首頁資訊微生物合成奇數(shù)鏈脂肪酸研究進(jìn)展*

微生物合成奇數(shù)鏈脂肪酸研究進(jìn)展*

來源：泰然健康網(wǎng) 時(shí)間：2024年12月25日 09:08

奇數(shù)鏈脂肪酸(odd-chain fatty acids,OCFA)是指碳鏈長度為奇數(shù)的脂肪酸,廣泛存在于微生物和動(dòng)植物中,但含量很低。在食品領(lǐng)域,OCFA可作為食品添加劑使用[1],有研究認(rèn)為OCFA是潛在的膳食必需化合物[2]。在醫(yī)藥健康領(lǐng)域,OCFA屬于高附加值脂肪酸,在人體健康和醫(yī)學(xué)領(lǐng)域有巨大的應(yīng)用潛力,如抗炎[3]、治療心肌病[4]或作為生物標(biāo)志物評(píng)估飲食攝入量、冠心病風(fēng)險(xiǎn)和Ⅱ型糖尿病[5-6]。目前針對(duì)OCFA生物活性和理化性質(zhì)的研究日益深入,因此未來可能發(fā)現(xiàn)OCFA更多的醫(yī)用功效。在生物能源領(lǐng)域,生物柴油中加入不同質(zhì)量和組分的OCFA可以改善生物柴油的質(zhì)量,提高經(jīng)濟(jì)效益。

目前,OCFA的生產(chǎn)方法主要包括化學(xué)合成[7]、天然產(chǎn)物提取和微生物發(fā)酵[8]。化學(xué)合成法和天然產(chǎn)物提取法需要使用大量化學(xué)品試劑,成本高且效率低,微生物發(fā)酵法則是最有潛力工業(yè)化生產(chǎn)OCFA的方法之一。微生物發(fā)酵法生產(chǎn)高附加值產(chǎn)物具有成本低、生產(chǎn)周期短和環(huán)境友好等優(yōu)勢,與基因工程和發(fā)酵調(diào)控工藝等手段相結(jié)合能夠?qū)崿F(xiàn)對(duì)微生物生產(chǎn)性能和發(fā)酵狀態(tài)的精確調(diào)控,從而達(dá)到高性能生產(chǎn)和高密度發(fā)酵的目的。運(yùn)用基因工程和發(fā)酵調(diào)控策略提升OCFA產(chǎn)量已有許多報(bào)道?；蚬こ滩呗灾饕性诖龠M(jìn)OCFA合成先導(dǎo)物丙酰輔酶A的合成和優(yōu)化關(guān)聯(lián)代謝途徑方面,包括表達(dá)關(guān)鍵酶[9]、阻斷旁路代謝途徑[10]、從頭模塊化構(gòu)建合成途徑[11]等;發(fā)酵調(diào)控策略則兼顧了OCFA合成所需最適碳源和發(fā)酵工藝調(diào)控等方面,如以丙酸等三碳物質(zhì)為碳源促進(jìn)丙酰輔酶A合成[12],分批補(bǔ)料發(fā)酵[13]優(yōu)化發(fā)酵狀態(tài)或兩相發(fā)酵促進(jìn)產(chǎn)物的及時(shí)分離[14]。本文綜述了多種能夠合成OCFA的微生物及其內(nèi)源合成途徑、基因工程改造和代謝調(diào)控策略,并對(duì)可深入研究的領(lǐng)域進(jìn)行討論。

1 OCFA的應(yīng)用

近年來,OCFA因藥理作用在醫(yī)藥與健康領(lǐng)域受到日益廣泛的關(guān)注。有研究發(fā)現(xiàn),奇數(shù)鏈脂肪酸可治療銀屑病、過敏或自身免疫性疾病,防止介質(zhì)釋放和抑制淋巴細(xì)胞激活[15]。Dojolvi(triheptanoin)是一種高純度的七碳脂肪酸甘油三酯,在體內(nèi)能夠代謝成丙酰輔酶A,進(jìn)而合成琥珀酰輔酶A參與到TCA循環(huán)(tricarboxylic acid cycle)中,現(xiàn)已作為治療兒童和成人長鏈脂肪酸氧化障礙(long chain fatty acids oxidation disorder,LC-FAOD)的藥物被FDA(Food and Drug Administration)批準(zhǔn),同時(shí)也被研究用于治療一系列其他代謝紊亂或涉及能量缺乏的疾病[16]。Avis等[17]報(bào)道了Pseudozyma flocculosa合成具有抗真菌活性的9-十七烯酸和6-甲基-9-十七烯酸,能夠誘導(dǎo)白粉病分生孢子鏈的快速崩解和一些真菌細(xì)胞的細(xì)胞質(zhì)解體,具有作為生物防治劑的應(yīng)用潛力。OCFA在健康領(lǐng)域可以作為評(píng)估膳食攝入的生物標(biāo)記物。OCFA在人體內(nèi)是由腸源性丙酸鹽內(nèi)源合成,而丙酸鹽又與膳食纖維的攝入呈正相關(guān),因此人體內(nèi)OCFA的含量與膳食攝入呈正相關(guān)[18-19]。Aglago等[20]探討了血清磷脂F(xiàn)As(serum phospholipid fatty acids,S-PLFAs)和肥胖指標(biāo)的關(guān)系。通過對(duì)372名墨西哥女性的對(duì)照研究發(fā)現(xiàn),S-PLFAs中的OCFA(C15:0,C17:0)與所有的肥胖指標(biāo)呈負(fù)相關(guān)。在工業(yè)應(yīng)用方面,OCFA及其衍生物作為原料或中間體被廣泛應(yīng)用于生產(chǎn)農(nóng)藥、香料、化妝品、涂料和工業(yè)化用品[21]。表1對(duì)OCFA的應(yīng)用進(jìn)行了簡要總結(jié)。

表1 OCFA應(yīng)用概括

Table 1 Summary of application of OCFA

OCFA的種類用途作用機(jī)理參考文獻(xiàn) 十三烷二酸(tridecanedioic acid) 用于合成透明聚酰胺與4,4'-二氨基二環(huán)己基甲烷(4,4'-diaminodicyclohexyl methane,PACM)成鹽進(jìn)行反應(yīng) [22] 壬二酸(azelaic acid) 治療黃褐斑、痤瘡病、惡性色素病競爭性酪氨酸酶抑制劑 [23-24] (9Z)-9-十七碳烯酸[(9Z)-9-heptadecenoic acid] 治療牛皮癬、過敏、自身免疫疾病阻止或減少TNF-α等介質(zhì)的釋放,抑制淋巴細(xì)胞活化,刺激巨噬細(xì)胞,使炎癥過程正?；?[15] 庚烷酸(heptanoate) 抗驚厥、治療癲癇病;長鏈脂肪酸氧化紊亂代謝成C5酮、β-酮戊酸鹽或β-羥基戊酸鹽,通過一元羧酸轉(zhuǎn)運(yùn)體進(jìn)入大腦;提供血管間質(zhì)代謝物,取代缺乏的三羧酸循環(huán)中間體;提高有效的能量代謝,顯著改善心臟結(jié)構(gòu)和功能 [4,25] 十五烷酸(pentadecanoic acid);十七烷酸(heptadecanoic acid) 與心血管疾病、肥胖癥、Ⅱ型糖尿病的發(fā)病呈負(fù)相關(guān) OCFA能夠降低患Ⅱ型糖尿病的風(fēng)險(xiǎn);血漿磷脂中的C15:0和C17:0濃度與心血管疾病和肥胖指標(biāo)呈負(fù)相關(guān) [25-26] 十五烷酸(pentadecanoic acid) 評(píng)估乳脂攝入的標(biāo)記物 OCFA來源于瘤胃微生物發(fā)酵或微生物從頭合成,然后轉(zhuǎn)入宿主動(dòng)物,表現(xiàn)為膽固醇、磷脂、血清和脂肪組織中的C15:0的相對(duì)含量與乳脂攝入呈正相關(guān) [27-28] 十五烷酸(pentadecanoic acid) 對(duì)人乳腺癌MCF-7/SC細(xì)胞具有選擇性的細(xì)胞毒性作用抑制IL-6誘導(dǎo)的JAK2/STAT3信號(hào)通路,誘導(dǎo)細(xì)胞周期阻滯在sub-G1期,并促進(jìn)MCF-7/SC中半胱天冬酶依賴性細(xì)胞凋亡 [29] 十五烷酸(pentadecanoic acid) 減輕炎癥、貧血、血脂異常和體內(nèi)纖維化可能是通過與關(guān)鍵代謝調(diào)節(jié)劑結(jié)合和修復(fù)線粒體功能 [30] 十一烷酸(undecanoic acid);十五烷酸(pentadecanoic acid) 抑制癌細(xì)胞增殖對(duì)組蛋白去乙?；妇哂幸种谱饔?能夠劑量依賴性地促進(jìn)MCF-7乳腺癌和A549肺癌細(xì)胞中α-微管蛋白的乙?；?[31]

