母乳是婴儿最优的营养物质,不仅支持新生儿生长,还含有促进适龄发育和提高免疫力的生物活性成分。世界卫生组织和美国儿科学会建议婴儿在出生后的第一个小时内便开始母乳喂养,而且建议在前6个月内纯母乳喂养,并持续2年[1-2]。母乳寡糖(human milk oligosaccharides, HMOs)是母乳中天然存在且结构多样的非结合聚糖,是仅次于乳糖和脂肪的第三大类固体成分[3]。HMOs的浓度在整个哺乳期存在变化,初乳中最高(平均质量浓度为 9～22 g/L),其次是过渡乳(平均质量浓度为8～19 g/L),然后随着泌乳的进行逐渐降低[4]。婴儿出生一个月内HMOs的质量浓度降至6～15 g/L,6个月后降至4～6 g/L[5]。HMOs具有多种生理功能,如防止病原体黏附、调节肠道屏障和免疫系统、促进益生菌生长、促进神经系统发育和修复[6-9]。

由于HMOs的安全性和重要功能特性,已有8种HMOs在美、欧等国家和地区获得批准为一般公认安全(generally recognized as safe, GRAS)食品。在国外,可作为新食品原料添加到食品中的HMOs分别是2′-岩藻糖基乳糖(2′-fucosyllactose, 2′-FL)、3-岩藻糖基乳糖(3-fucosyllactose, 3-FL)和二岩藻糖基乳糖(difucosyllactose, DFL)、乳酰-N-岩藻五糖Ⅰ(lacto-N-fucopentaose Ⅰ, LNFP Ⅰ)、乳酰-N-四糖(lacto-N-tetraose,LNT)、乳酰-N-新四糖(lacto-N-neotetraose,LNnT)、3′-唾液酸乳糖(3′-sialyllactose, 3′-SL)和6′-唾液酸乳糖(6′-sialyllactose, 6′-SL)。在商业应用方面,雅培公司在2016年率先推出添加2′-FL的婴幼儿配方奶粉,促进了配方奶粉的革新。此后,其他公司,如爱他美、惠氏、合生元、美赞臣、雀巢和杜邦等也相继推出添加HMOs的婴幼儿配方奶粉[9]。奶粉中添加HMOs推动了全球HMOs市场增长,预计到2028年HMOs市场总额将达到3.326亿美元,年复合增长率为14.1%[10]。我国作为全球第二大婴童消费市场,消费者对添加HMOs的高端婴幼儿配方奶粉需求很大。2023年10月7日,国内正式批准2′-FL和LNnT可作为食品营养强化剂添加到调制乳粉等食品中[11],这将大力推动国内生物合成HMOs的研究和应用。

1 HMOs基本组成及结构

近年来,分析方法和组学技术的快速发展有力推进了HMOs结构的解析[12]。目前已鉴定出200多种不同结构的HMOs,完全表征了160余种[13]。HMOs通常由葡萄糖(Glc)、半乳糖(Gal)、唾液酸(Sia)、L-岩藻糖(Fuc)和N-乙酰氨基葡萄糖(GlcNAc)五种单糖连接。5种单糖以不同组合形式出现,形成了结构复杂多样的HMOs[13]。依据单糖组成的不同,HMOs主要分为中性核心、岩藻糖基化和唾液酸化三大类,HMOs基本组成见图1。岩藻糖基化HMOs(fucosylated human milk oligosaccharides, FHMOs)的核心基团是Fuc,还原端总是由乳糖构成[14]。Fuc通过α-糖苷键连接到Gal、GlcNAc和Glc上[15],岩藻糖基化位点在位置2、3或4之间变化[14]。FHMOs约占HMOs的61%,主要包括2′-FL、3-FL、DFL、LNFPⅠ、乳酰-N-岩藻五糖Ⅱ(lacto-N-fucopentaose Ⅱ, LNFP Ⅱ)、乳酰-N-岩藻五糖Ⅲ(lacto-N-fuconeopentaose Ⅲ, LNFP Ⅲ)和乳酰-N-二岩藻六糖Ⅰ(lacto-N-difucohexaose Ⅰ, LNDFH Ⅰ),典型FHMOs在母乳中质量浓度见表1。中性核心HMOs约占HMOs的13%,以LNT、LNnT为主。唾液酸化HMOs在其末端含有唾液酸,约占HMOs的13%,以3′-SL和6′-SL为主[16]。

