2023, Vol. 39中国科学院微生物研究所、中国微生物学会主办
文章信息
- 张瑞元, 朱翊凡, 曾杜文, 魏士昊, 樊亚超, 廖莎, 赵心清, 张风丽, 张霖
- ZHANG Ruiyuan, ZHU Yifan, ZENG Duwen, WEI Shihao, FAN Yachao, LIAO Sha, ZHAO Xinqing, ZHANG Fengli, ZHANG Lin
- 利用酵母菌生产有机酸的研究进展
- Advances on the production of organic acids by yeast
- 生物工程学报, 2023, 39(6): 2231-2247
- Chinese Journal of Biotechnology, 2023, 39(6): 2231-2247
- 10.13345/j.cjb.230041
-
文章历史
- Received: January 16, 2023
- Accepted: March 31, 2023
- Published: March 31, 2023
引用本文
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2. 中石化(大连)石油化工研究院有限公司, 辽宁 大连 116045
2. SINOPEC Dalian Research Institute of Petroleum and Petrochemicals Co., Ltd., Dalian 116045, Liaoning, China
有机酸广泛应用于食品、农业、化工和医药等领域。随着石油等不可再生资源的日趋枯竭，以及全球对环境污染防治和环境保护意识的提高，可降解材料的生产和使用日益受到关注。全球生物基材料市场规模预计将从2021年的107亿美元增长至2026年的297亿美元，2021–2026年间的复合年增长率为22.7%[1]。有机酸可作为多种生物基可降解材料的中间体或者重要前体，其研究对促进可降解材料的生产具有重要意义[2-3]。根据恒州博智信息公司的统计及预测，2021年有机酸的全球市场销售额达到235亿美元，预计2028年将达到261亿美元，复合年增长率为1.1% (2022−2028年，https://bbs.csdn.net/topics/607684462 )。化学法合成有机酸存在使用有毒性的催化剂、中间体的产生影响生产效率及分离纯化复杂等缺点，因此利用微生物进行有机酸的生物合成意义重大[3-4]。多种有机酸在微生物如细菌、真菌和藻类等合成中的研究已有报道[4-6]；目前，已有柠檬酸、丁二酸、葡萄糖酸、苹果酸、衣康酸、富马酸、丙酮酸和丙酸等20多种有机酸采用微生物发酵法进行规模化生物制造[6-8]。
随着有机酸生物合成研究的深入和广泛的应用，国内外学者对于微生物合成有机酸的综述论文相继发表。国际上，澳大利亚学者Sauer等[7]对微生物合成的柠檬酸、乳酸和丁二酸生物合成进展进行了综述，并提出了有机酸生物合成研究的指导方针，不同菌株可利用不同碳源产生多种有机酸，菌株的代谢工程及性能改造可提高菌株的耐受性。瑞典学者Chen和Nielsen[4]对工业和商业中具有良好应用的柠檬酸、乳酸、丁二酸和3-羟基丙酸的微生物合成进行了总结，并且讨论了微生物合成生物有机酸的不足及面临的挑战主要是原料成本及下游产品的加工，因此发掘及构建高性能菌株是解决问题的关键。德国学者Becker等[9]综述了合成有机酸的菌株选育及工业方面的最新进展，重点介绍了乙醇酸、丙酮酸、乳酸、3-羟基丙酸、丁二酸、衣康酸、富马酸和己二酸的生物合成进展。利用可再生生物质生产有机酸，因其原料成本低、能源可持续利用等优点引起了关注。西班牙学者Alonso等[10]综述了利用可再生材料和废物低成本发酵产生有机酸的研究进展及工业前景。同时，国内学者对有机酸的微生物发酵也发表了综述，如江南大学李江华团队[11]综述了利用蛋白工程和代谢工程策略合成包括丙酸、丙酮酸、衣康酸、丁二酸、富马酸、苹果酸和柠檬酸等有机酸的研究进展，提出整合合成生物学、系统生物学及蛋白质工程从而建立细胞工厂的策略。南京工业大学姜岷团队[12]综述了微生物发酵产生四碳有机酸——富马酸、苹果酸和丁二酸的研究进展，并探讨了微生物高效合成四碳有机酸的可行策略及前景展望。