2 OCFA微生物合成途徑

OCFA廣泛分布于自然界中,包括植物、海洋生物和微生物等。植物中椴樹籽油OCFA的含量占總脂肪酸含量的4.95%~9.39%左右[32]。海洋生物中的OCFA含量略高于植物,如海參中的OCFA含量最高能達(dá)30%左右[33]。目前已報(bào)道的天然合成OCFA的微生物種類繁多,包括細(xì)菌類如大腸桿菌[9]、渾濁紅球菌[34]、瘤胃細(xì)菌[35];真菌類如念珠酵母(Candida sp.)、多孢克魯維酵母(Kluyveromyces polysporus)、粘紅酵母(Rhodotorula glutinis)、釀酒酵母(Saccharomyces cerevisiae)、圓孢酵母(Torulaspora delbrueckii)、皮狀絲孢酵母(Trichosporon cutaneum)、解脂耶氏酵母(Yarrowia lipolytica)[36]、裂殖壺菌[37];放線菌類如鏈霉菌(Streptomyces cinnamonensis)[38]等。微生物得益于其獨(dú)特的代謝機(jī)制,能夠天然合成更高比例的OCFA,因此微生物發(fā)酵生產(chǎn)OCFA具有先天的優(yōu)勢。越來越多基因編輯工具的開發(fā)和完善[39-40],也使對(duì)更多微生物遺傳特性的改造成為了可能。通過對(duì)微生物進(jìn)行合理設(shè)計(jì)改造,將突破原有生產(chǎn)能力的限制,展現(xiàn)巨大的生物合成潛力。圖1總結(jié)了一些常見脂肪酸的化學(xué)結(jié)構(gòu)式。

圖1 常見奇數(shù)鏈脂肪酸和偶數(shù)鏈脂肪酸的化學(xué)結(jié)構(gòu)

Fig.1 Chemical structures of common odd-chain and even-chain fatty acid

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在許多自然途徑中,碳骨架經(jīng)中心代謝流最先形成,然后從中生成不同化學(xué)結(jié)構(gòu)的化合物,進(jìn)一步被不同的修飾酶修飾后形成具有不同生物活性的物質(zhì)。葡萄糖經(jīng)糖酵解途徑(embden-meyerhof-parnas pathway,EMP)和磷酸戊糖途徑(hexose monophophate pathway,HMP)被轉(zhuǎn)化為丙酮酸,丙酮酸隨后進(jìn)入線粒體氧化脫羧形成乙酰輔酶A(acetyl-CoA)。乙酰輔酶A經(jīng)乙酰輔酶A羧化酶催化與二氧化碳反應(yīng)生成丙二酰輔酶A(malonyl-CoA)。丙二酰輔酶A由丙二酰輔酶A?；d體蛋白(malonyl-CoA-ACP)轉(zhuǎn)酰基酶催化生成丙二酰-ACP(malonyl-ACP),丙二酰-ACP和乙酰輔酶A在脂肪酸合成鏈延長循環(huán)中合成OCFA。丙酰輔酶A是OCFA合成的關(guān)鍵前體物,促進(jìn)丙酰輔酶A的合成被認(rèn)為是提升OCFA產(chǎn)量的有效策略[41],相關(guān)的研究包括改造局部代謝途徑增大流向丙酰輔酶A的代謝流,阻斷丙酰輔酶A的旁路代謝和全局代謝優(yōu)化增強(qiáng)總體代謝強(qiáng)度。圖2以Y. lipolytica為例闡明了脂肪酸內(nèi)源合成及代謝機(jī)制,并對(duì)相關(guān)功能基因進(jìn)行簡要說明。

圖2 Y. lipolytica中脂肪酸內(nèi)源合成及代謝途徑

Fig.2 Endogenous synthesis and metabolic pathways of fatty acids in Y. lipolytica

Bolded blue font indicates important intermediate metabolites of the odd-chain fatty acid synthesis pathway; Blue genes indicate overexpression targets; red genes indicate knockout targets; GPD1, encoding NAD+-dependent glycerol-3-phopshate dehydrogenase; GUT2, encoding glycerol-3-phosphate dehydrogenase; DGA1/DGA2, encoding diacylglycerol transferase; LRO1, encoding triacylglycerol synthases; TGL3/TGL4, encoding triacylglycerol lipases; FAA1, encoding acyl-CoA synthetases; PXA1/PXA2, encoding peroxisomal acyl-CoA transporter; POX1-6, encoding the six acyl-CoA oxidases; PEX10, encoding peroxisomal membrane E3 ubiquitin ligase; MFE1, encoding the multifunctional enzyme; POT1, encoding peroxisomal 3-oxoacyl-CoA-thiolase; ACS1, encoding acetyl-CoA synthetase; ACC1, encoding acetyl-CoA carboxylase; ACL1, encoding ATP-citrate lyase genes; PHD1, encoding 2-methylcitrate dehydratase

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在某些植物中存在α-氧化將偶數(shù)鏈脂肪酸脫氫、脫羧形成脂肪醛,然后在水的參與下氧化脫氫形成OCFA及其衍生物[42]。借鑒上述思路,有報(bào)道在大腸桿菌和酵母中表達(dá)α-雙加氧酶,將中長鏈的脂肪酸氧化為2-氫過氧脂肪酸,隨后脫羧形成奇數(shù)鏈的脂肪醛或脂肪醇[43-44]。OCFA的合成路徑中存在一些關(guān)鍵的限速酶,如乙酰輔酶A羧化酶、丙酰輔酶A合酶、β-酮脂酰-ACP合成酶[45],這些酶的表達(dá)水平對(duì)于OCFA的合成有著重要影響。為實(shí)現(xiàn)OCFA的高效合成,不僅要對(duì)關(guān)鍵酶和代謝途徑進(jìn)行改造,更要對(duì)全局或局部代謝網(wǎng)絡(luò)進(jìn)行調(diào)控與優(yōu)化,消除或弱化脅迫因子和平衡產(chǎn)物合成與微生物生長的關(guān)系,這也是目前相關(guān)研究的熱點(diǎn)。

3 工程策略促進(jìn)微生物合成OCFA

3.1 基因工程策略

在分子水平上對(duì)基因進(jìn)行操作改變遺傳特性是目前提升OCFA產(chǎn)量的強(qiáng)有力手段。丙酮酸是碳代謝流的重要中間物,也是OCFA合成的關(guān)鍵前體物。丙酮酸經(jīng)檸檬酸循環(huán)合成乙酰輔酶A,乙酰輔酶A與丙二酰-ACP在β-酮脂酰-ACP合酶催化下反應(yīng)合成乙酰乙酰ACP,這也是脂肪酸鏈延長的起始反應(yīng)。某些情況下,β-酮脂酰-ACP合酶也能催化丙酰輔酶A和丙二酰-ACP反應(yīng)生成乙酰乙酰ACP(圖3)。因此,OCFA的合成既受到細(xì)胞整體代謝強(qiáng)度的影響,也受到一些關(guān)鍵酶和調(diào)節(jié)基因的調(diào)控,并且一些支路代謝途徑也會(huì)影響OCFA的合成。針對(duì)這些影響因素,通過基因工程方法對(duì)微生物進(jìn)行理性改造是目前調(diào)控OCFA合成的重要手段[8]。利用基因工程增強(qiáng)OCFA合成的研究主要集中在四個(gè)方面: (1)增強(qiáng)重要前體物的合成和積累; (2)阻斷脂肪酸降解和推動(dòng)三酰甘油(triacylglycerol,TAG)合成;(3)模塊化重構(gòu)相關(guān)合成途徑;(4)關(guān)鍵酶的表達(dá)調(diào)控。

圖3 脂肪酸合成起始路徑

Fig.3 The initiation pathway of fatty acid synthesis

accABCD, encoding acetyl-CoA carboxylase; fabA, encoding 3-hydroxydecanoyl-ACP dehydratase; fabB, encoding beta-ketoacyl-ACP synthase; fabD, encoding malonyl-CoA: ACP transacylase; fabF, encoding 3-oxoacyl-ACP synthase Ⅱ; fabG, encoding 3-oxoacyl-ACP reductase; fabH, encoding beta-ketoacyl-ACP synthase III; fabI, encoding enoyl-ACP reductase

