND `status` = 1 [ RunTime:0.000159s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (95,38) [ RunTime:0.000193s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,38,%' or arrparentid like '0,95,%' ) [ RunTime:0.000169s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (47,74,75,76,77,78,79,80,81,82,83,99,95,38) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000890s ]
[ SQL ] SHOW COLUMNS FROM `yzn_article` [ RunTime:0.000663s ]
[ SQL ] SELECT `A`.*,`AD`.*,`C`.`catname` FROM `yzn_article` `A` INNER JOIN `yzn_article_data` `AD` ON `A`.`id`=`AD`.`did` INNER JOIN `yzn_category` `C` ON `C`.`id`=`A`.`catid` WHERE `A`.`id` = 5496 AND `A`.`status` = 1 LIMIT 1 [ RunTime:0.000276s ]
[ SQL ] SHOW COLUMNS FROM `yzn_category` [ RunTime:0.000534s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 35 AND `status` = 1 [ RunTime:0.000161s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` IN (36,37,38,39,40,41,42) AND `status` = 1 [ RunTime:0.000154s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 43 AND `status` = 1 [ RunTime:0.000129s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` IN (44,45,46,47,48,49,50) AND `status` = 1 [ RunTime:0.000154s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 6 AND `status` = 1 [ RunTime:0.000127s ]
[ DB ] CONNECT:[ UseTime:0.000259s ] mysql:host=127.0.0.1;port=3306;dbname=yzncms;charset=utf8
[ SQL ] SELECT `A`.`id`,`A`.`title` FROM `yzn_article` `A` INNER JOIN `yzn_category` `C` ON `C`.`id`=`A`.`catid` WHERE `A`.`status` = 1 AND `A`.`catid` IN (35,36,37,38,39,40,41,42) ORDER BY `A`.`flag` DESC,`A`.`listorder` ASC,`A`.`updatetime` DESC [ RunTime:0.020814s ]
[ SQL ] UPDATE `yzn_article` SET `hits` = `hits` + 1 WHERE `id` = 5496 [ RunTime:0.000339s ]
[ SQL ] SELECT * FROM `yzn_poster` WHERE `cate_id` = 10 AND `p_c_id` = 41 ORDER BY `listorder` ASC LIMIT 4 [ RunTime:0.000372s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 38 AND `a`.`status` = 1 [ RunTime:0.002778s ]
[ SQL ] SELECT `a`.*,`c`.`catname` FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 38 AND `a`.`status` = 1 ORDER BY `flag` DESC,`listorder` ASC,`updatetime` DESC LIMIT 0,6 [ RunTime:0.004763s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 37 AND `a`.`status` = 1 [ RunTime:0.002737s ]
[ SQL ] SELECT `a`.*,`c`.`catname` FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 37 AND `a`.`status` = 1 ORDER BY `flag` DESC,`listorder` ASC,`updatetime` DESC LIMIT 0,6 [ RunTime:0.005150s ]