FHMOs是HMOs中含量最为丰富的寡糖,主要包括2′-FL、3-FL和DFL等,三者约占HMOs总量的40%[16]。3种FHMOs的结构式见图2。2′-FL和3-FL是同分异构体,分子质量为488.44 Da,2′-FL 是末端Fuc以α-1,2-糖苷键连接在D-乳糖(Galβ1-4Glc)的Gal基形成的三糖(Fucα1-2Galβ1-4Glc)。3-FL和2′-FL结构上的差异在于3-FL是以α-1,3-糖苷键将Fuc连接至D-乳糖的还原端Glc基而形成的三糖[Galβ1-4(Fucα1-3)Glc]。DFL分子质量为634.58 Da,是α-1,3-糖苷键将Fuc连接至2′-FL的还原端Glc基而形成的四糖[Fucα1-2Galβ1-4(Fucα1-3)Glc]。

2 FHMOs的生理功能

FHMOs能提高神经元之间交流和突触发生,从而促进婴儿大脑发育[17]。在一项针对马拉维接受FUT2阳性母乳喂养婴儿的研究中发现,母乳中 2′-FL 和DFL的浓度与婴儿语言发育呈正相关;相反,母乳中缺乏2′-FL和DFL的婴儿则表现出语言发育迟缓的现象[18]。肠道微生物对HMOs的利用需要一系列转运蛋白和降解酶系,只有少数微生物有能力分解所有HMOs [19]。例如,长双歧杆菌婴儿亚种在内源的2′-FL转运体协助下,能利用岩藻糖苷酶分解2′-FL [20]。HMOs作为益生元可保护婴儿免受病原微生物的侵害,肠道中的共生菌能竞争性排斥对抗病原微生物的侵入[21]。此外,肠道中的益生菌,如长双歧杆菌婴儿亚种代谢HMOs产生的有机酸会塑造一个酸性环境,从而抑制病原微生物的增殖[22]。FHMOs作为可溶性诱饵受体发挥作用,能间接或直接阻止病原微生物进入体内[23-24]。HMOs可以调节肠上皮细胞的凋亡、增殖和分化[25]。已证明FHMOs可以改变肠上皮细胞相关基因的表达,导致细胞表面糖萼的改变。除充当可溶性诱饵受体外,FHMOs还可以通过重新编程上皮细胞来改变糖萼受体的表达,从而影响病原微生物与宿主之间的相互作用[26]。肠细胞对2′-FL的吸收会影响上皮细胞的蛋白质表达,2′-FL涉及受体途径的细胞旁或跨细胞途径运输[27-28]。严重且致命的肠道疾病“新生儿坏死性小肠结肠炎”(necrotizing enterocolitis,NEC)会影响5%～10%的低体重早产儿,导致超过25%的死亡率[29-30]。FHMOs促进了NEC实验模型中产生黏蛋白-2(Muc2)杯状细胞的发育[31]。动物实验研究表明,2′-FL显著降低NEC新生大鼠模型的病理评分[32]。膳食补充2′-FL增加内皮一氧化氮合酶的表达来维持肠系膜灌注,从而显著降低新生小鼠实验性NEC的严重程度[33]。肥胖是现代人类社会面临的主要健康问题之一,其特点是体内脂肪积累和能量失衡[34]。研究表明,膳食补充HMOs能有效改善肥胖。高脂饮食喂养的小鼠灌胃2′-FL后减缓了体重增加,减轻了肥胖小鼠的脂质积累,增加了线粒体DNA含量,并上调了产热标志物蛋白质的表达[35-36]。人群实验表明,补充2′-FL显著降低肥胖受试者的体重,改善超重久坐者相关健康指标[37]。FHMOs的生理活性如图3所示。

3 FHMOs的生物合成

随着FHMOs的功能不断被挖掘和利用,FHMOs 在国内外的市场需求逐步提升,其制备方法包括母乳分离提取、化学合成、酶法合成和精密发酵法合成[38-39]。受限于伦理道德和原料来源,从母乳中分离提取FHMOs常用于结构鉴定,不适合商业化大规模生产。而其他哺乳动物的乳汁中含有复杂的低聚糖,其含量和组成与人源的FHMOs差异较大。因此,直接从哺乳动物的乳汁中分离提取FHMOs也是不可行的[39]。化学合成FHMOs需要多个活化、保护和脱保护步骤,以实现所需的糖苷键形成的立体和区域选择性,反应条件苛刻,大量合成FHMOs效率很低。此外,反应过程中需要使用多种有机试剂,会造成环境污染,并具有一定的危险性,这限制了化学法合成FHMOs在乳品等食品行业的应用[40]。酶法合成及精密发酵法,具有操作方便、环境友好、反应条件温和等优点,已成为目前合成FHMOs的研究重点和热点[39, 41-45]。