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3.1.1 增強(qiáng)重要前體物的合成和積累

丙酰輔酶A是OCFA合成的重要前體物,因此所有提升丙酰輔酶A合成的手段都能促進(jìn)OCFA的合成。針對(duì)促進(jìn)OCFA合成的丙酰輔酶A相關(guān)代謝途徑包括甲基丙二酰輔酶A途徑、蘇氨酸途徑、丙酸途徑、檸檬酸/2-氧代丁酸途徑、天冬氨酸/2-氧代丁酸途徑、新型3-羥基丙酸鹽途徑等。有研究在培養(yǎng)基中補(bǔ)加0.5%~1.5%(v/v)的1-丙醇,使Rhodococcus opacus PD630的油脂產(chǎn)量由1.27 g/L增加至1.31~1.61 g/L,其中OCFA含量增加46.7%~55.1%[34]。檢測中間代謝產(chǎn)物和轉(zhuǎn)錄組分析表明,1-丙醇通過甲基丙二酰輔酶A途徑被R. opacus PD630同化。當(dāng)環(huán)境中氮源有限時(shí),丙酰輔酶A被轉(zhuǎn)化為丙酰-?；d體蛋白-ACP作為脂肪酸合成延長過程中的先導(dǎo)物,然后在循環(huán)中加入奇數(shù)個(gè)丙酰基-ACP時(shí),就能促進(jìn)OCFA的合成。蘇氨酸位于丙酰輔酶A合成途徑的上游,蘇氨酸合成基因的表達(dá)上調(diào)曾被用于提高大腸桿菌中丙酰輔酶A的積累。Lee等[46]發(fā)現(xiàn)通過在大腸桿菌中引入蘇氨酸合成途徑可以增加OCFA的水平,尤其在同時(shí)表達(dá)突變高絲氨酸脫氫酶時(shí),大腸桿菌中OCFA的比例從小于1%增至18%。Tseng等[47]通過過表達(dá)大腸桿菌thrAfrBC操縱子和ilvAfr基因,上調(diào)蘇氨酸的生物合成,構(gòu)建了一條由葡萄糖或甘油合成丙酰輔酶A的途徑。

通過丙酸途徑促進(jìn)丙酰輔酶A的積累是最早用于提升OCFA產(chǎn)量的前體工程策略。Ingram等[48]研究表明,外源性的丙酸可以作為OCFA合成的先導(dǎo)物,丙酸可以通過丙酸輔酶A合酶轉(zhuǎn)化為丙酰輔酶A,丙酰輔酶A與丙二酰輔酶A在OCFA合成的第一步縮合。有研究通過增強(qiáng)Y. lipolytica中三碳(丙酰輔酶A)和五碳(β-酮戊酰輔酶A)中間產(chǎn)物的積累以提高OCFA的合成。通過評(píng)估不同來源的丙酰輔酶A活化酶,如丙酰輔酶A轉(zhuǎn)移酶(Cppct和Repct)或丙酰輔酶A合成酶(SeprpE)對(duì)OCFA合成的影響,結(jié)合表達(dá)β-酮脂酰輔酶A硫解酶平衡乙酰輔酶A和丙酰輔酶A的前體,在優(yōu)化C/N比的培養(yǎng)基中獲得了1.87 g/L的OCFA產(chǎn)量,這也是目前為止在酵母中最高的產(chǎn)量報(bào)道[49]。在Y. lipolytica中,Park等[10]通過敲除編碼2-甲基檸檬酸脫水酶的合成基因PHD1,抑制2-甲基檸檬酸途徑增強(qiáng)丙酰輔酶A的積累,使OCFA在總脂肪酸中的比例提高至46.82%。此外,也有許多研究表明,丙酰輔酶A可經(jīng)檸檬酸/2-氧代丁酸途徑、天冬氨酸/2-氧代丁酸途徑、新型3-羥基丙酸鹽途徑合成,這也為后續(xù)增強(qiáng)丙酰輔酶A合成的研究方向提供了理論依據(jù)[50]。

3.1.2 推動(dòng)三酰甘油合成和阻斷脂肪酸降解

產(chǎn)油酵母具有強(qiáng)大的脂質(zhì)合成和積累能力,一直以來是微生物發(fā)酵合成OCFA的首選底盤菌。以Y. lipolytica為例,Y. lipolytica體內(nèi)可以產(chǎn)生大量脂質(zhì)體以積累脂肪酸,其中脂肪酸的降解完全通過過氧化物酶體中的β-氧化進(jìn)行[51]。Y. lipolytica中的脂肪酸由乙酰輔酶A作為起始分子合成。在脂肪酸合酶的催化下,乙酰輔酶A和丙二酰輔酶A產(chǎn)生?；o酶A,并延長脂肪酸鏈的兩個(gè)碳。酰基輔酶A也可由脂肪?；o酶A合成酶催化外界游離脂肪酸產(chǎn)生。?；o酶A經(jīng)肯尼迪途徑(Kennedy pathway)形成TAG。β-氧化途徑由四個(gè)反應(yīng)組成循環(huán),每個(gè)循環(huán)截?cái)嘀舅徭溨鞲傻膬蓚€(gè)碳,并釋放一分子乙酰輔酶A:第一步反應(yīng)由六個(gè)?；o酶A氧化酶POX催化,六個(gè)酶表現(xiàn)出不同鏈長脂肪酸的偏好;第二步和第三步反應(yīng)由多功能酶MFE催化;第四步反應(yīng)由硫代酶POT1催化。這幾種酶的缺失均可在不同程度上阻斷β-氧化途徑[52]。

OCFA的合成和代謝與常規(guī)脂肪酸相同,因此有研究嘗試在產(chǎn)油酵母中結(jié)合推動(dòng)TAG合成和阻斷脂肪酸β-氧化途徑來達(dá)到積累OCFA的目的。Park等[10]在工程Y. lipolytica的基礎(chǔ)上,通過敲除多功能酶編碼基因(MFE1)和三酰甘油脂肪酶編碼基因(TGL4),過表達(dá)二?；视王；D(zhuǎn)移酶基因(DGA2)和甘油-3-磷酸脫氫酶基因(GPD1),將阻斷β-氧化途徑、抑制TAG的再活化和推動(dòng)TAG合成相結(jié)合,使OCFA和總脂肪酸的積累量分別提升了3.35倍和3.78倍。這種調(diào)控策略常見于脂質(zhì)產(chǎn)物的工程菌構(gòu)建中,在OCFA相關(guān)研究中尚未廣泛出現(xiàn)。例如,Ghogare等[53]在Y. lipolytica中結(jié)合阻斷脂肪酸的β-氧化途徑、弱化脂肪酸活化能力和異源表達(dá)來源于細(xì)菌的硫酯酶三種策略,獲取更高產(chǎn)量的脂肪酸。類似的思路也被用于在釀酒酵母中提升TAG的水平。通過過表達(dá)乙酰輔酶A羧化酶(ACC1)及TAG形成的最后兩個(gè)步驟催化酶:磷脂磷酸酶(PAH1)和二酰基甘油?；D(zhuǎn)移酶(DGA1),破壞TAG脂肪酶基因(TGL3、TGL4、TGL5)和固醇?；D(zhuǎn)移酶基因(ARE1),在含2%葡萄糖的最低培養(yǎng)基中TAG水平達(dá)到了最大理論產(chǎn)量的27%,也是當(dāng)時(shí)釀酒酵母中報(bào)道的最高滴度[54]。目前產(chǎn)油酵母中針通過改造TAG和β-氧化途徑來調(diào)控OCFA合成的相關(guān)研究并不多,而這兩個(gè)途徑對(duì)脂肪酸的合成和降解均有直接的影響,因此或可深度挖掘相關(guān)基因?qū)CFA合成的影響。