与常用的细菌和霉菌产酸菌比较，酵母菌作为有机酸的产生菌一般具有更强的低pH耐受性，此外，酵母不存在噬菌体污染问题，而且酵母菌具有发酵技术及遗传改造技术相对成熟等独特优点，成为合成生物塑料及塑料有机酸单体的优质底盘生物。因此，近年来酵母发酵大规模工业生产的有机酸研究持续增长，并且正在呈逐步扩大的趋势[11-14]。但是，目前国内外还没有专门对酵母生产有机酸的综述报道，以往的综述均将不同宿主的相关研究放在一起总结，没有突出酵母菌的特色和优势。酵母菌较细菌霉菌等其他微生物具有独特的优越性[15]，有必要对其相关研究进行具体深入的总结和讨论。
本文参考美国能源部提出的高附加值生物化学品有机酸的种类[16]，聚焦以酵母菌作为底盘发酵生物，对代表性的有机酸，包括丁二酸、己二酸、乙醇酸、丙二酸、3-羟基丙酸、富马酸、苹果酸、衣康酸、木糖酸、葡萄糖酸和葡萄糖醛酸的特性和酵母生物合成情况进行综述，并对未来酵母合成有机酸的发展趋势及存在的局限性进行讨论和展望，本文概括了重要有机酸的特征、主要应用及合成酵母菌等信息。
1 酵母三羧酸循环中心代谢有机酸的合成丁二酸、苹果酸、富马酸这3种四碳酸在微生物体内主要通过3种途径合成，还原三羧酸循环途径、位于线粒体内的氧化三羧酸循环途径和乙醛酸途径[12, 17](图 1), 丁二酸、苹果酸、富马酸在食品、饲料和化工等领域具有比较普遍的应用价值，在酵母中生产这些内源的传统有机酸具有悠久的历史。
丁二酸和丁二醇(butanediol, BD)通过缩聚可产生聚丁二酸丁二醇酯(polybutylene succinate, PBS)，该材料是一种可生物降解的耐热材料。解脂耶氏酵母(Yarrowia lipolytica)是非传统酵母，具有生物安全、鲁棒性的特点，已报道有多株Y. lipolytica代谢葡萄糖合成SA[17-19]。
利用代谢工程改造可促进Y. lipolytica合成丁二酸的效率，其中围绕丁二酸脱氢酶基因(succinate dehydrogenase gene, SDH)的改造最为成功，阻断该酶基因表达或者削弱其活性，可以阻止丁二酸的进一步转化，从而积累丁二酸。例如，Babaei等[18]构建的工程菌Y. lipolytica ST8578中，SDH1的启动子序列被截短，从而导致丁二酸脱氢酶活性降低了77%，但不影响菌株在葡萄糖培养基中的生长，在pH值为5的培养基中，丁二酸产量、产率、生产率分别为35.3 g/L、0.26 g/g葡萄糖、0.60 g/(L·h)；辅酶A (coenzyme A, CoA)转移酶基因Ylach的缺失消除了乙酸的形成并提高了丁二酸的产量和细胞生长，来自酿酒酵母(Saccharomyces cerevisiae)的磷酸烯醇丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, ScPCK)基因和内源性琥珀酰辅酶合成酶(succinyl-CoA synthase beta subunit, YlSCS2)基因在Y. lipolytica中的超表达，使得菌株在不控制pH的情况下，丁二酸的产量和产率分别达到110.7 g/L和0.53 g/g甘油[19]，避免了细菌生产酸过程中需要控制pH为中性，碱的使用易造成发酵污染的问题。
Y. lipolytica中SDH的失活有利于丁二酸的产生，然而菌株代谢葡萄糖能力降低从而影响了菌株的应用，虽然用甘油作为唯一碳源能解决菌种生长的问题，但葡萄糖是自然界最丰富和廉价的碳源，因此，有必要提高利用葡萄糖产丁二酸的效率。Yang等[20]通过自适应进化策略(adaptive evolution strategy)在丁二酸脱氢酶基因Ylsdh5缺失的工程菌株Y. lipolytica PSA02004中，葡萄糖的利用率得到提高，丁二酸产量在酵母完全培养基(yeast extract peptone dextrose medium, YPD)和食品废弃水解物培养基中分别为65.7 g/L和87.9 g/L；Bondarenko等[21]通过对菌株Y.lipolytica Y3753的发酵优化，在pH为3.65时，丁二酸的产量和生产率分别为55.3 g/L和2.6 g/(L·h)。