3.1 酶法合成FHMOs

酶法合成具有良好的立体和区域选择性,能够特异性地将糖基基团逐个加入糖基受体上,实现FHMOs的多途径合成[46]。酶法合成FHMOs可以分为基于岩藻糖苷酶的“一步酶法”和基于岩藻糖基转移酶(fucosyltransferases,FTs)的“一锅多酶法”,其合成方式如图4所示。

3.1.1 FHMOs的一步酶法合成

岩藻糖苷酶具有广泛的底物特异性,能够将农副产品中岩藻糖基化合物作为供体,实现体外“一步酶法”合成FHMOs[图4(a)]。α-L-岩藻糖苷酶(EC 3.2.1.51)是一种糖苷水解酶,可催化低聚糖和糖缀合物中L-岩藻糖残基的释放,广泛分布在动物、植物和微生物中。目前大多数岩藻糖苷酶来源于细菌,如拟杆菌属、类芽孢杆菌属(Paenibacillus sp.)、坦纳菌属(Tannerella sp.)、黄杆菌属(Xanthomonas sp.)、嗜热菌属(Thermotoga sp.)和双歧杆菌属[47-48]。根据氨基酸序列同源性,岩藻糖苷酶分为3个糖苷水解酶(glycoside hydrolases,GH)家族:GH29、GH95和GH151,其中,只有GH29家族的岩藻糖苷酶具有转岩藻糖基活性。根据序列同源性和底物特异性,GH29家族进一步分为2个亚家族:GH29A和GH29B。GH29A亚家族的岩藻糖苷酶只作用于岩藻糖基化底物,而GH29B亚家族的糖苷酶具有区域特异性,作用于具有α-1,3-或α-1,4-半乳糖分支的底物。α-L-岩藻糖苷酶Mfuc5以4-硝基苯基-α-L-岩藻吡喃糖苷(pNP-FUC)为供体合成2′-FL的得率为6.4%[49]。GH29家族的禾谷镰刀菌(Fusarium graminearum)来源的α-L-岩藻糖苷酶FgFCO1在大肠杆菌中表达,能以柑橘果皮来源的木葡聚糖为岩藻糖基供体和以乳糖为受体,2′-FL得率为14%[48]。此外,Zeuner等[50]对两双歧杆菌表达的α-1,3/4-L-岩藻糖苷酶(EC 3.2.1.111,GH29, BbAfcB)进行loop区域改造,几乎完全消除了BbAfcB对3-FL的水解活性。采用半理性设计的方式对双歧杆菌来源的α-L-岩藻糖苷酶进行定点突变,提高了野生型酶的转糖苷活性,获得了2′-FL和3-FL等多种FHMOs,但部分产物的得率仍为17%[17]。本实验室从土壤中自主选育出地杆菌(Pedobacter sp.)CAU209,并对其进行了基因组测序和注释。从地杆菌CAU209克隆出α-L-岩藻糖苷酶基因(PbFuc),在大肠杆菌中表达了可溶性PbFuc。该酶与GH29家族中几种α-L-岩藻糖苷酶的同源性低,特别是与P. thiaminolyticus岩藻糖苷酶的氨基酸序列同源性最高,为36.8%。PbFuc最适温度为35 ℃,具有较宽的pH稳定范围(pH值为4.0～11.0)。此外,该酶能够以pNP-FUC为岩藻糖基供体和乳糖为受体合成2′-FL和3-FL,产物得率分别为14.5%和70.5%[47]。基于定向进化技术,采用易错PCR对PbFuc进行随机突变,经过两轮筛选得到转化率高的突变酶(mPbFuc29A1),比野生型的最适温度提高了5 ℃。此外,通过序列及定点突变分析,发现mPbFuc29A1中有2个氨基酸发生替换,分别是 Asp21Val和Glu266Lys,其中位于loop区的Glu266Lys在mPbFuc29A1c底物特异性和转糖苷作用中发挥重要作用[51]。