3.1.3 模塊化重構(gòu)相關(guān)合成途徑

關(guān)于脂肪酸合成代謝的研究一直受到廣泛的關(guān)注,既涉及許多功能基因的改造,也包含一些新型基因編輯工具的應(yīng)用[55]。因此,在OCFA合成領(lǐng)域,無論是潛在靶點(diǎn)基因或是研究理念,都有許多值得挖掘的地方。得益于合成生物學(xué)技術(shù)的發(fā)展,近些年有關(guān)OCFA的研究不只局限于單個(gè)基因的改造,模塊化重構(gòu)代謝途徑也是有力的改造手段之一。通過組合天冬氨酸合成模塊、高絲氨酸合成模塊和拓展強(qiáng)化的蘇氨酸合成模塊,將草酰乙酸依次轉(zhuǎn)化為天冬氨酸、高絲氨酸和蘇氨酸。蘇氨酸經(jīng)蘇氨酸脫水酶催化脫氨基生成α-酮丁酸,然后被丙酮酸脫氫酶復(fù)合物或丙酮酸氧化酶直接或間接轉(zhuǎn)化為丙酰輔酶A,作為OCFA合成的前體物強(qiáng)化OCFA合成,工程菌株OCFA產(chǎn)量相比對(duì)照提升了7.2倍[11]。Tseng等[47]通過構(gòu)建三個(gè)模塊:前體供應(yīng)、頂部通路和底部途徑來整合九步反應(yīng),從蘇氨酸合成起始積累前體物并在體內(nèi)合成奇數(shù)鏈底物,擴(kuò)大了從簡單碳水化合物通過脂肪酸生物合成和β-氧化途徑產(chǎn)生的酸和醇的底物池。使用旁路策略分析單個(gè)模塊,既能驗(yàn)證復(fù)雜的多步途徑的體內(nèi)功能,又能完全識(shí)別組裝途徑中的潛在瓶頸。結(jié)合Lee等人[46]通過引入蘇氨酸途徑提升OCFA產(chǎn)量的研究,進(jìn)一步拓展了模塊化重構(gòu)OCFA合成途徑的可能性。

3.1.4 關(guān)鍵酶的表達(dá)調(diào)控

除了上述手段,許多關(guān)鍵酶的表達(dá)調(diào)控也能起到正向的結(jié)果。乙酰輔酶A羧化酶作為催化乙酰輔酶A和二氧化碳反應(yīng)生成丙二酰輔酶A的關(guān)鍵酶,有相關(guān)研究顯示強(qiáng)化乙酰輔酶A羧化酶的表達(dá)可以增強(qiáng)脂肪酸的合成[56]。脂肪酸延伸循環(huán)的第一步是由β-酮脂酰-ACP合酶催化,將乙酰輔酶A和丙二酰-ACP轉(zhuǎn)化為乙酰乙酰-ACP,每個(gè)循環(huán)周期增加兩個(gè)碳原子。不同來源的β-酮脂酰-ACP合酶的底物特異性也有所差異,如表現(xiàn)出對(duì)異戊基輔酶A、異丁基輔酶A和2-甲基丁基輔酶A的特異性[57]。有研究在大腸桿菌中評(píng)估了不同來源的β-酮脂酰-ACP合酶對(duì)丙酰輔酶A的特異性,發(fā)現(xiàn)來源于Bacillus subtilis的β-酮脂酰-ACP合酶協(xié)同來源于Salmonella enterica的丙酰輔酶A合酶在48 h生成了最多的奇數(shù)鏈游離脂肪酸[9]。Jin等[44]結(jié)合Cao等[43]的研究基礎(chǔ),在釀酒酵母中整合胞質(zhì)硫酯酶、α雙加氧酶(α-dioxygenase,αDOX)和天然乙醇脫氫酶(alcohol dehydrogenase,ADH)生物合成途徑,在2%的葡萄糖培養(yǎng)基中,分別獲得了19.8 mg/L和20.3 mg/L的奇數(shù)鏈脂肪醛和奇數(shù)鏈脂肪醇。通過強(qiáng)化內(nèi)源關(guān)鍵酶的表達(dá)或異源表達(dá)特定功能的外源酶,對(duì)促進(jìn)OCFA的合成也能達(dá)到理想效果。圖4總結(jié)了以丙酰輔酶A為關(guān)鍵代謝中間物合成OCFA的不同底物轉(zhuǎn)化路徑。

圖4 微生物合成OCFA代謝途徑

Fig.4 Metabolic pathways for microbial synthesis of OCFA

Blue arrows indicate precursors for propionyl coenzyme A; Red arrows indicate the metabolic pathway for conversion of propionyl coenzyme A to OCFA; accABCD, encoding acetyl-CoA carboxylase; ADH, encoding alcohol dehydrogenase; ALDH, encoding aldehyde dehydrogenase; PduCDE, encoding adenosylcobalamin-dependent diol dehydratase; PduP, encoding propionaldehyde dehydrogenase; PCS, propionyl-CoA synthetase; fabA, encoding 3-hydroxydecanoyl-ACP dehydratase; fabB, encoding beta-ketoacyl-ACP synthase; fabD, encoding malonyl-CoA: ACP transacylase; fabF, encoding 3-oxoacyl-ACP synthase Ⅱ; fabG, encoding 3-oxoacyl-ACP reductase; fabH, encoding beta-ketoacyl-ACP synthase III; fabI, encoding enoyl-ACP reductase; αDOX, α-dioxygenases

Full size|PPT slide

3.2 發(fā)酵調(diào)控策略

利用基因工程的手段可以對(duì)工程菌株中的靶點(diǎn)基因直接進(jìn)行敲除、過表達(dá)或替換等操作,同時(shí)由于OCFA的合成特點(diǎn),某些物質(zhì)的外源補(bǔ)加對(duì)OCFA的合成也有著良好的效果。葡萄糖、果糖、乙酸、丙酸、丁酸、乳酸和甘油等均被用來探究對(duì)OCFA合成的影響,但只有丙酸等C3類物質(zhì)被證明能促進(jìn)OCFA的合成。Zhang等[34]在R. opacus PD630培養(yǎng)基中補(bǔ)加1-丙醇,使OCFA產(chǎn)量提升了46.7%~55.1%。Bhatia等[12]以丙酸為最佳碳源,采用響應(yīng)面優(yōu)化法設(shè)計(jì)的合成培養(yǎng)基中含有甘油、丙酸和氯化銨,Rhodococcus sp. YHY01可合成占總脂肪酸85%的OCFA。 ezanka等[58]分別以丙酸或乳酸為唯一碳源培養(yǎng)七種酵母,而只有以丙酸為唯一碳源的酵母能產(chǎn)生大量的OCFA。在丙酸培養(yǎng)基上培養(yǎng)念珠酵母,其中C17:1的產(chǎn)率達(dá)到111 mg/L。丙酸是目前報(bào)道的促進(jìn)OCFA合成最有效的碳源。丙酸被丙酰輔酶A合成酶轉(zhuǎn)化為丙酰輔酶A,作為合成起始先導(dǎo)物刺激OCFA的大量合成。但高濃度丙酸表現(xiàn)出對(duì)細(xì)胞生長明顯的抑制作用,如Park等[10]發(fā)現(xiàn)Y. lipolytica在10 g/L濃度的丙酸環(huán)境中生長狀況受到較大影響,而Fontanille等[13]發(fā)現(xiàn)在5 g/L的濃度時(shí)菌株生長便被抑制。在這種情況下發(fā)酵調(diào)控策略是被優(yōu)先考慮的策略,Park等[10]采用分批補(bǔ)料的策略,在16 h、23 h、40 h和47 h時(shí)分批補(bǔ)加葡萄糖和丙酸,Fontanille等[13]采取兩階段分批補(bǔ)料策略。這種類似的發(fā)酵調(diào)控方法,均在一定程度上減輕了丙酸對(duì)菌體生長的影響,達(dá)到了促進(jìn)OCFA合成的目的。表2為工程策略促進(jìn)微生物合成OCFA的研究進(jìn)展。

表2 工程策略促進(jìn)微生物合成OCFA研究進(jìn)展

Table 2 Advances in engineering strategies promoting OCFA synthesis by microorganisms

生產(chǎn)菌株 OCFA組成主要策略含量參考文獻(xiàn) Escherichia coli C11:0,C13:0 丙酸為碳源,并在大腸桿菌中耦合表達(dá)酰基-ACP硫酯酶、丙酰輔酶A合酶和β-酮酰-ACP合酶III 1 205 mg/L,占總脂肪酸的83.2% [9] Escherichia coli C11:0,C13:0,C15:0 引入硫酯酶基因,過表達(dá)來源于S. enterica的丙酰輔酶A合酶,并外源補(bǔ)加丙酸 297 mg/L [41] Escherichia coli C7-C13的mcl-PHA 引入丙酸同化和代謝途徑至反向脂肪酸β-氧化,敲除丙酮酸氧化酶和丙酮酸甲酸裂解酶,異源表達(dá)來源于Ralstonia eutropha的prpP和prpE基因奇數(shù)鏈mcl-PHA約占總產(chǎn)量的20.03% [59] Yarrowia lipolytica C15:0,C17:0,C17:1,C19:0 構(gòu)建包含七個(gè)基因的模塊化代謝途徑從頭合成奇數(shù)鏈脂肪酸 0.36 g/L [11] Yarrowia lipolytica C15:0,C17:0,C17:1,C19:0 評(píng)估不同來源的丙酸激活酶和丙酰輔酶A轉(zhuǎn)移酶,同時(shí)表達(dá)β-酮硫醇酶 1.87 g/L [49]