Li等[22-24]通过纤维床生物反应器(in situ fibrous bed bioreactor, isFBB)，利用农业残渣，通过优化发酵条件，Y. lipolytica PGC01003合成丁二酸的产量达到了209.7 g/L, Y. lipolytica PGC202通过喂养富含葡萄糖的混合食物垃圾，在pH 2.8的条件下，丁二酸的产量达到71.6 g/L，通过超表达木糖还原酶(xylose reductase, XR)、木糖醇脱氢酶(xylitol dehydrogenase, XDH)和木糖激酶(xylulose kinase, XK)基因，Y. lipolytica PSA02004以木糖作为唯一碳源，合成丁二酸的产量为22.3 g/L；Billerach等[25]研究发现，在氮缺乏的条件下，菌株PGC01003和PGC202丁二酸的产量提高，在PGC01003中，丁二酸产量、产率、生产率分别达到19.0 g/L、0.23 g/g甘油、0.23 g/(L·h)，在PGC202中，丁二酸产量、产率、生产率分别达到33.0 g/L、0.12 g/g甘油、0.57 g/(L·h)；利用零成本的城市有机生物垃圾，Y. lipolytica PSA02004的丁二酸产量、产率、生产率分别达到54.4 g/L、0.44 g/g葡萄糖、0.82 g/(L·h)[26]。以上研究为利用可再生的廉价生物质资源生产有机酸提供了借鉴。
丁二酸合成后积累在细胞内，但是工业生产希望产物分泌到培养基中。线粒体载体(mitochondrial carriers)和C4-二羧酸转运蛋白(C4‑dicarboxylic acid transporters)可以提高SA的分泌，Y. lipolytica PGC62‑SYF‑Mae通过表达转运子SpMae1和优化SA的生物合成路径，SA产量、产率、生产率分别达到101.4 g/L、0.37 g/g葡萄糖、0.70 g/(L·h)，比转运系统改造前菌株Y. lipolytica ST8578 [SA产量、产率、生产率分别为35.3 g/L、0.26 g/g葡萄糖、0.61 g/(L·h)]有明显提高[27]；重组表达乙酰辅酶A合成酶(acetyl-CoA synthase)的Y. lipolytica PSA02004PP可以在醋酸盐作为唯一碳源、高醋酸盐浓度条件下生产SA[28]。表达了来自黑曲霉(Aspergillus niger)的二羧酸转运蛋白(dicarboxylic acid transporter) DCT-02，酿酒酵母菌株UBR2 CBS - DHA -SA-AnDCT-02的SA产量和产率达到10.3 g/L、0.16 g/g甘油[29]。在不控制pH值的条件下，重组Y. lipolytica分别能产生22.3 g/L和46.9 g/L SA[30-31]。
除了解脂耶氏酵母，在重组毕赤酵母(Pichia kudriavzevii) CY902中，SA的合成也有报道[32]。但是，目前报道的最高SA产量(209.7 g/L)在解脂耶氏酵母中得到[22]，虽然和酿酒酵母比较，解脂耶氏酵母不产生大量乙醇，三羧酸循环(tricarboxylic acid cycle, TCA)通量更高，但是该酵母的弱点是强好氧，而利用还原三羧酸循环(reductive tricarboxylic acid cycle, rTCA)途径生产丁二酸的收率不高，因此利用其他兼性厌氧酵母宿主生产丁二酸的研究还需要进一步深入。
1.2 富马酸(fumaric acid, FA)富马酸是C4不饱和二元酸，广泛应用于食品、医药、化工等领域。米根霉(Rhizopus oryzae)是产生富马酸的主要微生物，但是根霉菌存在生长缓慢、产量低、遗传操作困难等缺点，在酿酒酵母中异源表达米根霉菌的苹果酸脱氢酶(malate dehydrogenase)和富马酸酶，重组菌株FMME-001的富马酸产量仅为3.2 g/L[33]。来自R. oryzae的富马酸还原途径酶在可利用木糖的酵母斯氏舍弗氏菌(Scheffersomyces stipitis)表达，工程菌株中富马酸产量仅为4.7 g/L，因此，改造的酵母菌株富马酸的产量与根霉菌中富马酸的最高产量(85.0 g/L)相比，要低很多[12, 34-35]。