3.1.2 FHMOs的一锅多酶法合成

FTs具有产率高、立体选择性强等优点,也广泛用于FHMOs的合成。基于FTs的“一锅多酶法”,L-岩藻糖在双功能酶L-岩藻激酶/二磷酸鸟苷(guanosine-5′-diphosphate,GDP)-L-岩藻糖焦磷酸化酶(fucokinase/GDP-L-fucose pyrophosphorylase, FKP)的催化下转化为GDP-L-岩藻糖,FTs将GDP-L-岩藻糖上的岩藻糖基转移到乳糖上合成FHMOs,如图4(b)至图4(c)所示。根据终产物中糖苷键的类型,FTs分为α-1,2-FTs、α-1,3-FTs、α-1,4-FTs和α-1,6-FTs,主要存在于植物、昆虫、蜗牛、寄生虫和哺乳动物等真核生物中。糖基转移酶11家族(glycosyltransferase family 11,GT11)包括α-1,2-FTs(FUT1和FUT2)以及其他许多受体底物未知的 α-1,2-FTs,但只有少数细菌GT11 FT能够在大肠杆菌中成功表达和发挥作用。在为数不多成功表达的α-1,2-FTs中,岩藻糖基的转移活性及底物特异性不够理想,如大肠杆菌O86来源的α-1,2-FT WbnK仅对Galβ-1,3GalNAc-α-OMe受体具有很高的亲和力,产物得率为75%[52]。大肠杆菌O126来源的α-1,2-FT WbgL仅对末端含β-1,4-半乳糖的底物具有良好的识别性[53]。α-1,3-FTs是合成3-FL和DFL的关键工具酶。DFL的合成则更为复杂,在合成GDP-L-岩藻糖和2′-FL的基础上,需要合适的 α-1,3-FTs将GDP-L-岩藻糖上的岩藻糖基转移到2′-FL上从而合成DFL。从哺乳动物或细菌中克隆表达的大多数α-1,3-FTs在大肠杆菌中的表达量很低,阻碍了3-FL的大规模合成。为提高α-1,3-FTs的溶解性和稳定性,删除幽门螺杆菌(Helicobacter pylori)NCTC 11639来源的FutA中含有疏水、带正电残基的α螺旋和部分七肽重复区,但α-1,3-FT的蛋白含量仍处于较低水平(4.4 ～15.0 mg/L)。另外一个幽门螺杆菌来源的α-1,3-FT具有高度严格的底物特异性,对N-乙酰氨基乳糖的特异性大于乳糖,这也限制了其在3-FL酶法合成中的应用[54]。截去C端66个氨基酸的FutA能够在大肠杆菌中成功表达,并可以利用不同底物合成FHMOs,但转岩藻糖基效率仅为30%[55]。

3.2 FHMOs的精密发酵法合成

精密发酵法合成HMOs主要基于代谢工程,利用微生物体内代谢途径和糖基转移酶的体内区域和立体选择活性,在微生物中过表达异源糖基转移酶基因和调节合成路径上其他相关酶基因的表达。近年来,由于合成生物学和系统生物学的快速发展,特别是分子生物学工具和代谢途径组装的进步,生物合成途径的改造更易于操作,从而使微生物细胞工厂生产HMOs从可能到实用。迄今,采用精密发酵法已合成了40多种HMOs,包括2′-FL、3-FL和DFL等 FHMOs。

3.2.1 FHMOs合成的底盘细胞

目前,FHMOs已在多种微生物底盘细胞中成功合成。大肠杆菌具有遗传背景清晰、生长迅速、培养简单等优点,是目前合成FHMOs研究中使用最多的底盘细胞[45],而这其中,又以大肠杆菌K-12和B系列菌株为主。实验室常用的大肠杆菌BL21(DE3)是通过将DE3原噬菌体整合到大肠杆菌BL21的λ位点而产生的。原噬菌体的DE3区域含有T7 RNA聚合酶,T7 RNA聚合酶合成RNA的速度是大肠杆菌自身RNA聚合酶的数倍。大肠杆菌BL21star(DE3)则是在大肠杆菌BL21(DE3)基础上进行了改造,mRNA稳定性增加,从而减少蛋白质降解,适用于高水平蛋白质生产[56]。大肠杆菌Nissle 1917是一种特殊的大肠杆菌菌株,无致病性,在医学上用作益生菌,用于治疗各种胃肠道疾病。目前已有少量利用大肠杆菌Nissle 1917合成HMOs的报道[57],Nissle 1917安全性高,在未来合成FHMOs的研究中具有优势。

除大肠杆菌外,其他常见微生物,如枯草芽孢杆菌(Bacillus subtilis)[58]、酿酒酵母(Saccharomyces cerevisiae)[59]、巴斯德毕赤酵母(法夫驹形氏酵母Komagataella phaffii,又名Pichia pastoris)[60]和谷氨酸棒杆菌(Corynebacterium glutamicum)[61]等也被用于FHMOs的生物合成。相较于大肠杆菌有产生内毒素的风险,这些微生物是GRAS菌株。然而,这些微生物体内大多数不具有GDP-L-岩藻糖合成通路,整体改造难度大于大肠杆菌。此外,酵母需要较长的发酵周期。