4 展望

OCFA具有獨(dú)特的理化性質(zhì),在食品、醫(yī)藥健康、工業(yè)材料和生物燃料等領(lǐng)域有著巨大的應(yīng)用潛力。OCFA在自然界中含量很低,傳統(tǒng)的化學(xué)合成法和提取法由于高成本、低產(chǎn)率, 無法實(shí)現(xiàn)大規(guī)模的工業(yè)化生產(chǎn)。

微生物憑借獨(dú)特的代謝機(jī)制,在合成OCFA方面具有先天優(yōu)勢。隨著基因工程和代謝工程的發(fā)展,眾多基因編輯工具的開發(fā)極大地拓展了改造微生物遺傳特性的方法,對(duì)其內(nèi)源代謝途徑也有了更深刻的了解。借助各種基因編輯工具對(duì)微生物內(nèi)源代謝途徑進(jìn)行理性設(shè)計(jì)和改造,結(jié)合微生物發(fā)酵周期短、不受場地限制等優(yōu)點(diǎn),并輔助發(fā)酵調(diào)控手段,能夠突破微生物原有生產(chǎn)能力的限制,以低成本獲得更高經(jīng)濟(jì)效益。

目前尚未有關(guān)于OCFA大規(guī)模工業(yè)化生產(chǎn)的報(bào)道,相關(guān)研究領(lǐng)域的問題仍局限于菌株生產(chǎn)能力差和轉(zhuǎn)化率低、培養(yǎng)條件和發(fā)酵工藝待優(yōu)化,相關(guān)代謝途徑也有待挖掘。針對(duì)上述問題,今后的研究重點(diǎn)可集中在以下幾個(gè)方面:(1)采用適應(yīng)性進(jìn)化或定向進(jìn)化等手段,結(jié)合高通量篩選技術(shù)選育抗高濃度丙酸的生產(chǎn)菌株;(2)基于基因組學(xué)和代謝組學(xué)等多組學(xué)手段,分析OCFA合成代謝流路,挖掘潛在的代謝回路關(guān)鍵調(diào)控節(jié)點(diǎn)和脅迫基因,實(shí)現(xiàn)OCFA生產(chǎn)菌株代謝網(wǎng)絡(luò)的局部優(yōu)化或全局優(yōu)化;(3)目前的相關(guān)研究局限在代謝調(diào)控和培養(yǎng)條件優(yōu)化等方向,對(duì)于OCFA的發(fā)酵分離等缺少深入的研究,可在發(fā)酵過程中對(duì)產(chǎn)物及時(shí)分離以解除反饋抑制,進(jìn)一步提升產(chǎn)率;(4)動(dòng)態(tài)調(diào)控策略的深入研究使得工程細(xì)胞能夠及時(shí)響應(yīng)外界環(huán)境變化,對(duì)自身相關(guān)基因表達(dá)進(jìn)行精確調(diào)控可在工程菌株中引入動(dòng)態(tài)調(diào)控策略,實(shí)現(xiàn)代謝流和物質(zhì)流的動(dòng)態(tài)分布,提高生產(chǎn)效率。隨著合成生物學(xué)領(lǐng)域的蓬勃發(fā)展和對(duì)微生物合成代謝途徑的明晰,從頭進(jìn)行人工設(shè)計(jì)并構(gòu)建新的生物合成途徑已成為可能。目前運(yùn)用合成生物學(xué)手段已在蛋白表達(dá)、基因調(diào)控和細(xì)胞互作方面取得了極大的研究進(jìn)展。未來將通過合成生物學(xué)的調(diào)控手段,建立更高效的微生物細(xì)胞合成工廠,以期早日實(shí)現(xiàn)OCFA的工業(yè)化生產(chǎn)。

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Amezquita E M

Ganuza T E

. Food additive useful in processed food product or food component, and treating gene metabolic disorders comprises microalgal anaplerotic oil rich in saturated tridecanoic, pentadecanoic, and heptadecanoic odd-chain fatty acids: US20210051988A1. 2021-02-25. [2021-11-20]. https://www.webofscience.com/wos/alldb/full-record/DIIDW:202119412Q.

{{custom_citation.annotation}6}https://doi.org/{{custom_citation.annotation}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citationList}8}{{custom_ref.citationList}4}本文引用 [{{custom_citation.content}5}]摘要{{custom_citation.doi}3}[2]Dornan K

Gunenc A

Oomah B D

, et al. Odd chain fatty acids and odd chain phenolic lipids (alkylresorcinols) are essential for diet. Journal of the American Oil Chemists’ Society, 2021, 98(8): 813-824.

{{custom_citation.doi}1}https://doi.org/{{custom_citation.doi}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.doi}3}{{custom_citation.doi}9}本文引用 [{{custom_citation.doi}0}]摘要{{custom_citation.pmid}8}[3]Liu H B

Yu H Y

Xia J

, et al. Topical azelaic acid, salicylic acid, nicotinamide, sulphur, zinc and fruit acid (alpha-hydroxy acid) for acne. The Cochrane Database of Systematic Reviews, 2020, 5(5): CD011368.

{{custom_citation.pmid}6}https://doi.org/{{custom_citation.pmid}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.pmid}8}{{custom_citation.pmid}4}本文引用 [{{custom_citation.pmid}5}]摘要{{custom_citation.pmid}3}[4]Gillingham M B

Heitner S B

Martin J

, et al. Triheptanoin versus trioctanoin for long-chain fatty acid oxidation disorders: a double blinded, randomized controlled trial. Journal of Inherited Metabolic Disease, 2017, 40(6): 831-843.

Observational reports suggest that supplementation that increases citric acid cycle intermediates via anaplerosis may have therapeutic advantages over traditional medium-chain triglyceride (MCT) treatment of long-chain fatty acid oxidation disorders (LC-FAODs) but controlled trials have not been reported. The goal of our study was to compare the effects of triheptanoin (C7), an anaplerotic seven-carbon fatty acid triglyceride, to trioctanoin (C8), an eight-carbon fatty acid triglyceride, in patients with LC-FAODs.A double blinded, randomized controlled trial of 32 subjects with LC-FAODs (carnitine palmitoyltransferase-2, very long-chain acylCoA dehydrogenase, trifunctional protein or long-chain 3-hydroxy acylCoA dehydrogenase deficiencies) who were randomly assigned a diet containing 20% of their total daily energy from either C7 or C8 for 4 months was conducted. Primary outcomes included changes in total energy expenditure (TEE), cardiac function by echocardiogram, exercise tolerance, and phosphocreatine recovery following acute exercise. Secondary outcomes included body composition, blood biomarkers, and adverse events, including incidence of rhabdomyolysis.Patients in the C7 group increased left ventricular (LV) ejection fraction by 7.4% (p = 0.046) while experiencing a 20% (p = 0.041) decrease in LV wall mass on their resting echocardiogram. They also required a lower heart rate for the same amount of work during a moderate-intensity exercise stress test when compared to patients taking C8. There was no difference in TEE, phosphocreatine recovery, body composition, incidence of rhabdomyolysis, or any secondary outcome measures between the groups.C7 improved LV ejection fraction and reduced LV mass at rest, as well as lowering heart rate during exercise among patients with LC-FAODs.Clinicaltrials.gov NCT01379625.

{{custom_citation.pmid}1}https://doi.org/{{custom_citation.url}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.url}3}{{custom_citation.url}9}本文引用 [{{custom_citation.url}0}]摘要{{custom_citation.url}8}[5]Imamura F

Sharp S J

Koulman A

, et al. A combination of plasma phospholipid fatty acids and its association with incidence of type 2 diabetes: the EPIC-InterAct case-cohort study. PLoS Medicine, 2017, 14(10): e1002409.