因此筛选高产富马酸天然酵母菌株至关重要，Wang等[36]分离到一株酵母菌出芽短梗霉(Aureobasidium pullulans var. aubasidanis) DH177，能产生微生物油和32.3 g/L富马酸，Wei等[37]研究发现鸟氨酸-尿素循环(ornithine- urea cycle)参与富马酸的生物合成，通过在菌株DH177中移除葡萄糖氧化酶GOX基因和3, 5-二羟基癸酸生物合成合酶基因，及超表达丙酮酸盐羧化酶的PYC基因，工程菌株能够产生(93.9±0.8) g/L的富马酸，是原始菌株DH177富马酸产量的2.9倍，产率达到0.63 g/g葡萄糖，是理论产率的49%。因此，结合优质高产原始菌株的合成生物学策略被用于优化代谢平衡必将有利于酵母菌的产酸量。
1.3 苹果酸(malic acid, MA)MA又名2-羟基丁二酸，由于分子中有一个不对称碳原子，有2种立体异构体，主要用于食品和医药领域。鲁氏酵母(Zygosaccharomyces rouxii) V19是分离自高糖发酵食品的糖耐受菌株，可以产生74.9 g/L苹果酸，产率为0.39 g/g葡萄糖[38]。Chen等[39]通过在S. cerevisiae异源表达还原三羧酸循环的丙酮酸羧化酶和苹果酸脱氢酶，及四碳二羧酸转运蛋白，苹果酸产量可以达到30.3 g/L。Zhang等[40]在巴斯德毕氏酵母(Pichia pastoris)表达还原三羧酸循环的丙酮酸羧化酶和苹果酸脱氢酶，同时可以合成0.8 g/L富马酸、42.3 g/L苹果酸和9.4 g/L的丁二酸。通过原位分离生物转化技术，在酵母细胞作为催化剂或者微反应器时，富马酸在渗透细胞液中富马酸酶作用下，由富马酸合成苹果酸[41-42]。
为了提升酵母菌生产四碳有机酸的产率，首先需要筛选天然的四碳有机酸高产酵母菌株，野生型酵母菌营养需求低，发酵条件简单，野生型酵母菌株的筛选是代谢工程菌株改造的基础；其次是要降低生产成本，可通过降低发酵原料成本、优化发酵工艺等完成。
2 酵母非传统有机酸的合成己二酸和丙二酸等有机酸通常在微生物中天然产量不高，但是具有非常重要的应用价值，因此通过代谢工程改造提高这些特殊有机酸的产量成为研究热点。
2.1 乳酸(lactic acid, 2-hydroxy propionic acid, LA)乳酸(分子式，C3H6O3)是合成当今最具潜力的生物可降解材料聚乳酸(polylactic acid, PLA)的单体，PLA在生物医药、食品包装以及电子电器等领域具有广阔的应用前景。乳酸可以通过化学合成或微生物发酵途径获得。乳酸是手性分子，化学合成导致外消旋体混合物(l-乳酸和d-乳酸)，微生物发酵生产乳酸既可以解决外消旋体混合物的问题，又可以消除中间体分子产生造成的环境污染等问题，酵母菌因为自身优点，成为首选的合成宿主。乳酸是丙酮酸在乳酸脱氢酶(lactate dehydrogenase, LDH)的作用下生成的，酵母自身不能有效产生乳酸，因此通过异源表达乳酸脱氢酶合成乳酸[43]。已有多种不同种属酵母菌改造后能够产生LA，如酵母属(Saccharomyces)[44-46]、接合酵母属(Zygosaccharomyces)[47]、假丝酵母(念珠菌属) (Candida)[48]、毕赤酵母属(Pichia)[49-50]、克鲁维酵母属(Kluyveromyces)[51]等。采用降低丙酮酸旁路代谢通量的策略，可以达到提高乳酸产量的目的，如对分离自葡萄皮的菌株库德里阿兹威(氏)毕赤酵母(Pichia kudriavzevii) NG7进行代谢工程改造，工程菌株P. kudriavzevii DKA用来自植物乳杆菌(Lactobacillus plantarum)的D‐LDH代替丙酮酸脱羧酶基因PDC1，在反应器中能产生154.0 g/L的D-LA，是迄今LA产量最高的酵母菌株，D-LA产率达到0.72 g/g葡萄糖[50]。