最近,美国加州大学伯克利分校Barnum等[62]改造模式植物本氏烟草(Nicotiana benthamiana)合成了2′-FL和LNFPⅠ。本氏烟草具有强大的糖代谢能力和较强的核苷酸糖合成能力,可利用光能将CO2转化为系列FHMOs。植物生产FHMOs后的生物质仍可用于生产乙醇,据计算,利用本氏烟草合成每公斤LNFPⅠ的成本为4.9～18.4美元,而微生物发酵制备每公斤LNFPⅠ的成本高达45美元[62]。因此,Barnum等[62]认为,利用本氏烟草等模式植物作为FHMOs生物制造平台具有成本优势。

3.2.2 FHMOs的合成通路

精密发酵法合成FHMOs的总体思路为过表达乳糖通透酶基因lacY和敲除半乳糖苷酶基因lacZ来提高底盘细胞对乳糖的转运能力,防止乳糖在胞内代谢流失;在微生物中构建合成GDP-L-岩藻糖通路以提供岩藻糖基受体;岩藻糖基转移酶将供体GDP-L-岩藻糖的岩藻糖基转移到乳糖受体上[63-65]。图5为在工程微生物中合成FHMOs的总体途径。在已知的合成途径中,GDP-L-岩藻糖和乳糖是生物合成FHMOs的直接前体[39]。GDP-L-岩藻糖在体内有两种合成途径:从头途径和补救途径[39, 63]。在从头合成途径中,来自中心碳代谢的果糖-6-磷酸通过5种内源性酶的催化转化为GDP-L-岩藻糖,所涉及的酶分别为甘露糖-6-磷酸异构酶(mannose-6-phosphate isomerase, ManA)、磷酸甘露糖突变酶(phosphomannomutase, ManB)、甘露糖-1-磷酸鸟苷转移酶(mannose-1-phosphate guanyltransferase, ManC)、GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-dehydratase, Gmd)和GDP-L-岩藻糖合酶(GDP-l-fucose synthase,WcaG/Fcl)[66]。补救途径由双功能酶FKP催化L-岩藻糖转化为GDP-L-岩藻糖[39]。与酶法合成FHMOs一样,从头途径或补救途径合成的GDP-L-岩藻糖在α-1,2-FTs(α-1,3-FTs)催化下将岩藻糖基转移到乳糖上从而合成2′-FL(3-FL)[40, 67]。之后,在胞内合成GDP-L-岩藻糖和2′-FL的基础上,异源表达合适的α-1,3-FTs将GDP-L-岩藻糖上的岩藻糖基转移到2′-FL上从而合成DFL[68-69]。

削弱或阻断旁路代谢对重要中间产物或底物的消耗是提高FHMOs合成的一个重要策略,这些中间产物或底物包括6-磷酸-果糖、GDP-L-岩藻糖、乳糖和岩藻糖(补救合成途径)。由于6-磷酸-果糖参与微生物碳中央代谢,完全阻断其到1,6-二磷酸-果糖的通路会影响菌株的生长。因此,以葡萄糖为碳源时,一般采用的策略是筛选合适强度的启动子调控6-磷酸果糖激酶Ⅰ PfkA或6-磷酸果糖激酶Ⅱ PfkB表达强度[70];而以甘油为碳源时,磷酸二羟丙基酮会部分进入碳中央代谢,此时则会敲除pfkA或pfkB。GDP-L-岩藻糖会参与可拉酸的合成,在合成FHMOs时敲除编码UDP糖脂载体转移酶基因wcaJ阻断GDP-L-岩藻糖到可拉酸的合成通路[43]。另外,转录因子RcsA和RcsB会调控GDP-L-岩藻糖合成通路基因的表达水平,而蛋白酶Lon则降解该转录因子。因此,要提高胞内GDP-L-岩藻糖的积累,还需敲除lon并过表达rcsA或rcsB。底物乳糖通常会被微生物降解用于生长,敲除代谢乳糖的基因lacZ,以及代谢岩藻糖的基因fucI(L-岩藻糖异构酶)、fucK(L-岩藻糖激酶)、rhaA(鼠李糖异构酶)和araA(D-阿拉伯糖异构酶),是提高FHMOs合成的有效策略[63]。