{{custom_citation.url}6}https://doi.org/{{custom_citation.url}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citationIndex}8}{{custom_citationIndex}4}本文引用 [{{custom_ref.citationList}5}]摘要{{custom_citation.annotation}3}[6]Weitkunat K

Schumann S

Nickel D

, et al. Odd-chain fatty acids as a biomarker for dietary fiber intake: a novel pathway for endogenous production from propionate. The American Journal of Clinical Nutrition, 2017, 105(6): 1544-1551.

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黃霄霄. 從天然偶碳脂肪酸合成奇碳脂肪酸的研究. 無錫: 江南大學(xué), 2013.

Huang X X

. Synthesis of odd-numbered fatty acids from natural even-numbered fatty acids. Wuxi: Jiangnan University, 2013.

{{custom_ref.citationList}6}https://doi.org/{{custom_ref.citationList}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.id}8}{{custom_ref.id}4}本文引用 [{{custom_ref.citedCount}5}]摘要{{custom_citationIndex}3}[8]Zhang L S

Liang S

Zong M H

, et al. Microbial synthesis of functional odd-chain fatty acids: a review. World Journal of Microbiology & Biotechnology, 2020, 36(3): 35.

{{custom_citationIndex}1}https://doi.org/{{custom_ref.citationList}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citationList}3}{{custom_ref.bodyP_ids}9}本文引用 [{{custom_ref.bodyP_ids}0}]摘要{{custom_bodyP_id}8}[9]Wu H

San K Y

. Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli. Biotechnology and Bioengineering, 2014, 111(11): 2209-2219.

{{custom_bodyP_id}6}https://doi.org/{{custom_bodyP_id}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.annotation}8}{{custom_citation.annotation}4}本文引用 [{{custom_citation.annotation}5}]摘要{{custom_citation.annotation}3}[10]Park Y K

Dulermo T

Ledesma-Amaro R

, et al. Optimization of odd chain fatty acid production by Yarrowia lipolytica. Biotechnology for Biofuels, 2018, 11: 158.

Ledesma-Amaro R

Nicaud J M

. De novo biosynthesis of odd-chain fatty acids in Yarrowia lipolytica enabled by modular pathway engineering. Frontiers in Bioengineering and Biotechnology, 2020, 7: 484.

Gurav R

Choi T R

, et al. A clean and green approach for odd chain fatty acids production in Rhodococcus sp. YHY 01 by medium engineering. Bioresource Technology, 2019, 286: 121383.

Kumar V

Christophe G

, et al. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresource Technology, 2012, 114: 443-449.

The valorization of volatile fatty acids into microbial lipids by the oleaginous yeast Yarrowia lipolytica was investigated. Therefore, a two-stage fed-batch strategy was designed: the yeast was initially grown on glucose or glycerol as carbon source, then sequential additions of acetic acid under nitrogen limiting conditions were performed after glucose or glycerol exhaustion. The typical values obtained with an initial 40 g/L concentration of glucose were close to 31 g/L biomass, a lipid concentration of 12.4 g/L, which correspond to a lipid content of the biomass close to 40%. This cultivation strategy was also efficient with other volatile fatty acids (butyric and propionic acids) or with a mixture of these three VFAs. The lipids composition was found quite similar to that of vegetable oils. The study demonstrated the feasibility of simultaneous biovalorization of volatile fatty acids and glycerol, two cheap industrial by-products.Copyright ? 2012 Elsevier Ltd. All rights reserved.

Dulermo R

Niehus X

, et al. Combining metabolic engineering and process optimization to improve production and secretion of fatty acids. Metabolic Engineering, 2016, 38: 38-46.

Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW.Copyright ? 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Jacob J

Steckel F

. Use of cis-9-heptadecenoic acid for treating psoriasis and allergies: EP, EP19940912534. 1993-03-24[2021-11-20]. https://europepmc.org/article/PAT/EP0690710.

Boulanger R R

Bélanger R R

. Synthesis and biological characterization of (Z)-9-heptadecenoic and (Z)-6-methyl-9-heptadecenoic acids: fatty acids with antibiotic activity produced by Pseudozyma flocculosa. Journal of Chemical Ecology, 2000, 26(4): 987-1000.

Bishop C A

Wittmüss M

, et al. Effect of microbial status on hepatic odd-chain fatty acids is diet-dependent. Nutrients, 2021, 13(5): 1546.

Hervás G

Badia A D

, et al. Effect of dietary lipids and other nutrients on milk odd- and branched-chain fatty acid composition in dairy ewes. Journal of Dairy Science, 2020, 103(12): 11413-11423.

Milk odd- and branched-chain fatty acids (OBCFA) are largely derived from bacteria leaving the rumen, which has encouraged research on their use as biomarkers of rumen function. Targeted research has examined relationships between these fatty acids (FA) and dietary components, but interactions between the effects of lipids and other nutrients on milk OBCFA are not well characterized yet. Furthermore, factors controlling milk OBCFA in sheep are largely unknown. Thus, the present meta-analysis examined relationships between diet composition and milk OBCFA using a database compiled with lot observations from 14 trials in dairy ewes fed lipid supplements. A total of 47 lots received lipid supplements, whereas their respective controls (27 lots) were fed the same basal diets without lipid supplementation. Relationships between milk OBCFA and dietary components were first assessed through a principal component analysis (PCA) and a correlation analysis. Then, responses of milk OBCFA to variations in specific dietary components (selected on the basis of the PCA) were examined in more detail by regression analysis. According to the loading plot, dietary unsaturated C18 FA loaded opposite to major milk OBCFA (e.g., 15:0, 15:0 anteiso, and 17:0) and were strongly correlated with principal component 1, which described 46% of variability. Overall, regression equations supported this negative, and generally linear, relationship between unsaturated C18 FA levels and milk OBCFA. However, the influence of C20-22 n-3 polyunsaturated FA and saturated FA was more limited. The PCA also suggested that dietary crude protein is not a determinant of milk OBCFA profile in dairy ewes, but significant relationships were observed between some OBCFA and dietary fiber or starch, consistent with a potential role of these FA as biomarkers of rumen cellulolytic and amylolytic bacteria. In this regard, regression equations indicated that iso FA would show opposite responses to increasing levels of acid detergent fiber (positive linear coefficients) and starch (negative linear coefficients). Lipid supplementation would not largely affect these associations, supporting the potential of OBCFA as noninvasive markers of rumen function under different feeding conditions (i.e., with or without lipid supplementation). Because consumption of these FA may have nutritional benefits for humans, the use of high-fiber/low-starch rations might be recommended to maintain the highest possible content of milk OBCFA in dairy sheep.Copyright ? 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Biessy C

Torres-Mejía G

, et al. Association between serum phospholipid fatty acid levels and adiposity in Mexican women. Journal of Lipid Research, 2017, 58(7): 1462-1470.

Fatty acids (FAs) have been postulated to impact adiposity, but few epidemiological studies addressing this hypothesis have been conducted. This study investigated the association between serum phospholipid FAs (S-PLFAs) and indicators of obesity. BMI and waist-to-hip ratio (WHR) were collected from 372 healthy Mexican women included as controls in a case-control study. S-PLFA percentages were determined through gas chromatography. Desaturation indices, SCD-16, SCD-18, FA desaturase (FADS)1, and FADS2, biomarkers of endogenous metabolism, were proxied respectively as 16:1n-7/16:0, 18:1n-9/18:0, 20:4n-6/20:3n-6, and 22:6n-3/20:5n-3. Multiple linear regressions adjusted for relevant confounders and corrected for multiple testing were conducted to determine the association between S-PLFA, desaturation indices, and indicators of adiposity. SCD-16 (β = 0.034, = 0.001, q = 0.014), palmitoleic acid (β = 0.031, = 0.001, q = 0.014), and dihomo-γ-linolenic acid (β = 0.043, = 0.000, q = 0.0002) were positively associated with BMI. Total n-6 PUFAs (β = 1.497, = 0.047, q = 0.22) and the ratio of n-6/n-3 PUFAs (β = 0.034, = 0.040, q = 0.19) were positively associated with WHR, while odd-chain FAs (pentadecanoic and heptadecanoic acid) showed negative associations with all the adiposity indicators. In conclusion, increased endogenous synthesis of palmitoleic acid and a high n-6/n-3 ratio are associated with increased adiposity, while odd-chain FAs are associated with decreased adiposity. Further studies are needed to determine the potential causality behind these associations.Copyright ? 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Nicaud J M

. Metabolic engineering for unusual lipid production in Yarrowia lipolytica. Microorganisms, 2020, 8(12): 1937.