2.2 己二酸(adipic acid, AA)生物基己二酸是工业中的一种重要平台化学品，是生产尼龙66的重要单体，是当前石化路线的一种有前途的替代品。在大肠杆菌(Escherichia coli)中，合成生物学和代谢工程策略大大提高了AA产量[52]。然而，在酵母细胞中，关于AA生物合成的研究很少。热带假丝酵母(Candida tropicalis) KCTC 7212在最佳搅拌速率、pH和底物月桂酸甲酯中缺乏脂酰辅酶A氧化酶AOX基因，获得12.1 g/L的己二酸，生产率为0.1 g/(L·h)[53]。AA分别由β-酮硫酶(Tfu_0875)、3-羟基酰基-CoA脱氢酶(Tfu_2399)、3-羟己二酸-CoA脱氢化酶(Tfu_0067)、5-羧基-2-戊烯酰基-CoA还原酶(Tfu_1647)和己二酸-CoA合成酶(Tfu_2576-7)催化，通过5个反应步骤由乙酰辅酶A和琥珀酰辅酶A合成AA (图 2)[54]。在酿酒酵母中共表达了来自褐色嗜热裂孢菌(Thermobifida fusca)的反己二酸降解途径(reverse adipate degradation pathway, RADP)基因(Tfu_0875、Tfu_2399、Tfu_0067、Tfu_1647、Tfu2576和Tfu_2576)，在优化条件下获得了10.1 mg/L的AA[54]，在酿酒酵母中表达RADP途径基因，在YPD培养基中获得3.4 mg/L的AA[55]。在酿酒酵母中，异源表达凝结芽孢杆菌(Bacillus coagulans)的烯醇化还原酶ER基因，可以利用可再生饲料生物合成AA[56]。
与其他有机酸类似，高浓度的己二酸会对生产菌造成毒害。有研究证明，与细菌、丝状真菌相比，酵母菌具有更高的己二酸耐受性[57]，Fletcher等[58]的研究发现，删除三羧酸循环的关键基因KGD1能提高菌株对己二酸及毒性前体-邻苯二酚的耐受性，因此，提高生产菌对不同有机酸和低pH的耐受性是改善细胞性能和提高己二酸生产效率的关键。
2.3 乙醇酸(glycolic acid, GA)乙醇酸是一种双碳α-羟基酸，具有醇和酸基团，应用广泛，如可作为染色剂用于纺织工业、作为防腐剂和香料用于食品工业、作为皮肤护理剂用于制药工业等[59]，预计到2024年，GA市场预计将达到4.15亿美元( https://www.grandviewresearch.com/press-release/global-glycolic-acid-market )。
利用可再生木质纤维素类生物质资源生产有机酸具有良好的前景。木糖是该类生物质水解液中除了葡萄糖外的第二大丰富的可发酵糖。多种非常规酵母天然可以利用木糖，因此可以作为宿主利用木质纤维素水解液生产有机酸。例如，克鲁维酵母可以天然利用木糖，在生物反应器中，以D-木糖和乙醇为碳源，工程酵母乳酸克鲁维酵母(Kluyveromyces lactis)产生15.0 g/L的乙醇酸，这是工程酵母首次通过乙醛酸在乙醛酸还原酶的作用下合成乙醇酸的报道[60]，实际产率达到0.52 g/g乙醇，是理论产率的32%[60]。酿酒酵母通常不具备天然利用木糖的能力，需要首先引入木糖利用途径。在工程酵母S. cerevisiae中，Salusjärvi等[61]通过异源表达多种酶[木糖脱氢酶(d-xylose dehydrogenase, XylB)、木糖脱水酶(d-xylonate dehydratase, XylD)、醛缩酶(aldolase, YagE或YjhH)、乙醛脱氢酶(aldehyde dehydrogenase, aldA)] (图 3)，利用木糖合成了1.0 g/L的乙醇酸，提高木糖脱水酶的活性和优化发酵条件将有利于乙醇酸的产生。
3-HP末端羟基和羧基能参与多种化学反应，作为一种重要的化工原料应用广泛，生物法合成3-HP具有高效、绿色、可持续发展等优势，将取代化学合成方法，而成为今后研究重点[62]。Kildegaard等[63]通过适应性进化提高了S. cerevisiae对3-HP的耐受性，并发现谷胱甘肽会增强菌株对3-HP的耐受性。北京化工大学的刘子鹤团队[64]通过在酿酒酵母中重构乙酰辅酶A和NADPH再生途径实现了相较于出发菌株24倍的3-HP产量提升，产量达到864.5 mg/L。中国科学院大连化学物理研究所周雍进团队[65]通过在酿酒酵母中，重构丙二酰辅酶A还原酶，在前体物质丙二酰辅酶A和NADPH充足条件下，补料分批发酵条件下，生物反应器中3-HP产量达到56.5 g/L，是迄今在工程微生物中利用葡萄糖生产3-HP的最高产量，产率达到0.31 g/g葡萄糖，是理论产率的41.3%。作为丙烯酸及其衍生物的前体，在工程酵母菌S. cerevisiae中表达丙二酰辅酶A还原酶(malonyl‑CoA reductase, MCR)合成产生(9.8±0.4) g/L的3-HP[66]，在P. pastoris中，过表达丙二酰辅酶A还原酶和删除产生副产物D-阿拉伯糖醇的主要基因D-阿拉伯糖醇脱氢酶(D-arabitol dehydrogenase)基因ArDH，在补料分批发酵生物反应器中，3-HP的产量提高到37.1 g/L[67]，因此，降低副产物产量仍然是提高3-HP产量的有效手段。通过代谢工程改造酵母应用于3-HP的生产将为生物合成进一步替代传统的石油工业打下基础，为工业绿色化创造可能。