3.2.3 FHMOs合成的关键酶FTs

FHMOs生物合成的一个关键因素是FTs,乳糖的半乳糖残基均可被 α-1,2-FTs岩藻糖基化,末端葡萄糖残基可被α-1,3-FTs岩藻糖基化,从而形成不同聚合度和同分异构体的FHMOs。哺乳动物来源的FTs具有区域特异性和立体特异性,已被广泛表征和鉴定用于 FHMOs的合成。然而,这类FTs通常只能在真核表达系统中表达,细菌来源的FTs更容易在原核表达系统中表达,尤其是大肠杆菌。因此,研究人员广泛挖掘与哺乳动物酶具有相似催化活性和特异性的细菌来源FTs,目前大规模生产FHMOs的FTs几乎都来自细菌[58]。这些特异性FTs的筛选、结构分析、分子改造和强化可溶性表达是提高FHMOs生物合成的重点。

3.2.3.1 α-1,2-FTs

α-1,2-FTs在生物合成2′-FL中尤为重要。至少有10余种细菌来源的α-1,2-FTs主要被用于合成2′-FL,如来自幽门螺杆菌26695的FutC[71]、脆弱拟杆菌(Bacteroides fragilis)NCTC 9343的WcfB[72]、Azospirillum lipoferum的SAMT[73]、蜡样芽孢杆菌(B. cereus)VD107的FutBc[74]、大肠杆菌O126 的WbgL[53]、幽门螺杆菌11S02629-2的BKHT[75]、Thermosynechococcus elongatus的Te2FT[76]和奈瑟菌属(Neisseria sp.)的NsFutC[77]。这些α-1,2-FTs的氨基酸序列同源性比较低,为20%～60%[78]。大多报道的α-1,2-FTs具有一个明显的特征,即含有4个序列高度保守的基序,其中基序Ⅰ、Ⅱ和Ⅲ靠近C端,基序Ⅳ位于N端。基序I HxRRxD在哺乳动物来源的α-1,6-FTs中也是保守的,其晶体结构表明其作为供体 GDP-L-岩藻糖结合结构域的作用[79]。但目前尚无α-1,2-FTs晶体结构的报道,这些高度保守的基序与FTs催化机制的关系仍不清楚。

大多数α-1,2-FTs的催化活性较低,难以在微生物系统中以可溶性形式表达,从而限制了工程菌株合成2′-FL的效果[80-81]。研究人员采取多种策略来解决这些问题,包括优化核糖体结合位点(ribosomebinding site,RBS)[82]、筛选合适的蛋白质标签与α-1,2-FTs融合表达[83]以及优化α-1,2-FTs表达强度[64]。这些策略能在一定程度上提高工程菌合成2′-FL的能力。以催化效率较好的α-1,2-FTs为模板,通过序列比对和结构可视化分析从数据库筛选的方式是挖掘高催化效率的α-1,2-FTs的一种有效方式[84]。如SAMT、BKHT和NsFutC等都是通过这种方式挖掘得到的,基于这3种酶的工程菌株合成的2′-FL产量分别能达到112.6、94.7、141.3 g/L[73, 75, 77]。对α-1,2-FTs进行分子改造,提高其可溶性表达水平或催化能力,也是增加2′-FL合成产量的另一种有效方法[85]。由于缺乏α-1,2-FTs晶体结构,采用理性设计方法分子改造α-1,2-FTs较困难。目前,仅有少数报道采用半理性设计方法分子改造α-1,2-FTs[85-86],但得到的突变体在体内促进2′-FL合成效果有限。

α-1,2-FTs底物特异性直接决定了产物的2′-FL纯度和底物转化效率。由于2′-FL和3-FL是同分异构体,且与DFL难以有效分离,通过常规液相色谱或质谱法很难准确检测。最近,Zhu等[75]使用高效阴离子交换色谱同时检测这3种 FLs,证明FucT2和WbgL具有α-1,3-FTs活性,在合成2′-FL的同时会产生DFL或3-FLs作为次要产物。通过这种方式,研究人员证明SAMT和BKHT具有高效的α-1,2-FTs活性,同时无α-1,3-FTs活性。