沙莎, 鄭玉斌. 利用奇數(shù)碳二元酸制備透明聚酰胺的研究. 塑料科技, 2016, 44(8): 37-41.

Sha S

Zheng Y B

. Study on transparent polyamide with odd carbon dicarboxylic acid. Plastics Science and Technology, 2016, 44(8): 37-41.

{{custom_citation.annotation}6}https://doi.org/{{custom_citation.annotation}2}https://www.ncbi.nlm.nih.gov/pubmed/{{referenceList}8}{{referenceList}4}本文引用 [{{custom_ref.id}5}]摘要{{custom_index}3}[23]Blaskovich M A T

Elliott A G

Kavanagh A M

, et al. In vitro antimicrobial activity of acne drugs against skin-associated bacteria. Scientific Reports, 2019, 9(1): 14658.

Acne is a common skin affliction that involves excess sebum production and modified lipid composition, duct blockage, colonization by bacteria, and inflammation. Acne drugs target one or more of these steps, with antibiotics commonly used to treat the microbial infection for moderate to severe cases. Whilst a number of other acne therapies are purported to possess antimicrobial activity, this has been poorly documented in many cases. We conducted a comparative analysis of the activity of common topical acne drugs against the principal etiological agent associated with acne: the aerotolerant anaerobic Gram-positive organism Propionibacterium acnes (recently renamed as Cutibacterium acnes). We also assessed their impact on other bacteria that could also be affected by topical treatments, including both antibiotic-sensitive and antibiotic-resistant strains, using broth microdilution assay conditions. Drugs designated specifically as antibiotics had the greatest potency, but lost activity against resistant strains. The non-antibiotic acne agents did possess widespread antimicrobial activity, including against resistant strains, but at substantially higher concentrations. Hence, the antimicrobial activity of non-antibiotic acne agents may provide protection against a background of increased drug-resistant bacteria.

{{custom_index}1}https://doi.org/{{custom_ref.nian}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.nian}3}{{custom_ref.citedCount}9}本文引用 [{{custom_ref.citedCount}0}]摘要{{custom_ref.citationList}8}[24]Searle T

Ali F R

Al-Niaimi F

. The versatility of azelaic acid in dermatology. The Journal of Dermatological Treatment, 2022, 33(2): 722-732.

{{custom_ref.citationList}6}https://doi.org/{{custom_ref.citationList}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.content}8}{{custom_citation.content}4}本文引用 [{{custom_citation.doi}5}]摘要{{custom_citation.doi}3}[25]Vockley J

Charrow J

Ganesh J

, et al. Triheptanoin treatment in patients with pediatric cardiomyopathy associated with long chain-fatty acid oxidation disorders. Molecular Genetics and Metabolism, 2016, 119(3): 223-231.

Long-chain fatty acid oxidation disorders (LC-FAOD) can cause cardiac hypertrophy and cardiomyopathy, often presenting in infancy, typically leading to death or heart transplant despite ongoing treatment. Previous data on triheptanoin treatment of cardiomyopathy in LC-FAOD suggested a clinical benefit on heart function during acute failure. An additional series of LC-FAOD patients with critical emergencies associated with cardiomyopathy was treated with triheptanoin under emergency treatment or compassionate use protocols. Case reports from 10 patients (8 infants) with moderate or severe cardiomyopathy associated with LC-FAOD are summarized. The majority of these patients were detected by newborn screening, with follow up confirmatory testing, including mutation analysis; all patients were managed with standard treatment, including medium chain triglyceride (MCT) oil. While on this regimen, they presented with acute heart failure requiring hospitalization and cardiac support (ventilation, ECMO, vasopressors) and, in some cases, resuscitation. The patients discontinued MCT oil and began treatment with triheptanoin, an investigational drug. Triheptanoin is expected to provide anaplerotic metabolites, to replace deficient TCA cycle intermediates and improve effective energy metabolism. Cardiac function was measured by echocardiography and ejection fraction (EF) was assessed. EF was moderately to severely impaired prior to triheptanoin treatment, ranging from 12-45%. Improvements in EF began between 2 and 21days following initiation of triheptanoin, and peaked at 33-71%, with 9 of 10 patients achieving EF in the normal range. Continued treatment was associated with longer-term stabilization of clinical signs of cardiomyopathy. The most common adverse event observed was gastrointestinal distress. Of the 10 patients, 7 have continued on treatment, 1 elected to discontinue due to tolerability issues, and 2 patients died from other causes. Two of the case histories illustrate that cardiomyopathy may also develop later in childhood and/or persist into adulthood. Overall, the presented cases suggest a therapeutic effect of triheptanoin in the management of acute cardiomyopathy associated with LC-FAOD.Copyright ? 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Wittenbecher C

Eichelmann F

, et al. Association of the odd-chain fatty acid content in lipid groups with type 2 diabetes risk: a targeted analysis of lipidomics data in the EPIC-Potsdam cohort. Clinical Nutrition, 2021, 40(8): 4988-4999.

Aoun M

Feillet-Coudray C

, et al. The dietary total-fat content affects the in vivo circulating C15: 0 and C17: 0 fatty acid levels independently. Nutrients, 2018, 10(11): 1646.

{{custom_citation.url}1}https://doi.org/{{custom_citation.url}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.url}3}{{custom_citation.url}9}本文引用 [{{custom_citation.url}0}]摘要{{custom_citationIndex}8}[28]Poppitt S D

. Cow’s milk and dairy consumption: is there now consensus for cardiometabolic health. Frontiers in Nutrition, 2020, 7: 574725.

{{custom_citationIndex}6}https://doi.org/{{custom_citationIndex}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citationList}8}{{custom_ref.citationList}4}本文引用 [{{custom_citation.annotation}5}]摘要{{custom_citation.annotation}3}[29]To N B

Nguyen Y T K

Moon J Y

, et al. Pentadecanoic acid, an odd-chain fatty acid, suppresses the stemness of MCF-7/SC human breast cancer stem-like cells through JAK2/STAT3 signaling. Nutrients, 2020, 12(6): 1663.

{{custom_citation.annotation}1}0}9}!=''" class="new_full_rich_cankaowenxian_zuozhe new_full_rich_cankaowenxian_lianjie">https://doi.org/{{custom_ref.citedCount>0}7}0}6} && {{custom_ref.citedCount>0}5}!=''" class="new_full_rich_cankaowenxian_zuozhe new_full_rich_cankaowenxian_lianjie">https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citedCount>0}3}0}2} && {{custom_ref.citedCount>0}1}!=''" class="new_full_rich_cankaowenxian_zuozhe new_full_rich_cankaowenxian_lianjie">{{custom_citationIndex}9}本文引用 [{{custom_citationIndex}0}]摘要{{custom_ref.id}8}[30]Venn-Watson S

Lumpkin R

Dennis E A

. Efficacy of dietary odd-chain saturated fatty acid pentadecanoic acid parallels broad associated health benefits in humans: could it be essential. Scientific Reports, 2020, 10: 8161.

Dietary odd-chain saturated fatty acids (OCFAs) are present in trace levels in dairy fat and some fish and plants. Higher circulating concentrations of OCFAs, pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0), are associated with lower risks of cardiometabolic diseases, and higher dietary intake of OCFAs is associated with lower mortality. Population-wide circulating OCFA levels, however, have been declining over recent years. Here, we show C15:0 as an active dietary fatty acid that attenuates inflammation, anemia, dyslipidemia, and fibrosis in vivo, potentially by binding to key metabolic regulators and repairing mitochondrial function. This is the first demonstration of C15:0's direct role in attenuating multiple comorbidities using relevant physiological mechanisms at established circulating concentrations. Pairing our findings with evidence that (1) C15:0 is not readily made endogenously, (2) lower C15:0 dietary intake and blood concentrations are associated with higher mortality and a poorer physiological state, and (3) C15:0 has demonstrated activities and efficacy that parallel associated health benefits in humans, we propose C15:0 as a potential essential fatty acid. Further studies are needed to evaluate the potential impact of decades of reduced intake of OCFA-containing foods as contributors to C15:0 deficiencies and susceptibilities to chronic disease.

{{custom_ref.id}6}https://doi.org/{{custom_ref.id}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.citedCount}8}{{custom_ref.citedCount}4}本文引用 [{{custom_citationIndex}5}]摘要{{custom_ref.citationList}3}[31]Ediriweera M K

To N B

Lim Y

, et al. Odd-chain fatty acids as novel histone deacetylase 6 (HDAC6) inhibitors. Biochimie, 2021, 186: 147-156.

