大肠杆菌L-丝氨酸脱水酶的表达改善甘氨酸营养缺陷型毕赤酵母L-丝氨酸的生长  

Expression of Escherichia coli L-serine dehydratase improves growth of glycine auxotroph Pichia pastoris on serine

作　　者：刘璐瑶 李康[1,2,3] 王世杰 白仲虎 杨艳坤 金坚 LIU Luyao;LI Kang;WANG Shijie;BAI Zhonghu;YANG Yankun;JIN Jian(The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;Jiangsu Provincial Engineering Research Center for Bioactive Product Processing,Jiangnan University,Wuxi 214122,China;School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室(江南大学),江苏无锡214122 [2]粮食发酵与食品生物制造国家工程研究中心(江南大学),江苏无锡214122 [3]江苏省生物活性制品加工工程技术研究中心,江苏无锡214122 [4]江南大学生命科学与健康工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2024年第13期16-24,共9页Food and Fermentation Industries

基　　金：国家自然科学基金项目(32370054)。

摘　　要：氨基酸作为一种迟效碳源无法被微生物快速利用。L-丝氨酸脱水酶(L-serine dehydratase,L-SerDH)可以将L-丝氨酸一步催化为丙酮酸和氨进入中心碳代谢生成生物量,且此过程不需要消耗ATP和还原力。L-丝氨酸是甲酸利用途径(还原性甘氨酸途径)的关键中间体。因此,改善L-丝氨酸的利用可以为甲酸和丝氨酸作为碳源利用提供参考价值。该文对巴斯德毕赤酵母(Pichia pastoris)进行了丝氨酸耐受性实验,结果显示毕赤酵母可以耐受20 g/L丝氨酸。对内源L-丝氨酸脱水酶在毕赤酵母中的表达进行了优化。在甘氨酸营养型毕赤酵母内分别表达了8种异源L-丝氨酸脱水酶,结果表示大肠杆菌tdcG基因编码的L-SerDH对丝氨酸利用改善效果最好,终OD 600提高至出发菌株的1.6倍。该研究验证了巴斯德毕赤酵母对L-丝氨酸的耐受性,并筛选得到了较优的L-丝氨酸脱水酶来源,为毕赤酵母的丝氨酸利用提供了更优的酶来源。Amino acids cannot be rapidly utilized by microorganisms as a gradually metabolized carbon source.L-serine dehydratase(L-SerDH)catalyzes the one-step conversion of L-serine into pyruvate and ammonia,facilitating central carbon metabolism to generate biomass without the consumption of ATP and reducing power.L-serine is a key intermediate in the formate utilization pathway(reductive glycine pathway).Therefore,improving the utilization of L-serine can provide valuable insights into the utilization of formate and serine as carbon sources.This paper explored serine tolerance of Pichia pastoris.Results demonstrated that P.pastoris exhibited tolerance to 20 g/L of L-serine.Expression optimization of endogenous L-serine dehydratase was carried out in P.pastoris.Eight heterologous L-serine dehydratases were expressed separately in glycine auxotroph P.pastoris.The results indicated that L-SerDH encoded by the E.coli tdcG gene had the best effect on the improvement of serine utilization,with the final OD 600 increased to 1.6-fold that of the original strain.This study represents the validation of L-serine tolerance in P.pastoris and screens a superior source of L-serine dehydratase.These findings offer valuable insights into enhancing enzyme sources for serine utilization in P.pastoris.

关 键 词：L-丝氨酸脱水酶 巴斯德毕赤酵母 丝氨酸耐受性 甘氨酸营养缺陷型菌株 L-丝氨酸 

分 类 号：TQ922[轻工技术与工程—发酵工程]