2.5 衣康酸(2-甲基丁二酸，itaconic acid, IA)衣康酸由于结构中存在一个乙烯基键和2个羧基，在生物聚合物工业中有广泛的应用，合成衣康酸的微生物主要是丝状真菌土霉菌(Aspergillus terreus)，产量达到150.0 g/L[68-70]；除了A. terreus，黑粉菌(Ustilago maydis)也是一种很有前途的衣康酸生产宿主，产量可达到220.0 g/L[71]。然而，在工程化酵母中，衣康酸产量一直很低，不超过10.0 g/L，如工程酵母Y. lipolytica的产量是4.6 g/L[69, 72]，在酵母P. kudriavzevii中，通过突变异柠檬酸脱氢酶基因和异源超表达线粒体三羧酸转运蛋白和顺式乌头酸脱羧酶，IA在反应器中产量达到1.2 g/L[73], Young等[74]通过优化多步途径，酿酒酵母衣康酸的合成量达到815.0 mg/L。然而最近，在工程菌株Y. lipolytic中，以地沟油作为唯一碳源，衣康酸的产量达到54.6 g/L，这是以工程酵母作为细胞工厂获得的最高衣康酸产量，产率达到0.30 g/g地沟油[75]。Xu和Li[76]通过在酿酒酵母中超表达线粒体ATP/ADP转运蛋白，从而利用酒精合成了衣康酸。由于酵母菌的耐酸特性，在发酵过程中不需要pH调节，酵母菌作为生产衣康酸的工业底盘，具有巨大潜力。
2.6 其他高值有机酸丙二酸(malonic acid)是一种平台化学品，可以用来生产高附加值的化合物，也可以作为聚合物和聚酯的前体。作为一种具有高附加值的化合物，丙二酸被应用于制药、电子和香精香料等行业，江南大学邓禹团队[77]在酿酒酵母中通过丙二酰-CoA途径构建了丙二酸生物合成途径，通过对来源于的3-羟基异丁酰-CoA水解酶(由EHD3编码)的F121和E124位点突变从而使Ehd3获得丙二酰-CoA水解酶的活性，在分批补料发酵反应器中，丙二酸产量达到1.6 g/L。木糖酸(xylonic acid, XA)是化合物的重要中间体，重组酵母P. kudriavzevii VTT C-79090T在低pH发酵条件下，能合成146.0 g/L的D-xylonate，产率达到1.00 g/g D-木糖[78]。葡萄糖酸作为一种温和有机酸，葡萄糖酸及其衍生物得到了广泛的应用和关注，表达葡萄糖氧化酶的酵母S. cerevisiae细胞壁作为转化平台，可用于蔗糖转化高效转化为葡萄糖[79]；隐球酵母(Cryptococcus podzolicus) OY‑1产生20.7 g/L葡萄糖酸[80]。D-葡糖醛酸(glucuronic acid)的游离形式不稳定，一般以更稳定的呋喃环3, 6-内酯形式存在，Gupta等[81]通过在S. cerevisiae中异源表达肌醇氧化酶，第一次实现了在工程化酵母中，葡萄糖醛酸的从头合成；重组酵母S. cerevisiae Bga-4异源表达透明颤菌(Vitreoscilla)血红蛋白，提高了葡糖醛酸的合成量至6.4 g/L[82]。以上这些有机酸目前在酵母中的合成研究较少，但是这些有机酸都具有一定的应用前景，值得进一步挖掘。
3 总结与展望利用酵母产有机酸具有良好的优势和发展前景，来自不同种属的酵母，包括模式酵母S. cerevisiae和非常规酵母A. pullulans、C. sonorensis、C. tropicalis、C. podzolicus、K. lactis、S. stipitis、P. kudriavzevii、P. pastoris、Y. lipolytica、Z. bailii和Z. rouxii，这些酵母中既有天然酵母，也有工程酵母(表 1)。