3.2.3.2 α-1,3-FTs

最早鉴定的α-1,3-FTs来源于幽门螺杆菌,分别为FutA和FutB,两者之间的同源性高达97%。FutA对LNnT受体具有更好的活性,主要产物为Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,而FutB对Lex[Galβ1,4(Fuc a 1,3)GlcNAc]合成的LacNAc表现出更好的活性[87]。部分α-1,3-FTs有一个显著的特征,在C端附近有2～10个七肽重复序列,而C端则富含疏水的氨基酸残基,形成的2个α-螺旋充当膜锚,疏水表面嵌入膜内[14]。将2个α-螺旋截断后,α-1,3-FTs在胞内的可溶性表达有所提高,并显著提高3-FL产量[88-89]。目前,用于3-FL合成的α-1,3-FTs相对较少。最具代表性的是FutA、FutM2和FucTa[90]。FucTa与FutA序列同源性较高,底物谱较窄,因此适合在大肠杆菌中合成DFL而非 3-FL[69, 91]。 最近的一项研究中,研究者选择了9种α-1,3-FTs,发现来自B. gallinaceum的FutM2合成3-FL效果最佳[92]。同样,分子改造α-1,3-FTs是增加3-FL合成产量的另一种有效方法。采用配体结合位点多样性和蛋白质结构模拟的分析相结合方式对FucTa进行分子改造,得到最佳突变体Y218K。基于突变体Y218K的工程菌在摇瓶中3-FL产量为7.55 g/L,在5 L发酵罐中3-FL的产量达36.98 g/L。活性增强归因于Lys残基侧链为乳糖结合提供结构支持并形成盐桥,从而增加了FucTa对其底物的亲和力[93]。

一对匹配良好的FTs是实现DFL的高效生物合成的关键,由于WbgL和FucTa受体选择性较窄,适用于合成DFL。通过表达基因lacY和L-岩藻糖转运蛋白基因fucP增强底物的转运,优化发酵条件后,工程菌产DFL达5.1 g/L[69]。此外,还可利用α-1,2-FTs的底物混杂性合成DFL。在大肠杆菌BL21star(DE3)中异源表达基因fkp和fucT2,从而构建了2′-FL的补救合成途径,此时,工程菌在合成2′-FL时会产生少量的DFL。为了减少胞内2′-FL的积累,同时增加底物乳糖和L-岩藻糖与酶的相互作用,将工程菌的天然信号肽PelB替换为P-PLA2,部分FKP和FucT2分泌到培养基中。在胞外的FKP和FucT2可从乳糖和L-岩藻糖合成DFL,工程菌ΔLFAR-YA/FF+P-PLA产DFL为1.22 g/L[94]。在大肠杆菌 BL21(DE3)中过表达基因簇manBC、gmd-wcaG并异源表达一对FTs基因fucT和fucTa,从而实现了DFL的从头合成。为进一步提高工程菌合成DFL的水平,基于计算机辅助筛选和基于结构的合理设计对FucTa进行分子改造,表达最佳突变体MH5(F49K/Y131D/Y197N/E338D/R369A)的工程菌BLC09-58在5 L发酵罐中产DFL可达45.8 g/L[95]。 近期,研究人员采用SAMT和Fut3Bc合成DFL,过表达转录因子基因簇rcsAB可增加GDP-L-岩藻糖的积累,得到的工程菌CS-10在5 L发酵罐中产DFL可达53.2 g/L[96]。此外,合成通路精细调控也能实现高产DFL的同时尽量减少FL的残留,通过优化2′-FL合成,增加α-1,3-FTs的表达,减少2′-FL的分泌,工程菌BSF41在5 L发酵罐中可产55.3 g/L的DFL,残留的FL仅为2.59 g/L[65]。

3.2.4 其他合成策略

生物合成FHMOs需要消耗GTP和NADPH,因此,提高GTP和NADPH的再生速率是提升FHMOs生物合成的重要策略[64, 97]。过表达Zwf后,NADPH的再生得到改善,2′-FL和3-FL产量分别增加了82.2%和87.9%[97]。同时过表达Gsk和Zwf后,可有效促进GTP和NADPH的再生,2′-FL产量提高了53.8%[98]。此外,通路酶的合理空间组装也能显著提高FHMOs的生物合成。使用短肽对 RIAD-RIDD与2′-FL补救合成途径Fkp和FutC融合表达,可形成自组装多酶复合物。优化肽对空间定位和酶组装体的化学计量,进一步优化合成体系,2′-FL 产量提高了1.56倍。采用基于Mind C-tag的方式能将通路酶定向锚定在细胞膜上,当Mind C-tag 与WbgL融合表达时,2′-FL 产量提高了24%[61]。此外,优化转运蛋白改善FHMOs的胞外分泌是提高其生物合成的有力策略。过表达大肠杆菌内源性转运蛋白基因SetA,加快工程菌分泌2′-FL和3-FL,其产量显著增加[70, 99]。