{{custom_ref.citationList}1}https://doi.org/{{custom_ref.bodyP_ids}7}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_ref.bodyP_ids}3}{{custom_ref.id}9}本文引用 [{{custom_ref.id}0}]摘要{{custom_citation.annotation}8}[33]Wen J

Hu C Q

Fan S G

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{{custom_citation.annotation}6}https://doi.org/{{custom_citation.annotation}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_citation.annotation}8}{{custom_citation.annotation}4}本文引用 [{{custom_citation.annotation}5}]摘要{{custom_citation.annotation}3}[34]Zhang L S

Xu P

Chu M Y

, et al. Using 1-propanol to significantly enhance the production of valuable odd-chain fatty acids by Rhodococcus opacus PD630. World Journal of Microbiology & Biotechnology, 2019, 35(11): 164.

Lassen J

Noel S J

, et al. Impact of the rumen microbiome on milk fatty acid composition of Holstein cattle. Genetics, Selection, Evolution: GSE, 2019, 51(1): 23.

Fatty acids (FA) in bovine milk derive through body mobilization, de novo synthesis or from the feed via the blood stream. To be able to digest feedstuff, the cow depends on its rumen microbiome. The relative abundance of the microbes has been shown to differ between cows. To date, there is little information on the impact of the microbiome on the formation of specific milk FA. Therefore, in this study, our aim was to investigate the impact of the rumen bacterial microbiome on milk FA composition. Furthermore, we evaluated the predictive value of the rumen microbiome and the host genetics on the composition of individual FA in milk.Our results show that the proportion of variance explained by the rumen bacteria composition (termed microbiability or [Formula: see text]) was generally smaller than that of the genetic component (heritability), and that rumen bacteria influenced most C15:0, C17:0, C18:2 n-6, C18:3 n-3 and CLA cis-9, trans-11 with estimated [Formula: see text] ranging from 0.26 to 0.42. For C6:0, C8:0, C10:0, C12:0, C16:0, C16:1 cis-9 and C18:1 cis-9, the variance explained by the rumen bacteria component was close to 0. In general, both the rumen microbiome and the host genetics had little value for predicting FA phenotype. Compared to genetic information only, adding rumen bacteria information resulted in a significant improvement of the predictive value for C15:0 from 0.22 to 0.38 (P?=?9.50e-07) and C18:3 n-3 from 0 to 0.29 (P?=?8.81e-18).The rumen microbiome has a pronounced influence on the content of odd chain FA and polyunsaturated C18 FA, and to a lesser extent, on the content of the short- and medium-chain FA in the milk of Holstein cattle. The accuracy of prediction of FA phenotypes in milk based on information from either the animal's genotypes or rumen bacteria composition was very low.

{{fundList_cn}6}https://doi.org/{{fundList_cn}2}https://www.ncbi.nlm.nih.gov/pubmed/{{custom_fund}8}{{custom_fund}4}本文引用 [{{custom_ref.citedCount}}]摘要{{custom_citation.annotation}}[36]Kolouchová I

Schreiberová O

Sigler K

, et al. Biotransformation of volatile fatty acids by oleaginous and non-oleaginous yeast species. FEMS Yeast Research, 2015, 15(7): fov076.

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Diao J J

, et al. Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31. Biotechnology for Biofuels, 2019, 12: 141.

Jackson M A

Hartman T M

, et al. Branched chain lipid metabolism as a determinant of the N-acyl variation of Streptomyces natural products. ACS Chemical Biology, 2021, 16(1): 116-124.

Branched-chain fatty acids (BCFA) are encountered in Gram-positive bacteria, but less so in other organisms. The bacterial BCFA in membranes are typically saturated, with both odd- and even-numbered carbon chain lengths, and with methyl branches at either the ω-1 () or ω-2 () positions. The acylation with BCFA also contributes to the structural diversity of microbial natural products and potentially modulates biological activity. For the tunicamycin (TUN) family of natural products, the toxicity toward eukaryotes is highly dependent upon -acylation with -2,3-unsaturated BCFA. The loss of the 2,3-unsaturation gives modified TUN with reduced eukaryotic toxicity but crucially with retention of the synergistic enhancement of the β-lactam group of antibiotics. Here, we infer from genomics, mass spectrometry, and deuterium labeling that the -2,3-unsaturated TUN variants and the saturated cellular lipids found in TUN-producing are derived from the same pool of BCFA metabolites. Moreover, non-natural primers of BCFA metabolism are selectively incorporated into the cellular lipids of TUN-producing and concomitantly produce structurally novel -branched TUN -acyl variants.

. The CRISPR tool kit for genome editing and beyond. Nature Communications, 2018, 9(1): 1911.

Huang H

Kerkhoven E J

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. Engineering Escherichia coli for odd straight medium chain free fatty acid production. Applied Microbiology and Biotechnology, 2014, 98(19): 8145-8154.

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Schrader J

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Liu D

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Foo J L

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Marsafari M

Wang F

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Hu Z H

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Gabba E J

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Exogenous propionate is incorporated in vivo by Escherichia coli as a primer to produce lipids with fatty acids of odd chain lengths. This provides a method for the specific labeling of the three terminal carbons in the fatty acyl chains of phospholipids.

Bordes F

Letisse F

, et al. Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica. Metabolic Engineering Communications, 2021, 12: e00158.

Hou J

Zhang F

, et al. Multiple propionyl coenzyme A-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferax mediterranei. Applied and Environmental Microbiology, 2013, 79(9): 2922-2931.

Verbeke J

Beopoulos A

, et al. A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica. Applied Microbiology and Biotechnology, 2017, 101(11): 4605-4616.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of β-oxidation (pox1-6?), the inability to store lipids as triglycerides (dga1? dga2? are1? lro1?), and the overexpression of the Y. lipolytica ?12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating β-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Nicaud J M

. Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Progress in Lipid Research, 2016, 61: 40-50.

One of the most promising alternatives to petroleum for the production of fuels and chemicals is bio-oil based chemistry. Microbial oils are gaining importance because they can be engineered to accumulate lipids enriched in desired fatty acids. These specific lipids are closer to the commercialized product, therefore reducing pollutants and costly chemical steps. Yarrowia lipolytica is the most widely studied and engineered oleaginous yeast. Different molecular and bioinformatics tools permit systems metabolic engineering strategies in this yeast, which can produce usual and unusual fatty acids. Usual fatty acids, those usually found in triacylglycerol, accumulate through the action of several pathways, such as fatty acid/triacylglycerol synthesis, transport and degradation. Unusual fatty acids are enzymatic modifications of usual fatty acids to produce compounds that are not naturally synthetized in the host. Recently, the metabolic engineering of microorganisms has produced different unusual fatty acids, such as building block ricinoleic acid and nutraceuticals such as conjugated linoleic acid or polyunsaturated fatty acids. Additionally, microbial sources are preferred hosts for the production of fatty acid-derived compounds such as γ-decalactone, hexanal and dicarboxylic acids. The variety of lipids produced by oleaginous microorganisms is expected to rise in the coming years to cope with the increasing demand.Copyright ? 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chen S L

Xiong X C

. Metabolic engineering of oleaginous yeast Yarrowia lipolytica for overproduction of fatty acids. Frontiers in Microbiology, 2020, 11: 1717.

Teixeira P G

Gossing M

, et al. Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols. Metabolic Engineering Communications, 2018, 6: 22-27.

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase,, as well as the last two steps of TAG formation: phosphatidic phosphatase () and diacylglycerol acyltransferase (), ultimately leading to 129?mg?gCDW of TAGs. Disruption of TAG lipase genes,, and sterol acyltransferase gene increased the TAG content to 218?mg?gCDW. Further disruption of the beta-oxidation by deletion of, as well as glycerol-3-phosphate utilization through deletion of, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter led to accumulation of 254?mg?gCDW. The TAG levels achieved here are the highest titer reported in, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.

Fan J

Luo S

, et al. Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids. Nature Communications, 2021, 12(1): 4976.

Gu Q

Wang W

, et al. Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nature Communications, 2013, 4: 1409.

Middelhaufe S

Moore K

, et al. Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(19): 7636-7641.

Kolouchová I

Sigler K

, Precursor directed biosynthesis of odd-numbered fatty acids by different yeasts. Folia Microbiologica, 2013, 110(19): 7636-7641.

Qi Q S

. Engineering the pathway in Escherichia coli for the synthesis of medium-chain-length polyhydroxyalkanoates consisting of both even- and odd-chain monomers. Microbial Cell Factories, 2019, 18(1): 135.

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