| Organic acid | Structural formula | Molecular formula | Main applications | Substrate | Maximum production (g/L)* | Theoretical yield | Actual yield | Synthetic yeast host and related references |
| Glycolic acid |  |
C2H4O3 | Cosmetics and biopolymer precursor | Ethanol[60] | 15.0[60] | 1.65 g/g ethanol[60] | 0.52 g/g ethanol[60] | Kluyveromyces lactis[60], Saccharomyces cerevisiae[60-61] |
| Acrylic acid |  |
C3H4O2 | Coating, adhesives and detergents | – | – | – | – | – |
| Malonic acid |  |
C3H4O4 | Polymers and polyesters Precursors | Glucose[77] | 1.6[77] | - | - | S. cerevisiae[77] |
| 3- hydroxypropionic acid |
 |
C3H6O3 | Plastics, coatings, adhesives, and chemical precursor | Glucose[65] | 56.5[65] | 0.75 g/g glucose[65] | 0.31 g/g glucose[65] | S. cerevisiae[63-66], Pichia pastoris[67] |
| Lactic acid |  |
C3H6O3 | Food additives and polymer precursor | Glucose[50] | 154.0[50] | - | 0.72 g/g glucose[37] | Candida sonorensis[48], Kluyveromyces lactis[51], S. cerevisiae[43-46], Pichia kudriavzevii[50], P. pastoris[49] Zygosaccharomyces bailii[47] |
| Glyceric acid |  |
C3H6O4 | Drugs, surfactants, and polymers precursor | – | – | – | – | – |
| Butyric acid |  |
C4H8O2 | Food additive and feed supplement | – | – | – | – | – |
| Fumaric acid |  |
C4H4O4 | Polymer building block, food and feed additive | Glucose[37] | 93.9[37] | 1.29 g/g glucose[37] | 0.78 g/g glucose[37] | Aureobasidium pullulans[36], Scheffersomyces stipitis[34], S. cerevisiae[33], P. pastoris[37] |
| Succinic acid |  |
C4H6O4 | Polymer building block and chemical precursor | Crude glycerol[22] |
209.7[22] | - | - | S. cerevisiae[29], P. kudriavzevii[32], Yarrowia lipolytica[19-28, 30-31], Z. rouxii[38] |
| Malic acid |  |
C4H6O5 | Polymer intermediate and food additive | Glucose[38] | 74.9[38] | - | 0.39 g/g glucose[38] | S. cerevisiae[39, 41-42], P. pastoris[40], Z. rouxii[38] |