4 结论与展望

尽管FHMOs的生物合成已取得很大进展,许多品种实现了工业化生产,但仍存在一些不足。2′-FL合成时由于部分α-1,2-FTs具有底物混杂性,合成的产物中混杂着DFL等杂质,不利于2′-FL的下游分离纯化,同时也会影响乳糖的转化效率。3-FL的生物合成报道较少且产量偏低。此外,缺乏更高效的α-1,3-FTs是制约3-FLs合成的关键因素之一。DFL的生物合成更为复杂,近几年生物合成DFL的报道增加,其产量明显提升,但DFL合成过程中有较多的中间产物和副产物残留,制约了其产量提升,并影响了乳糖转化效率。

未来,针对生物合成FHMOs的不足,需挖掘高效的α-1,3-FTs,进一步提升3-FLs的合成,同时可避免DFL生物合成时产生3-FL。另外,还需将合成通路插入底盘细胞基因组中,从而可减少抗生素使用和提高菌株的稳定性。此外,需开发基于机器学习的代谢建模,并结合多组学数据分析精确识别关键基因,以改善目标FHMOs的生产。同时,还需要解析更多岩藻糖基转移酶的晶体结构,阐明岩藻糖基化反应的确切机制。进一步优化FHMOs生物合成的发酵和纯化工艺,降低生产成本,将有助于 FHMOs 在功能性食品和医学领域的广泛应用。

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Advances in Biosynthesis of Fucosylated Human Milk Oligosaccharides

JIANG Zhengqiang, LIANG Shanquan, YANG Shaoqing

(College of Food Science and Nutritional Engineering/Key Laboratory of Food Bioengineering, China National Light Industry, China Agricultural University, Beijing 100083, China)

Abstract：Fucosylated human milk oligosaccharides (FHMOs), mainly including 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), and difucosyllactose (DFL), are the most abundant neutral human milk oligosaccharides in breast milk. FHMOs have multiple physiological functions, such as preventing pathogen adhesion, regulating the immune system, promoting probiotic growth, and promoting neurological development. The biosynthesis methods, including enzymatic and precision fermentation, have the advantages of easy operation, environmental friendliness, and mild reaction conditions, and have become a research focus and hotspot for the synthesis of FHMOs. Herein, the latest research progress in the biosynthesis of FHMOs by enzymatic and precision fermentation was systematically summarized. The contents focused on the microbial chassis cells used for FHMOs synthesis, FHMOs synthesis pathways and bypass inhibition, as well as the exploration and modification of fucosyltransferases. The microbial chassis cells used for FHMOs synthesis were introduced. Escherichia coli was the most commonly used chassis cell, followed by microorganisms such as Bacillus sp. and yeasts, and emerging chassis cells were also widely developed, such as Nicotiana benthamiana. The FHMOs synthesis pathway and bypass inhibition were analyzed. The overall idea was to enhance the gene expression level of the FHMOs synthesis pathway and weaken the consumption of carbon resources by bypass metabolism, while balancing the coordinated expression of genes in the synthesis pathway and carbon central metabolism pathway. Subsequently, the exploration and modification of fucosyltransferases were discussed. Various bioinformatics methods were used to discover efficient and highly specific fucosyltransferases, which were effective methods to enhance the biosynthesis of FHMOs. Rational/semi rational molecular modification was used to further improve the performance of fucosyltransferases, which could maximize the production and product singularity of FHMOs. Finally, the future trends of FHMOs biosynthesis were proposed.

Keywords：fucosylated human milk oligosaccharides; biosynthesis; enzymatic synthesis; precision fermentation; chassis cells; fucosyltransferase; molecular modification

doi：10.12301/spxb202500386

文章编号：2095-6002(2025)05-0001-14

引用格式：江正强,梁山泉,杨绍青.岩藻糖基化母乳寡糖的生物合成研究进展[J]. 食品科学技术学报,2025,43(5):1-14.

JIANG Zhengqiang, LIANG Shanquan, YANG Shaoqing. Advances in biosynthesis of fucosylated human milk oligosaccharides[J]. Journal of Food Science and Technology, 2025,43(5):1-14.

中图分类号：TS201.3; TS252.1

文献标志码：A

收稿日期：2025-07-21

基金项目：国家自然科学基金重点项目(22338013)。

Foundation：National Natural Science Foundation of China (22338013).

第一作者：江正强,男,教授,博士,主要从事食品生物技术方面的研究。

(责任编辑：叶红波)