| Itaconic acid |  |
C5H6O4 | Coatings, detergents and polymer building block | Waste cooking oil[75] | 54.6[75] | - | 0.30 g/g waste cooking oil[75] | S. cerevisiae[73, 75], P. kudriavzevii[72], Y. lipolytica[71, 74] |
| a-ketoglutaric acid |  |
C5H6O5 | Chemicals precursor | – | – | – | – | – |
| Xylonic acid |  |
C5H10O6 | Polymer precursor | D xylose[78] | 171.0[78] | - | 1.00 g/g D xylose[78] | P. kudriavzevii[78] |
| Adipic acid |  |
C6H1004 | Nylon and polymer precursor | Glucose[53] | 12.1[53] | - | - | C. tropicalis[53], S. cerevisiae[54-56] |
| Galactonic acid |  |
C6H1207 | Detergents, solvents and paints | – | – | – | – | – |
| Gluconic acid |  |
C6H12O7 | Food additive and pharmaceutical ingredient | Glucose[80] | 20.7[80] | - | - | Cryptococcus podzolicus[80], S. cerevisiae[79] |
| Glucaric acid |  |
C6H10O8 | Detergent builder and polymer building block | Glucose[82] | 6.4[82] | - | - | S. cerevisiae[81-82] |
| Lactobionic acid |  |
C12H22O12 | Cosmetics, personal care and pharmaceutical products | – | – | – | – | – |
| –: Represents no research reported on organic acid production in yeast; *: Represents maximum organic acid production when yeast as host cell. | ||||||||
目前利用酵母菌产有机酸存在的问题是与其他微生物比较，有机酸浓度较低，副产物较多，未来可以在以下几个方面聚焦研究。
(1) 开发高效的有机酸生产底盘酵母细胞，提高非常规酵母遗传操作效率。多种非常规酵母具有天然较好的耐酸性，是良好的有机酸出发菌株，但很多非常规酵母的遗传改造效率还有待提高，未来随着精准基因组编辑技术的进展，有望快速提高酵母菌产酸效率。
(2) 深入理解酵母菌产酸的分子调控机制。目前对酵母菌产有机酸的研究多停留在途径酶和转运蛋白等研究上[83]，但是对酵母菌合成有机酸的分子调控机制还了解甚少，限制了代谢工程的高效改造。未来多组学技术的应用和酵母功能基因组学的进展将有助于进一步加深对酵母产有机酸的调控机制认识，提高代谢工程改造效率。
(3) 酵母有机酸生产目前很多停留在实验室研究，工业放大研究相关的认知还不够深入，比如，很多研究利用酵母粉蛋白胨等丰富培养基进行产酸评价，但工业生产的培养基相对贫瘠，利用木质纤维素水解液产有机酸还涉及到水解液中抑制物的影响等，因此需要进行系统性的菌种改造和工艺优化。
(4) 随着合成生物学、计算技术的应用发展，通过代谢网络计算模拟评估酵母产生有机酸的潜力，构建产酸菌株，必将有利于工业菌种的定向合成。
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